Bioresource Technology 166 (2014) 201–210

Contents lists available at ScienceDirect

Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Processing anaerobic sludge for extended storage as anaerobic digester

inoculum
Jiajia Li a,b, Steven M. Zicari b, Zongjun Cui a, Ruihong Zhang b,⇑
a
College of Agronomy and Biotechnology, China Agricultural University, Beijing 100193, China
b
Department of Biological and Agricultural Engineering, University of California, Davis, CA 95616, United States

h i g h l i g h t s

 Different methods were used to reduce MC of sludge for storage as digester inoculum.
 The methanogenic activity of sludge at different MC levels was assessed and compared.
 Effect of storage time of up to 4 months on methanogenic activity was evaluated.
 Processed and stored sludge retained between 76% and 99% of fresh sludge methane yield.

a r t i c l e i n f o a b s t r a c t

Article history: Thermophilic anaerobic sludge was processed to reduce the volume and moisture content in order to
Received 12 March 2014 reduce costs for storing and transporting the sludge as microbial inoculum for anaerobic digester startup.
Received in revised form 29 April 2014 The moisture content of the sludge was reduced from 98.7% to 82.0% via centrifugation and further to
Accepted 2 May 2014
71.5% via vacuum evaporation. The processed sludge was stored for 2 and 4 months and compared with
Available online 17 May 2014
the fresh sludge for the biogas and methane production using food waste and non-fat dry milk as
substrates. It was found that fresh unprocessed sludge had the highest methane yield and the yields of
Keywords:
both unprocessed and processed sludges decreased during storage by 1–34%, however processed sludges
Anaerobic sludge
Concentrated sludge
seemed to regain some activity after 4 months of storage as compared to samples stored for only
Storage time 2 months. Maximum methane production rates obtained from modified Gompertz model application also
Methanogenic activity increased between the 2-month and 4-month processed samples.
Biogas production Ó 2014 Published by Elsevier Ltd.

1. Introduction
Anaerobic digestion (AD) is becoming increasingly popular as
an efficient method for converting a wide range of organic
materials into biogas (primarily methane) and digested sludge
(or digestate). Many countries have developed incentive policies
to promote the development of anaerobic digesters for waste
Abbreviations: OS, original sludge effluent from an operational anaerobic
digester; CS, centrifuged original sludge; CES, centrifuged and further evaporated
original sludge; MC, moisture content; FW, food waste; NFDM, non-fat dry milk;
BMP, biochemical methane potential; BD, biodegradability; Fresh sludge, sludge
without storage; TS, total solids; VS, volatile solids; F/M ratio, food to microorgan-
ism ratio; EBY, experimental biogas yield; EMY, experimental methane yield; TMY,
theoretical methane yield; DBY, daily biogas yield; DMY, daily methane yield.
⇑ Corresponding author. Address: Biological and Agricultural Engineering Depart-
ment, University of California at Davis, One Shields Avenue, Davis, CA 95616, United
States. Tel.: +1 530754 9530.
E-mail address: rhzhang@ucdavis.edu (R. Zhang).
treatment and energy recovery. For example, the European Union
requires at least 25% of bioenergy to be produced via biogas in
the future (Holm-Nielsen et al., 2009). In the United States, there
are various incentive programs at both federal and state levels
for anaerobic digestion projects. The California Bioenergy Action
Plan recently has set a goal of producing 95 MW electricity from
dairy manure and 250 MW electricity from other organic wastes.
Successful development of anaerobic digesters involves the
startup of new digesters, which requires establishment of suitable
environment and microbial populations needed to achieve the
desired performance. Commonly, anaerobic sludge from an exist-
ing anaerobic digester is used as inoculum for a new digester.
When there are no digesters nearby, animal manure or wastewater
lagoon sludge are used as sources of microbes but the enrichment
process can take considerable time as the methanogenic popula-
tions and activities are low, requiring a period of weeks to months
to cultivate a healthy population of methanogens and other
microorganisms needed for efficient full-scale digester operation

http://dx.doi.org/10.1016/j.biortech.2014.05.006
0960-8524/Ó 2014 Published by Elsevier Ltd.
202 J. Li et al. / Bioresource Technology 166 (2014) 201–210

