Mesophilic and Thermophilic Anaerobic

Digestion of Municipal Sludge and

Fat, Oil, and Grease
John C. Kabouris1*, Ulas Tezel2, Spyros G. Pavlostathis2, Michael Engelmann3,
James A. Dulaney3, Allen C. Todd1, Robert A. Gillette1

ABSTRACT: The anaerobic biodegradability of municipal primary

sludge, thickened waste activated sludge (TWAS), and fat, oil, and grease
(FOG) was assessed using semi-continuous-feed, laboratory-scale anaerobic
digesters and compared with the ultimate degradability obtained from 120day batch digestion at 358C. In run 1, combined primary sludge and TWAS
(40/60%, volatile solids [VS] basis) were fed to digesters operated at
mesophilic (358C) and thermophilic (528C) temperatures at loading rates of
0.99 and 1.46 g-VS/L
d for primary sludge and TWAS, respectively, and
a hydraulic retention time (HRT) of 12 days. The volatile solids destruction
values were 25.3 and 30.7% (69 and 83% biodegradable volatile solids
destruction) at 358C and 528C, respectively. The methane (CH4) yields were
159 and 197 mL at the standard temperature and pressure (STP) conditions
of 08C and 1 atm/g-VS added or 632 and 642 mL @ STP/g-VS destroyed at
358C and 528C, respectively. In run 2, a mix of primary sludge, TWAS, and
FOG (21/31/48%, volatile solids basis) was fed to an acid digester operated
at a 1-day HRT, at 358C, and a loading rate of 52.5 g-VS/L
d. The acidreactor effluent was fed to two parallel methane-phase reactors operated at
an HRT of 12 days and maintained at 358C and 528C, respectively. After an
initial period of 20 days with near-zero gas production in the acid reactor,
biogas production increased and stabilized to approximately 2 mL CH4 @
STP/g-VS added, corresponding to a volatile solids destruction of 0.4%.
The acid-phase reactor achieved a 43% decrease in nonsaturated fat and a
16, 26, and 20% increase of soluble COD, volatile fatty acids, and ammonia,
respectively. The methane-phase volatile solids destruction values in run 2
were 45 and 51% (85 and 97% biodegradable volatile solids destruction) at
358C and 528C, respectively. The methane yields for the methane-phase
reactors were 473 and 551 mL @ STP/g-VS added, which is approximately
3 times larger compared with run 1, or 1040 and 1083 mL @ STP/g-VS
destroyed, at 358C and 528C, respectively. The results indicate that,
when co-digesting municipal sludge and FOG, a large FOG organic load
fraction could have a profound effect on the methane gas yield. Water
Environ. Res., 81, 476 (2009).
KEYWORDS: acid phase, anaerobic digestion, mesophilic, thermophilic,
FOG, lipids, methane production, municipal sludge, fatty acids, codigestion.
doi:10.2175/106143008X357192

Carollo Engineers, Sarasota, Florida.

School of Civil & Environmental Engineering, Georgia Institute of

Technology, Atlanta, Georgia.

Pinellas County Utilities, Clearwater, Florida.

* Carollo Engineers, 401 North Cattlemen Road, Sarasota, FL 34232;

e-mail: jkabouris@carollo.com.
476

Introduction
The addition and co-digestion of fat, oil, and grease (FOG) in
municipal sludge digesters have been implemented in several fullscale facilities for improving biogas production. Cotrell (2008) reported a 50% increase in biogas production at a full-scale digestion
facility accepting and co-digesting sludge and FOG at an FOG
loading rate of 0.48 kg-VS/m3
d. The potential for an increase in
methane production by 100% or more through the addition and codigestion of FOG with municipal sludge has been demonstrated in
laboratory studies (e.g., Kabouris et al., 2008; Suto et al., 2006).
However, long-chain fatty acids (LCFAs) low solubility, adsorption,
and inhibition of anaerobic microorganisms have been listed as causes
for several operational problems in anaerobic systems treating waste
streams with relatively high lipid contents (Angelidaki and Ahring,
1992; Hanaki et al., 1981; Hwu et al., 1998; Lalman and Bagley,
2002; Pereira et al., 2001).
Acid-methane-phased digestion has been used to reduce the risk
of toxicity from lipids. A two-phase anaerobic system successfully
treated a synthetic wastewater that contained a mixture of LCFAs
at loading rates up to 1.72 kg LCFA-chemical oxygen demand
(COD)/m3
d (Kim et al., 2004). Eastman and Ferguson (1981)
studied the acid-phase digestion of primary sludge in laboratoryscale reactors. Batch acid-phase digestion at 358C for 2.75 days
following continuous acid digestion and before the onset of active
methane formation resulted in a 70% reduction of carbohydrates,
which were mostly cellulose; a 55% reduction of nitrogenous
material; and no reduction of lipids.
In addition to reducing the potential for lipid toxicity, acidmethane-phased digestion has the potential to significantly accelerate reaction rates and increase volatile solids reduction and process
stability by providing the optimal environment for the growth of
acid- and methane-producing organisms (Andryszak et al., 2004;
Ghosh et al., 1975, 1995; Harrison et al., 2005). Ghosh (1987)
performed a comparison of a laboratory-scale conventional and
phase digestion of a primary sludge and WAS mix at mesophilic
(358C) and thermophilic (558C) conditions and 15 days overall HRT
with 2 days acid HRT. The ultimate anaerobic degradability was
58% of volatile solids, and the methane yield at 358C single-stage
digestion was 0.225 m3 at the standard temperature and pressure
(STP) conditions of 08C and 1 atm per kg-VS added. The methane
yield increased by 24, 82, and 34% when digestion was performed
at single stage at 558C, acid-methane-phase reactors at 358C, and
acid phase at 358C with methane phase at 558C, respectively.
The volatile solids destruction values based on the gas yield were
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Kabouris et al.

estimated as 29.7, 39.4, 54.9, and 42% for the single stage at 358C,
single stage at 558C, acid-methane at 358C, and acid at 358C with
methane at 558C, respectively. The associated biodegradable volatile solids reduction values were 51.2, 67.9, 94.9, and 72.4%, and
the overall carbohydrate-lipids-protein sum degradation values were
26.6, 51.5, 57.1, and 46%, respectively. Crude protein and lipids
were degraded more efficiently than carbohydrates under phased
digestion and thermophilic conditions, but all three components
were degraded at approximately the same extent in the 358C singlestage digester.
The municipal sludge and FOG used in this study was provided
by Pinellas County, Florida. The county operates the South Cross
Bayou Water Reclamation Facility (SCBWRF) at an average daily
flow of 125 3 106 L/d (33 mgd). To increase biogas production and
provide a beneficial reuse for FOG, in addition to SCBWRF sludge,
the county has been feeding the SCBWRF egg-shape digesters
partially dewatered FOG from the countys offsite FOG-receiving
facility. The SCBWRF primary clarifiers have traditionally been
partially bypassed and, in combination with the operation of a long
solids retention time (SRT) modified Lutzack-Ettinger activated
sludge process with alum addition before the secondary clarifiers,
results in a relatively difficult-to-degrade sludge, which has traditionally been a 20/80% (total solids basis) primary sludge/WAS
sludge mix (Kabouris et al., 2007). When the samples for this study
were collected, SCBWRF was operated at an SRT of approximately
18 to 22 days. The countys FOG dewatering facility receives
FOG obtained from grease traps associated with, among others,
restaurants, hospitals, schools, assisted-living facilities, and catering
services. The facility uses a FOG dewatering process based on
polymer addition and gravity water drainage to generate partially
dewatered FOG.
The objective of this study was to assess and quantify the
anaerobic biodegradation of municipal sludge when co-digested
with large quantities of FOG, under a completely mixed semicontinuously fed mesophilic-acid phase followed by mesophilic
and thermophilic methane-phase digestion. Such information is very
valuable for establishing the feasibility and associated cost savings
from the co-digestion of municipal sludge and dewatered FOG. To
provide baseline data, ultimate degradability batch digestion testing
was used for the sludge and FOG components, with mesophilic and
thermophilic semi-continuous feed methane-phase digestion of
sludge without and with FOG.
Materials and Methods
Samples. Three samples were obtained from the Pinellas
County SCBWRF, as follows: primary sludge (PS), thickened
waste activated sludge (TWAS), and polymer-dewatered FOG. The
sludge and FOG samples were shipped to the laboratory at the
Georgia Institute of Technology (Atlanta, Georgia) overnight, and
upon delivery, they were stored at 48C. Because the primary sludge
was relatively dilute (1.2% total solids, 0.9% volatile solids), the
following procedure was followed to prepare a more concentrated
primary sludge. The sludge was sequentially passed through a 5 3
5-mm square mesh screen, a 2-mm sieve (U.S. Standard No. 10),
and, finally, a 1.4-mm sieve (U.S. Standard No. 14). Screening
resulted in a minor loss of sludge solids. The screened primary
sludge was stored at 48C for 2 days, and then the supernatant was
decanted, thus creating two sub-samplesprimary supernatant and
primary concentrate. The FOG sample was placed in a plastic
container, spread thin and mixed thoroughly using a spatula, and the
container was firmly sealed to avoid water losses. Diluted samples
May 2009

of TWAS and FOG were prepared by mixing 10 g dry sample in 1 L

of deionized water (1% total solids) in wide-mouth glass bottles,
mechanically mixed for 1 hour, and then used to measure soluble
COD, volatile fatty acids (VFAs), and ammonia after sample centrifugation (5000 rpm for 15 minutes) and filtration (0.2-lm membrane filter). Total COD was measured by directly adding a known
mass of wet sample to replicate COD reflux flasks. All other
analyses were performed with undiluted sludge and FOG samples.
Details on the analytical methods are described below.
Batch Biodegradability Test. A batch biodegradability test
was performed with the primary sludge, TWAS, and FOG separately, and with combinations of these components (PS 1 TWAS
and PS 1 TWAS 1 FOG) using 160-mL serum bottles. Seed for
this test was digester liquor obtained from the Pinellas County
SCBWRF mesophilic anaerobic digesters, which was then further
digested in the laboratory under batch conditions at 358C for 90
days. Primary sludge and TWAS were tested separately at a volatile
solids concentration of 3 g/L (3 kg/m3). The FOG volatile solids
concentration was 2.39 g/L, as the result of an initial underestimation of the total and volatile solids contents of this sample. The
combined PS 1 TWAS sample had volatile solids concentrations of
1.16 and 1.84 g/L, respectively (3 g-VS/L total), whereas the combined PS 1 TWAS 1 FOG sample had volatile solids concentrations of 1.16, 1.84, and 0.48 g/L, respectively (3.48 g VS/L total).
Incubation was carried out in the dark at 358C, and the bottles were
shaken manually once per day. Details of the batch biodegradability
testing have been presented by Kabouris et al. (2007).
Digestion System. The complete digestion system included
two 4-L and two 1-L glass reactors. All reactors had a water jacket,
and their temperature was controlled with water recirculation using
two heated water-circulating baths. One 4-L reactor was maintained
at 358C (mesophilic), and the other 4-L reactor was maintained at
528C (thermophilic). A 1-L reactor served as the feed reservoir,
was maintained at room temperature (22 to 248C), and was cleaned
every 1 to 2 days. The other 1-L reactor was used as the acid-phase
reactor and was maintained at 358C. Mixing in all reactors was
achieved with Teflon (DuPont, Wilmington, Delaware) shafts
carrying three flat paddles perpendicularly positioned and rotating
at 90 rpm driven by adjustable-speed motors, which were magnetically coupled to the shafts. Wasting and then feeding of the acidphase reactor was achieved for 5 minutes on every hour with two
peristaltic pumps. Both methane-phase reactors were wasted manually once per day by syringe, 5 minutes before feeding. Feeding of
the two methane-phase reactors was achieved either manually once
per day using a 60-mL syringe (run 1) or with a peristaltic pump
for 5 minutes every 3 hours (run 2). The peristaltic pumps were
controlled by an electronic timer. Gas produced in both the acidphase and methane-phase reactors was collected in graduated
cylinders filled with an acid brine solution (10% NaCl w/v, 2%
H2SO4 v/v). Gas samples were taken directly from the reactors by
syringe and analyzed as described below.
Reactors Startup. The mesophilic, methane-phase reactor
was started with a digester liquor sample obtained from the Pinellas
County SCBWRF mesophilic anaerobic digesters. The digester
sample was screened through a U.S. Standard No. 20 sieve
(850 lm) and then kept in a sealed, plastic bottle with minimal
headspace volume at room temperature (22 to 248C) for 2 months,
before it was transferred to the mesophilic, methane-phase reactor.
The thermophilic, methane-phase reactor was started following
the same procedure as that of the mesophilic reactor with a digester
liquor sample obtained from the thermophilic methane-phase
477