Table 1 mesophilic inoculum to thermophilic conditions (De la Rubia

Characteristics of anaerobic sludge and substrates. et al., 2013). For low solids digesters, recommended inoculum rates
Sample OS CS CES FW NFDM may range from 0.5% to 15% of the operating total solids (TS)
MC (%) a
98.7 ± 0.2 82.0 ± 0.4 71.5 ± 3.2 76.6 ± 0.4 4.1 ± 0.1 content by mass, and research on high solids anaerobic digestion
TS (%)a 1.3 ± 0.2 18.0 ± 0.4 28.5 ± 3.2 23.4 ± 0.4 95.9 ± 0.1 indicates that up to 50% of TS may be required as inoculum to
VS (%)a 0.9 ± 0.1 14.7 ± 0.3 22.4 ± 2.6 22.0 ± 0.3 88.0 ± 0.1 achieve a rapid start-up (Li et al., 2011; Brown and Li, 2013; Zhu
VS/TS (%) 67.4 ± 0.2 81.5 ± 0.4 78.6 ± 0.5 94.0 ± 0.2 91.8 ± 0.1 et al., 2014). Therefore, it would mark a significant time and cost
Ash (%)a 0.4 ± 0.1 3.3 ± 0.1 6.1 ± 0.6 1.4 ± 0.2 7.8 ± 0.1
C (%)b ND ND ND 48.9 ± 0.2 42.9 ± 0.1
savings to develop a proper method to produce concentrated
H (%)b ND ND ND 7.7 ± 0.1 6.0 ± 0.1 anaerobic sludge that can both be stored and transported econom-
O (%)b ND ND ND 34.7 ± 0.1 44.1 ± 0.3 ically while retaining the methanogenic activities in the sludge for
N (%)b ND ND ND 2.7 ± 0.1 1.8 ± 0.1 efficient start-up of new digesters.
ND, not determined; OS, original sludge; CS, centrifuged original sludge; CES, Considerable past research has focused on treatment options for
centrifuged and further evaporated original sludge; FW, food waste; NFDM, non-fat separation and dewatering of anaerobic sludge or digestate, such
dry milk. as centrifugation and vacuum evaporation (Mihoubi, 2004; Rehl
a
As total weight of sample.
b
and Müller, 2011; Chiumenti et al., 2013). It was also reported by
As TS of sample.
Agrawal et al. (1997) that dried pelletized anaerobic sludge con-
taining only 12% moisture content (MC) achieved stable perfor-
mance during mesophilic wastewater digestion with variations in
(Zeeman et al., 1988; Zhao et al., 2013). A recent review of thermo- organic loading rates. Other studies have reported that reducing
philic anaerobic digestion systems indicated that many operational MC in anaerobic digestion systems can lead to decreasing metha-
problems associated with thermophilic systems might be nogenic activities. Lay et al. (1997) found methanogenic activity
attributed to inconsistencies in the common practice of adapting of mesophilic sludge dropped by 47% when MC decreased from

Fig. 1. Daily and cumulative biogas production from FW and NFDM with fresh and stored original sludge. FW, food waste; NFDM, non-fat dry milk; OS, original sludge; C,
cumulative gas yield; D, daily gas yield.
 J. Li et al. / Bioresource Technology 166 (2014) 201–210 203

96% to 90% by adding dry sludge cake in batch digesters. However,

DBYmax, maximum daily biogas yield; DMYmax, maximum daily methane yield; TM90, the digestion time needed to produce 90% of maximum 21 day methane yield; EBY, experimental biogas yield; EMY, experimental methane
555.9bd
there were few reports on the effect of reduced MC on the preser-