Kabouris et al.

digesters of Regional Plant 1, operated by Inland Empire Utilities

Agency and located in Ontario, California. Both reactors were
equilibrated at their respective temperature without any feeding for
4 days before regular feeding started (see run 1, below). The initial
pH values, before any feeding, of the mesophilic and thermophilic
reactor were 7.4 and 7.6, respectively. The acid-phase reactor was
started by transferring 0.5 L of a mix of primary sludge, TWAS, and
FOG to this reactor, and batch incubating it at 358C for 2 days.
Digestion Runs. Continuous testing of primary sludge, TWAS,
and FOG under both mesophilic and thermophilic conditions was
conducted in two runs. Run 1 was a direct comparison of mesophilic versus thermophilic digestion, and the feed was a mix of
primary sludge and TWAS without preacidification in an acid-phase
reactor (i.e., conventional). For run 2, the feed was a mix of primary
sludge, TWAS, and FOG and was pre-acidified by the use of a
mesophilic acid-phase reactor. Portions of the acid digester effluent
were fed to two side-by-side methane-phase digesters operating at
mesophilic and thermophilic temperatures, respectively. For each
run, the following analyses were performed on both the feed and
reactor effluent: total and volatile solids, total and soluble COD,
VFAs, ammonia, pH, gas production, gas composition (CH4 and
CO2), carbohydrate, protein, fat, total Kjeldahl nitrogen (TKN), and
total and water-soluble phosphorus.
Analytical Methods. The pH, total and volatile solids, COD,
TKN, ammonia, and total and water-soluble phosphorus were measured following procedures outlined in Standard Methods (APHA
et al., 2005). The VFAs (C2 to C7) were measured using an HP 5890
Series II GC (Hewlett Packard, Palo Alto, California) equipped with
a flame ionization detector and a 35-m Stabilwax-DA, 0.53-mm
internal diameter column (Restek, Bellefonte, Pennsylvania). Samples used for the measurement of soluble COD, VFAs, and ammonia
were prepared by centrifugation at 10 000 rpm for 30 minutes and
filtration through 0.2-lm syringe filters.
The fat content of sludge and FOG samples was measured following procedures outlined in AOAC International (Gaithersburg,
Maryland) method 922.06/948.15 (AOAC, 2005). Although single
measurements were performed for total fat analyses using 5-day
composite sampling, the coefficient of variation for these analyses
was not expected to exceed 12%. The crude protein content
was estimated by multiplying the organic nitrogen (TKN minus
ammonia nitrogen) by 6.25 (for a typical 16% nitrogen content of
proteins). The difference of the volatile solids and the sum of fat
plus protein was designated as carbohydrate. As a result, the carbohydrate estimates are influenced by the uncertainty in volatile solids,
fat, and protein estimates and are expected to be more uncertain
compared with the fat and protein estimates. The saturated, polyunsaturated, monounsaturated, and trans fat fractions of the total fat
were determined following AOCS method Ce-1h-05 (AOCS, 1971).
Total gas production was measured by displacing an acidified
brine solution (10% NaCl w/v, 2% H2SO4 v/v) in a graduated
burette and recording the volume of displaced solution after correcting to atmospheric pressure. To establish the gas composition,
methane, carbon dioxide, and nitrogen were determined by a gas
chromatography unit (model 6890N, Agilent Technologies, Santa
Clara, California) equipped with two columns and two thermal
conductivity detectors. Methane and nitrogen were separated with
a 25-m Chrompack Molsieve (Varian, Palo Alto, California) 5A
fused silica, 0.53-mm internal diameter column. Carbon dioxide and
hydrogen sulfide were separated with a 25-m Chrompack PoraPlot
Q fused silica, 0.53-mm internal diameter column. Both columns
were operated with helium as the carrier gas at a constant flowrate
478

of 6 mL/min. The 10:1 split injectors were maintained at 1508C, the

oven was set at 408C, and the detectors temperature was set to
1508C. Molecular hydrogen was measured with the above-described
gas chromatography unit and Molsieve column, except the carrier
gas was nitrogen. The minimum gas-phase detection limits for CH4,
CO2, N2, H2S, and H2 were 0.5, 0.8, 0.05, 0.1, and 0.5 mL/L,
respectively.
Results and Discussion
Sample Characterization. The results of the analysis of the
sludge and FOG samples are shown in Table 1. All sludge and FOG
samples were acidic, with the TWAS sample having the highest pH
value of 6.61 and the FOG sample having the lowest value of 4.03.
The TWAS sample had the lowest COD/VS value (approximately
1.94), and the FOG COD/VS value was 2.96. A significant level
of soluble COD and VFAs was found in the liquid portion of all
samples, indicating that a degree of sample solubilization and preacidification had taken place during sample transport and/or storage.
The primary sludge samples soluble-COD-to-total-COD ratio was
equal to 0.1 and was significantly higher compared with that of the
TWAS and FOG samples. The VFA-COD-to-soluble-COD ratio for
the primary sludge sample at 0.30 also was significantly higher than
the ratios for the TWAS and FOG samples. Acetate, followed by
propionate, were the major fractions of the total VFAs COD measured, and the remainder was approximately equally divided among
isobutyric, butyric, isovaleric, and valeric acids.
The primary sludge contents in carbohydrate, protein, and fat
were 39, 30, and 31% of volatile solids, respectively, and over 75%
of the fat was saturated. The predominant saturated fat components
were palmitic acid (16:0), followed by stearic acid (18:0) and
myristic acid (14:0). The TWAS contents in carbohydrate, protein,
and fat were 48, 46, and 6% of volatile solids, respectively. Over
71% of the TWAS fat was saturated and comprised of (in
descending order) palmitic acid (16:0), myristic acid (14:0), stearic
acid (18:0), and very low levels of lauric acid (12:0) and capric acid
(10:0). The dewatered FOG contents in carbohydrate, protein, and
fat were 15, 7, and 78% of volatile solids, respectively. As expected,
the dewatered FOG fat content was very high (78% of volatile
solids), and its protein content was low (7% of volatile solids). The
majority of the fat in the FOG sample was saturated and monounsaturated, followed by trans fat and polyunsaturated fat. Palmitic
acid (16:0) was the major component of the FOG saturated fat,
followed by stearic acid (18:0) and arachnidic acid (20:0). Oleic
acid (18:1) was the major component of the FOG monounsaturated
fat, followed by eicosenoic acid (20:1), linoleic acid (18:2W6), and
palmitoleic acid (16:1).
All samples had high (above 200 mg/L) ammonia concentrations,
consistent with a degree of sample solubilization and ammonification having taken place during sample transport and/or storage. The
TKN-to-VS ratio for FOG was equal to 0.01 and was significantly
lower compared with the values of 0.06 and 0.08 for primary sludge
and TWAS, respectively, consistent with the low protein content
of the FOG sample. All samples had relatively low water-soluble
phosphorus content. The TWAS sample had the highest total
phosphorus content. The soluble and total phosphorus content is
consistent with the full-scale practice of alum addition to the
SCBWRF sludge for phosphorus removal to average effluent levels
below 1 mg/L.
Ultimate Biodegradability. The biodegradability test was
carried out for 120 days. Table 2 summarizes the results of the
batch test for the three individual samples. Table 2 also includes the
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Kabouris et al.

Table 1Results of sludge and FOG sample analysis.a

Parameter
pH
Total solids, g/kg wet sampleb
Volatile solids, g/kg wet sample
VS/TS, %
Total COD, g/kg wet sample
Soluble COD, g/L
Soluble COD/total COD, mg/mg
VFAs, mg COD/L
VFA COD/soluble COD, mg/mg
Total COD/volatile solids, g/g
Carbohydrate, g/kg wet sample (% of VS)
Protein, g/kg wet sample (% of VS)
Total fat, g/kg wet sample (% of VS)
Saturated fat, g/kg (% of total fat)
Polyunsaturated fat, g/kg (% of total fat)
Monounsaturated fat, g/kg (% of total fat)
Trans fat, g/kg (% of total fat)
Total nitrogen, g-N/kg wet sample
Total Kjeldahl nitrogen (TKN), g-N/kg wet sample
Ammonia, mg-N/L
Ammonia/TKN, mg-N/mg-N
TKN/VS, mg/mg
Organic nitrogen, mg-N/mg-VS
Total phosphorus, mg-P/kg wet sample
Water soluble phosphorus, mg-P/kg wet sample

Primary sludge
supernatant

Primary sludge
concentrate

Primary
sludge mix

5.49
3.9 6 0.1c
2.3 6 0.1
59.0
11.6 6 0.2
4.4 6 0.1
0.38
1295
0.29
5.04

5.63
58.2 6 0.4
47.0 6 0.4
80.8
96.8 6 9.3
4.8 6 0.44.56
0.05
1566
0.33
2.06

123 6 2

431 6 32

5.55
25.0
19.7
78.8
44.8
2.5 6 0.1
0.10
1400
0.30
2.27
7.6 (39)
6.0 (30)
6.1 (31)
4.6 (75.5)
0.1 (2.1)
0.7 (10.9)
0.7 (11.5)
1.3
1.2
243
0.20
0.06
0.05
780
78

TWAS

FOG

6.61
67.0 6 1
44.0 6 1
65.7
85.4 6 1.2
13.7 6 1.3
0.03
567
0.23
1.94
21.2 (48)
20.3 (46)
2.6 (6)
1.9 (71.6)
0.05 (1.8)
0.4 (15.5)
0.3 (11.1)

4.03
424.0 6 8
409.0 6 7
96.5
1211 6 31

3.7
460 6 13
0.12
0.08
0.07
4910
, 10

0.01
3473
0.25
2.96
60.1 (15)
29.7 (7)
319.3 (78)
121 (37.9)
23.6 (7.4)
126.1 (39.5)
48.5 (15.2)
5.4
5.1
356 6 10
0.07
0.01
0.01
670
77

Primary supernatant and concentrate measured directly; primary sludge mix values are for a mixture of primary sludge supernatant and
concentrate (61/39% by volume, respectively), resulting in a mix of 2.5% total solids; TWAS and FOG samples were diluted (1% TS; see text),
and reported values are for the undiluted samples.
b
1 g/kg wet sample 5 1000 mg/L.
c
Mean 6 standard deviation (n  3).

results for the PS391TWAS61 and PS211TWAS311FOG48 combinations, with subscripts indicating the volatile solids percentage for
each component, which represents the feed mix for runs 1 and 2,
respectively. For all samples tested, the amount of methane produced normalized to the COD destroyed varied from 338 to 343
mL-CH4 @ STP/g-COD destroyed, which is close to the theoretical
value of 350 mL-CH4 @ STP/g-COD destroyed. Based on these
results, the ultimate biodegradability of the three individual samples
follows the descending series FOG, primary sludge, and TWAS.
The same descending order is applicable to the observed methane
yield per unit volatile solids mass added for the three samples.
The results of the biodegradability test for several additional
combinations of sludge and FOG were reported by Kabouris et al.
(2007), who also observed that the results for a mix of components
could be reasonably predicted by assuming that each waste
components contribution to the overall volatile solids and COD
destructions was additive (i.e., remained the same in systems where
samples are combined). Based on the ultimate biodegradability
results of each component (i.e., primary sludge, TWAS, and FOG)
and taking into account the volatile solids fraction of each component in the PS211TWAS311FOG48 combination, the projected
mix volatile solids and COD destructions and the methane and
biogas yields were computed and are presented in Table 2.
Digestion Run 1. This run was conducted to directly compare
mesophilic versus thermophilic digestion of a mix of primary sludge
and TWAS. Both reactors were maintained under exactly the same
May 2009

conditions, except for their temperatures (35 versus 528C). The

reactor characteristics during run 1 are listed in Table 3. Table 4
shows the feed composition during run 1. A significant fraction
(approximately 9%) of the total feed COD was soluble COD,
indicating that a certain degree of sludge acidification had taken
place, which also is reflected by the relatively low feed pH of 6.4
and high VFA value of 3500 mg/L (approximately 73% of the
soluble COD)values compatible with those observed in some
full-scale acid-phase digesters (Andryszak et al., 2004). In addition,
further acidification may have occurred in the feed vessel that was
maintained at room temperature (22 to 248C) and for 1 to 2 days.
The protein and fat fractions of the feed volatile solids were 40 and
16%, respectively. Saturated fat comprised more than 80% of the
total feed fat.
Run 1 lasted for 42 days, which corresponds to 3.5 retention times
for both methane-phase reactors. The total gas production and pH in
the mesophilic and thermophilic reactors, with the feed pH during the
entire run 1, are shown in Figure 1. Following the first HRT of 12
days, the reactors gas production became significantly more stable
and further stabilized following the second 12-day HRT period. A
gradual decrease of the pH in the mesophilic reactor was observed,
and, after approximately one 12-day HRT period, the pH varied
between 6.90 and 7.05. The thermophilic reactor had consistently
higher and less stable pH, which varied between 7.24 and 7.42.
The effluent characteristics measured after the two reactors had
reached three HRT periods and the reactors performance data are
479

Kabouris et al.

Table 2Batch anaerobic ultimate biodegradability test results and projections.

Parameter

Primary sludgea

PS VS loading, g/L (% of total VS loading)

TWAS VS loading, g/L (% of total VS loading)
FOG VS loading, g/L (% of total VS loading)
Total volatile solids loading, g/L
Volatile solids destructionf,g, %
COD destructionc, %
Methane, %
Total gas produced, mL @ STP/g component
volatile solids addedf
Methane, mL @ STP/g-VS destroyedh
Methane, mL @ STP/g-VS addedh

TWASa

FOGa

PS39 + TWAS61a,b

PS21 + TWAS31 + FOG48c,d

1.16 (39)
1.84 (61)

NAe (21)
NA (31)
NA (48)
NA
53
55
73.8
855

3 (100)
3 (100)
3
56.7
58.5
69.2
680

3
23.3
26.3
72.3
248

3 (100)
3
70.7
70.8
75.0
1324

3
36.8
40.0
71.5
407

830
470

767
179

1404
993

792
291

1190
630

Experimental results.
PS/TWAS mix, 39/61% volatile solids basis (run 1 mix).
c
PS/TWAS/FOG mix, 21/31/48% volatile solids basis (run 2 mix).
d
Predicted, based on additivity of results for the individual components and volatile solids composition in the mix.
e
NA 5 not applicable.
f
Seed-corrected, corresponds to the individual component or mix of components.
g
Based on the mass-balance method.
h
1 mL/g 5 0.01602 ft3/lb.
b

The low value for the COD balance, which is based on feed and
effluent COD and methane COD values, indicates good agreement
between these measurements and further demonstrates that the COD
destroyed was converted to methane as opposed to being consumed
via alternative routes (i.e., sulfate reduction). Hydrogen sulfide
was not detected in the gas phase of these reactors. Decreases of
approximately 28 and 68% in the fat content were observed in the
mesophilic and thermophilic reactors, respectively, although saturated fat was the major fat component in both reactors effluent. The
protein reductions in the mesophilic and thermophilic reactors were
11 and 23%, respectively (Table 4). Carbohydrate was reduced by
approximately 37 and 24% in the mesophilic and thermophilic
reactors, respectively. The increased degradation of protein and fat
under thermophilic conditions is consistent with the observations of
Ghosh (1987).
Digestion Run 2. For this run, the acid reactor was started with
a mix of primary sludge, TWAS, and FOG, and incubated at 358C
for 2 days before continuous feeding started. The acid-phase reactor
was initially operated on a semi-continuous-feed, stand-alone mode
for a period of 7 days. Starting on the 7th day, every hour, the acid
reactor effluent was either fed to the mesophilic or thermophilic

shown in Table 4. The thermophilic reactors volatile solids

destruction exceeded that of the mesophilic reactor by 5.4
percentage points. An excellent agreement between volatile solids
and COD destruction was achieved in this run. The higher volatile
solids and COD destruction in the thermophilic reactor resulted in
a higher methane production, by approximately 24%. Both reactors
had comparable methane content in the gas produced and very low
VFA concentrations. The methane yields were 159 and 197 mL @
STP/g-VS added or 632 and 642 mL @ STP/g-VS destroyed for the
mesophilic and thermophilic reactors, respectively, at the standard
temperature and pressure (STP) conditions of 08C and 1 atm.
Effluent ammonia concentrations of 495 and 719 mg-N/L corresponded to net ammonia productions of 196 and 420 mg-N/L for
the mesophilic and thermophilic reactors, respectively, when the
feed ammonia concentration of 299 mg/L was subtracted. The
soluble phosphorus concentration remained below 30 mg/L under
both mesophilic and thermophilic conditions. The relatively low
soluble phosphorus could be the result of the alum included in the
TWAS. The thermophilic reactor effluent had a soluble COD
concentration of 1446 mg/L, which was more than twice that in the
mesophilic reactor effluent (601 mg/L).
Table 3Reactor characteristics for digestion runs 1 and 2.

Run 2
Parameter
Volume, L
Retention time, days
Flowrateb, mL/d
Primary solids loading, g-VS/L
db (% of total loading)
TWAS loading, g-VS/L
d (% of total loading)
FOG loading, g-VS/L
d (% of total loading)
Total loading, g-VS/L
d

Run 1

Acid phase

Methane phasea

2.0
12
166.7
0.99 (40.5)
1.46 (59.5)

2.45

0.5
1
500
11.1 (21.1)
16.4 (31.3)
25.0 (47.6)
52.5

2.0
12
166.7

4.35c

Mesophilic and thermophilic reactors operating in parallel using acid-phase reactor effluent.
1 lb/ft3
d 5 16.02 g/L
d.
c
Nominal, based on feed to acid-phase reactor.
b

480

Water Environment Research, Volume 81, Number 5

Kabouris et al.

Table 4Feed and efuent characteristics and reactors performance for digestion run 1.
Methane phase
Parameter
Total solids, g/L
Volatile solids, g/L
VS/TS, g/g
Total COD, g/L
Soluble COD, mg/L
VFAs, mg COD/ L
pH
Carbohydrate, g/L (% of VS)
Crude protein, g/L (% of VS)
Total fat, g/L (% of VS)
Saturated fat, g/L (% of total fat)
Polyunsaturated fat, g/L (% of total fat)
Monounsaturated fat, g/L (% of total fat)
Trans fat, g/L (% of total fat)
Total carbohydrate reduction, %
Total protein reduction, %
Total fat reduction, %
Total Kjeldahl nitrogen, mg-N/Lc
Ammonia, mg-N/L
Total phosphorus, mg-P/L
Water soluble phosphorus, mg-P/L
Total gas, mL @ 228C/d
Methane, %
Methane, mL @ 228C/d
VS destructionc, %
Degradable VS destructiond
Total COD destructiond, %
Degradable COD destructione, %
COD balance, %d
Biogas, mL @ STP/g-VS addedf
Methane, mL @ STP/g-VS addedf
Methane, mL @ STP/g-VS destroyedf
Methane, mL @ STP/g-COD destroyed

Feed

Mesophilic

Thermophilic

41.5 6 0.1a
29.4 6 0.1
70.8
53.1 6 3.7
4820 6 150
3500 6 35
6.40 6 0.02
13.2 (45)
11.5 (39)
4.7 (16)

33.8 6 0.1
22.0 6 0.1
65.1
39.7 6 3.4
601 6 9
50 6 5
6.95 6 0.04
8.4 (38)
10.2 (47)
3.4 (15)

32.8 6 0.1
20.4 6 0.1
62.2
37.5 6 1.0
1446 6 106
75 6 7
7.30 6 0.04
10.1 (49)
8.8 (43)
1.5 (7)

2.2 (66)
0.2 (5)
1 (29)
NDb
37
11
28
2130 6 58
495 6 16
2490
22
1274 6 25
66.1 6 2.1
842 6 25
25.2
68.5
25.3
63.3
0.2
241
159
632
347

1 (69)
0.1 (8)
0.4 (24)
ND
24
23
68
2130 6 58
719 6 58
2460
26
1577 6 44
66.0 6 2.8
1041 6 30
30.7
83.2
29.4
73.5
21.6
298
197
642
369

3.8 (81)
0.05 (1)
0.5 (11)
0.3 (7)

2130 6 58
299 6 16
2450
ND

Mean 6 standard deviation (n  5).

ND 5 not detected.
c
Based on averaging single measurements of feed and mesophilic and thermophilic reactors.
d
Based on the mass-balance method.
e
COD balance 5 (CODin 2 CODout 2 CODCH4) 3 100/CODin.
f
1 mL/g 5 0.01602 ft3/lb.
b

methanogenic reactors or wasted to maintain the desired acid- and

methane-phase HRTs of 1 and 12 days, respectively. Before being
used in run 2, the mesophilic and thermophilic reactors were
acclimated to combined sludge and FOG feed for several weeks.
Subsequently, the reactors were kept without any feeding for 31
days and maintained at 35 and 528C, respectively, to allow for
degradation of all degradable substrate in them. The three reactors
characteristics during run 2 are listed in Table 3. Table 5 shows the
feed composition during run 2. The soluble COD was 4.9% of the
total feed COD, and the VFAs comprised approximately 56% of
the soluble feed COD. Sludge acidification had taken place, which,
combined with the low FOG pH value of approximately 4 (Table 1),
resulted in a very low feed pH of 5.6. The feed pH and VFAs were
comparable with those observed in some full-scale acid-phase
digesters (Andryszak et al., 2004). In addition, further acidification
had likely occurred in the feed vessel that was maintained at room
temperature (22 to 248C) for 1 to 2 days. The protein and fat
fractions of the feed volatile solids were 28 and 56%, respectively.
May 2009

Saturated and monounsaturated fat were more than 79% of the total
feed fat. The increase in the monounsaturated fraction of the fat in
this feed reflects the fat component distribution in the FOG (Tables
1 and 5).
Run 2 lasted for 36 days, that is, 7 days with the acid reactor
alone and 29 days with all three reactors. Thus, the acid reactor
completed 36 retention times, whereas the two methane-phase
reactors completed almost 2.5 retention times each. The total gas
production, pH, and gas composition in the acid-phase reactor, with
the feed pH during the entire run 2, are shown in Figure 2. During
the first 5 days of operation of the acid reactor, the gas production
decreased and, for approximately 10 days, was below 50 mL/d.
After 21 days of operation, the gas production increased rapidly
and, during the last 6 days, averaged 137 mL/d. Initially, the acid
reactor headspace was predominantly carbon dioxide, but, after 16
days of operation, a gradual decrease in carbon dioxide and an
increase in methane was observed. At the end of this run, the reactor
headspace relative composition (i.e., considering only CO2 and
481

Kabouris et al.

CH4) was 64% CO2 and 36% CH4. Upon further investigation of
the gas composition at the end of this run, H2 and N2 were also
found at 10 and 7%, respectively. Taking into account all four gas
components, the absolute gas composition towards the end of run 2
was 53% CO2 and 30% CH4. The feed pH varied between 5.36 and
5.59. The acid reactor pH increased initially from 5.0 to 5.3 and
then stabilized to approximately 5.1 (Figure 2). The initial pH increase may be associated with a low rate of acid production and/or
consumption of feed VFAs, resulting in the observed relatively high
initial gas production. A comparison of the acid-phase reactor influent and effluent characteristics shows a minimal decrease in total
COD and volatile solids and an increase of soluble COD, VFAs,
and ammonia by approximately 16, 25, and 20%, respectively
(Table 5). Although a modest decrease (approximately 5%) in total
fat content of the acid reactor effluent was observed, both the monounsaturated and polyunsaturated fat fractions decreased, and the
saturated fat fraction increased from 40 to 64% of the total fat content (Table 5). The major saturated fat components in the effluent of
the acid reactor were (in descending order) palmitic (16:0), stearic
(18:0), and myristic acids (14:0).
The total gas production and pH in the mesophilic and thermophilic reactors during the entire run 2 are shown in Figure 3. During
the first 10 days, the gas production increased steadily in both
reactors, and, by the 12th day, the gas production became very
stable. The pH in both reactors decreased initially and remained
at approximately 7.0 in the mesophilic reactor, whereas it increased
in the thermophilic reactor and stabilized at approximately 7.35
after 15 days of operation. The effluent characteristics of the two
methane-phase reactors, measured after they had reached two
retention times, and their performance data are shown in Table 5.
The thermophilic reactor achieved 5.8 percentage points higher
volatile solids destruction compared with the mesophilic reactor.
The calculated COD destruction in this run was approximately 5.1
and 7.0 percentage points higher than the volatile solids destruction
in the mesophilic and thermophilic reactors, respectively. The
higher volatile solids and COD destruction in the thermophilic
reactor resulted in a higher methane production by approximately
16% compared with the mesophilic reactor. The methane content in
the gas produced in the thermophilic reactor was slightly higher
than in the mesophilic reactor. The VFA concentrations were very
low in both reactors.
The methane yields were 473 and 551 mL @ STP/g-VS added or
1049 and 1083 mL @ STP/g-VS destroyed for the mesophilic and
thermophilic reactors, respectively. Compared with run 1, the run 2
methane yields per gram volatile solids added for mesophilic and
thermophilic conditions were 300 and 280% larger, respectively.
The high values of methane produced per gram volatile solids
destroyed were the result of the high proportion of FOG digested,
combined with the higher methane yield from FOG and the likely
acceleration of TWAS degradation kinetics, as discussed in the
following subsection. Effluent ammonia concentrations of 504 and
728 mg-N/L corresponded to net ammonia productions of 84 and
308 mg-N/L for the mesophilic and thermophilic reactors, respectively, based on the acid-phase reactor effluent ammonia concentration of 420 mg-N/L. The soluble phosphorus concentration
remained below 20 mg/L under both mesophilic and thermophilic
conditions, likely as a result of the alum included in the TWAS.
The thermophilic reactor effluent had a soluble COD concentration almost twice that in the mesophilic reactor effluent. The COD
balance was very good for all three reactors. Based on the composition of the feed to the acid-phase reactor and the effluent of the two
482

Figure 1(a) Total gas production and (b) pH in the

mesophilic and thermophilic reactors and feed during
digestion run 1.

methanogenic reactors, the overall fat removals were 78 and 85%

for the mesophilic and thermophilic reactors, respectively. The associated crude protein reductions for the mesophilic and thermophilic
reactors were 6 and 16%, respectively, and carbohydrate increased
by approximately 1 and 6%, respectively. The increased degradation of protein and fat compared with carbohydrates is consistent
with the observations of Ghosh (1987) for acid-methane-phased
digestion. The calculated small increase in carbohydrate concentration could be the result of underestimating the crude protein
destruction, as the result of an error introduced by the relatively high
coefficient of variation (0.12) in the TKN measurements.
A slight further increase (approximately 3 to 6%) of the saturated
fat fraction was observed after going through the methanogenic
reactors. The saturated fat fraction was the major fat component in
both reactors effluent, followed by monounsaturated and trans fat.
The major saturated fat components in the effluent of both reactors
were (in descending order) palmitic (16:0), stearic (18:0), and
myristic acids (14:0), which is similar to the saturated fat components distribution in the feed. Thus, degradation of the saturated
fat components was relatively uniform, that is, there was not any
clear preference towards certain fatty acids.
Comparison of Runs 1 and 2 with Batch Mesophilic
Digestion. The destruction values of the degradable COD under
mesophilic batch digestion conditions for primary sludge, TWAS,
FOG, and PS391TWAS61 are presented in Figure 4. Simulation of
the destruction of the degradable COD and its conversion to
methane of the primary sludge, TWAS, and FOG samples based on
first-order kinetics resulted in the following rate constants: primary
sludge, 0.2 day21; TWAS, initial 0.10 and then 0.03 to 0.05 day21;
and FOG, from 0.10 to 0.15 day21 (Kabouris et al., 2007).
Similarly, the rate constants for the PS391TWAS61 sample were as
follows: initial 0.15 and then 0.04 to 0.08 day21. Kabouris et al.
(2007) also reported that, for a PS331TWAS531FOG14 sample, the
rate constants initial 0.15 and then 0.06 to 0.10 day21, implying that
Water Environment Research, Volume 81, Number 5

Kabouris et al.

Table 5Feed and efuent characteristics and reactors performance for digestion run 2.
Methane phase
Parameter
Total solids, g/L
Volatile solids, g/L
VS/TS, g/g
Total COD, g/L
Soluble COD, mg/L
VFAs, mg COD/ L
pH
Carbohydrate, g/L (% of VS)
Crude protein, g/L (% of VS)
Total fat, g/L (% of VS)
Saturated fat, g/L (% of total fat)
Polyunsaturated fat, g/L (% of total fat)
Monounsaturated fat, g/L (% of total fat)
Trans fat, g/L (% of total fat)
Reduction based on feed, %
Total carbohydrate
Total protein
Total fat
Total Kjeldahl nitrogen, mg-N/Lb
Ammonia, mg-N/L
Total phosphorus, mg-P/L
Water soluble phosphorus, mg-P/L
Total gas, mL @ 228C/d
Methane, %
Methane, mL @ 228C/d
VS destructionb, %
Degradable VS destructionc
Total COD destructionc, %
Degradable COD destructionc, %
COD balance, %d
Biogas, mL @ STP/g-VS added
Methane, mL @ STP/g-VS added
Methane, mL @ STP/g-VS destroyede
Methane, mL @ STP/g-COD destroyed

Feed

Acid phase

Mesophilic

Thermophilic

64.8 6 0.1a
52.5 6 0.2
81.0
129.9 6 8.7
6340 6 80
3580 6 80
5.57 6 0.03
8.3 (16)
14.8 (28)
29.4 (56)
12 (40)
2 (7)
11 (39)
4 (14)

64.8 6 0.3
52.3 6 0.9
80.7
129.5 6 6.0
7372 6 272
4500 6 22
5.12 6 0.03
10 (19)
14.4 (28)
27.9 (53)
18 (64)
1 (3)
5 (17)
4 (16)

41.1 6 0.5
28.7 6 0.2
69.8
64.5 6 4.2
705 6 85
20 6 5
7.03 6 0.02
8.3 (29)
13.9 (48)
6.5 (23)
5 (70)
0.13 (2)
1 (15)
1 (13)

38.6 6 0.2
25.7 6 0.2
66.6
54.5 6 1.8
1525 6 60
70 6 5
7.34 6 0.01
8.7 (34)
12.5 (49)
4.5 (18)
3 (67)
0.14 (3)
1 (17)
1 (13)

221
3
5
2725 6 335
420 6 10
2310
33
137 6 9
30.0 6 3.0
41.1 6 3
0.4

21
6
78
2725 6 335
504 6 5
2460
14
6775 6 43
65.8 6 1.8
4458 6 40
45.1
85.3
50.2
87
24.4
719
473
1049
381

26
16
85
2725 6 335
728 6 15
2540
19
7550 6 73
68.7 6 1.2
5187 6 50
50.9
96.2
57.9
100
25.6
802
551
1083
384

2725 6 335
350 6 10
1780
112

0.3
0.2
5
2
381
189

Mean 6 standard deviation (n  5).

Based on averaging single measurements of feed and mesophilic and thermophilic reactors.
c
Based on the mass-balance method.
d
COD balance 5 (CODin 2 CODout 2 CODCH4) 3 100/CODin.
e
1 mL/g 5 0.01602 ft3/lb.
b

the addition of FOG resulted in an overall faster destruction of the

composite sample.
The fraction of the ultimately degradable COD that was degraded
in runs 1 and 2 under mesophilic and thermophilic methane-phase
conditions also is shown in Figure 4. Theoretically, based on firstorder degradation kinetics, batch digestion is expected to be
approximately 20 to 25% more efficient, in terms of COD
destruction, at an average digestion time of 10 to 20 days compared
with continuous-flow digestion (Kabouris et al., 2007). This effect
was not observed in our case. The fractional destruction of
degradable COD under the 12-day HRT of the semi-continuousfeed mesophilic conditions in run 1 was approximately 0.64 and
almost equal to the fractional degradable COD destruction obtained
under batch conditions for the PS401TWAS60 sample, estimated at
approximately 0.65. This is attributed to the higher loading and
associated increased microbial activity in the semi-continuous-feed
testing and the samples pre-acidification described above. The
May 2009

enhanced kinetics under thermophilic conditions during run 1

resulted in a higher fractional destruction of degradable COD to
approximately 0.74.
An even larger enhancement of COD destruction kinetics was
observed in run 2, which reached a fractional degradable COD
destruction of 0.87 under mesophilic methane-phase conditions.
This performance exceeds the fractional degradable COD destruction obtained under batch mesophilic digestion for 12 days for
any of the samples in the mix (i.e., primary sludge, TWAS, and
FOG). This enhancement is attributed to the higher loading and
associated increased microbial activity in the semi-continuous-feed
system and to the effect of the acid-phase digestion and indicates
that the initial retardation of the batch FOG degradation may have
not materialized in run 2, as a result of biomass acclimation and
because the degradation of WAS in run 2 may have been accelerated
to match the faster degradation rates of the FOG and primary sludge
components. A further enhancement of the degradation kinetics under
483

Kabouris et al.

Figure 3(a) Total gas production and (b) pH in the

mesophilic and thermophilic reactors during digestion
run 2.

Figure 2(a) Total gas production, (b) pH, and (c) relative
reactor headspace gas composition neglecting hydrogen
and nitrogen in the acid-phase reactor during digestion
run 2.
thermophilic conditions during run 2 resulted in complete, within
experimental uncertainty, destruction of degradable COD.
The run 2 mesophilic reactors methane production of 1049 mLCH4/g-VS destroyed is a slightly less than that predicted in Table 2
for the run 2 feed mix (PS211TWAS311FOG48) (1190 mL-CH4/
g-VS destroyed). This indicates that the run 2 volatile solids
degradation was not dominated by the degradation of the FOG
component, because a preferential degradation of FOG would
increase the methane production to a value closer to the 1404 mLCH4/g-VS presented in Table 2 for the FOG sample.
Conclusions
The results of this study indicate that a mix of primary sludge,
TWAS, and FOG, corresponding to 21/31/48%, respectively, on
a volatile solids basis, or 16/28/56% on a carbohydrate/protein/fat
basis, could efficiently be digested based on an acid digester
operated at a 1-day HRT, at 358C, and a loading rate of 52.5 g-VS/
L
d, with the acid reactor effluent fed to methane-phase reactors
operated at an HRT of 12 days, and at 35 or 528C. The acid reactor
exhibited an initial period of 20 days with near-zero biogas
production before it stabilized to a biogas production of
approximately 2 mL-CH4 @STP/g-VS added, corresponding to
a minimal volatile solids destruction of 0.4%. The acid-phase
reactor received a partially solubilized and acidified feed and
achieved a 43% decrease in non-saturated fat and a 16, 26, and 20%
484

increase of soluble COD, VFAs, and ammonia, respectively. An

acid-phase HRT higher than 1 day could accelerate the establishment of an acid-phase-reactor steady-state under a high FOG
volatile solids fraction load.
The methane-phase volatile solids destruction values in run 2
were 45 and 51% (85 and 97% biodegradable volatile solids
destruction) at 35 and 528C, respectively. Stable gas production and
pH level were reached within only one 12-day HRT period, at 35 or
528C. The methane yields for the methane-phase reactors were
473 and 551 mL @ STP/g-VS added, at 35 and 528C, respectively,
representing approximately 3 times larger methane yield per gram
volatile solids added compared with the digestion of the sludge
without the FOG. The results indicate that operation at 528C is

Figure 4Normalized COD destruction of primary sludge,

TWAS, FOG and a PS/TWAS mix sample over a 120-day
mesophilic batch incubation period with superimposed
results of digestion runs 1 and 2 (12-day HRT).
Water Environment Research, Volume 81, Number 5

Kabouris et al.

advantageous in cases when there is a need for the near-complete

destruction of degradable volatile solids and for increased methane
production. However, financial and treatment process design
considerations should be taken into account because of the increase
in released nutrients at the digestion temperature of 528C compared
with 358C. This is of particular concern for treatment facilities with
low effluent nutrient requirements. With reference to run 1, the
addition of FOG and acid-methane-phased digestion in run 2
significantly increased methane production, resulting in methane
gas yield ratios of 3.0 and 3.4 at 358C on a volatile and total solids
basis, respectively, while, at 528C, the methane yield ratios were 2.8
and 3.2 on a volatile and total solids basis, respectively.
These results indicate that that the co-digestion of FOG with
sludge could significantly increase the methane gas yield and
significantly improve a facilitys energy balance. The increase in
methane yield could be profound for sludge with relatively low
biodegradability (i.e., sludge from plants with long SRT values) and
a large FOG organic load fraction, as was the case in this study.
Credits
The authors thank Pinellas County Utilities (Clearwater, Florida)
for funding this study and the staff of South Cross Bayou WRF for
their assistance during the project.
Submitted for publication January 29, 2008; revised manuscript
submitted August 25, 2008; accepted for publication September 4,
2008.
The deadline to submit Discussions of this paper is August 15,
2009.
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485