349.0i

82.2ps
424.6
7.85
7.89

43.5
63.0
vation of thermophilic biogas activity, especially in combination

13
4
with storage duration. Storage temperature is also known to be
an important factor in the preservation of sludge methanogenic

314.2kl
514.8d

424.6
74.0r
7.19
7.15

33.5
85.0
activity. One study showed that after 10-months, the methanogen-

13
CES + NFDM
2
ic activity of granular sludge stored at room temperature
(10–26 °C) was reduced by 60% of its original value, while sludge

395.5h
702.5e
158.8

424.6
Fresh

93.1o
8.22
8.17

65.0
stored at 4 °C was only 20% of the original (Bae et al., 1995).
9
However, no literature was found on the evaluation of different
storage times on the methanogenic activity of anaerobic sludge
408.8m
626.8a

424.6
96.3t
that has undergone different processes for moisture removal.
7.72
7.68
72.9
45.7
14

yield; TMY, theoretical methane yield; BD, biodegradability; Within each feedstock (FW or NFDM), values represented by different letters are significantly different at a level of a = 0.05.
4

The measurement of biochemical methane potential (BMP), ini-

tially developed by Owen et al., is used not only as a fast and valu-
334.7il
529.8d

424.6
78.8s

able method for determination of methane production rate and

7.17
7.21
95.4
44.1
12
2

potential with various biomass substrates, but also to evaluate

CS + NFDM

the methanogenic activity of different inoculum sources (Owen

517.7d
313.9k
175.2

424.6
Fresh

73.9r

et al., 1979; Li et al., 2013). However, there is little agreement upon

7.58
7.60

80.8
7

a standard protocol defined for BMP assays leading to a wide range

of parameters such as food/microorganism (F/M) ratio and sug-
431.2c
273.3j
424.6
64.4q

gested standard substrates such as acetic acid, glucose, cellulose,

8.45

84.3
39.3
8.20
Comparison of different batch digestion parameters using fresh sludge and stored sludge as inoculum and using food waste and non-fat dry milk as substrates.

18
4

or non-fat dry milk used in literature (Raposo et al., 2006;

Angelidaki et al., 2009). The procedures used for performing batch
596.5b
351.6i
147.7

424.6
82.8p

digestion tests to determine BMP at the UC Davis Bioenvironmen-

7.97
7.83

59.3
12
2

tal Research Laboratory were reported by Zhang et al. (2006).

OS + NFDM

Through this study, we attempted to identify changes in methano-

414.8hm
662.7ae

genic activity of unprocessed and processed sludges with respect

97.7ot
162.6

424.6
Fresh
8.19
8.15

69.1

to storage time and selected two sample substrates, both with rel-
11

atively high organic content and easy degradation potential, but

473.2K
671.6E

with one representing a more complex substrate, food waste

568.3
83.3R
7.77
7.79
88.9
64.7

(FW); and the other being more uniform in composition and

13
4

widely available, non-fat dry milk (NFDM).

614.6CD

The objectives of the present study were to: (1) assess the effect
411.9I
568.3
72.5P
65.7
44.7
7.05
7.05

of reducing the MC of thermophilic anaerobic sludge by centrifuga-

12
2

tion and evaporation on methanogenic activity as measured by

CES + FW

using batch anaerobic digestion tests with FW and NFDM as sub-

735.5F
435.1J
568.3
76.6Q
Fresh
8.15
8.13
92.4
58.9

strates; and (2) evaluate the effect of storage time on the BMP of
11

the condensed sludge as compared to the fresh sludge.

695.9AE
495.7H
115.3

568.3
87.2O
7.56
7.48

80.6

2. Methods
12
4

624.7D

2.1. Anaerobic sludge and preparation of condensed sludge

431.9J
568.3
76.0Q
7.13

49.5
7.10
70.6

12
2

Digester effluent was collected from an industrial thermophilic

CS + FW

anaerobic digester in Sacramento, California, fed with various food

629.7BD
403.0 I
568.3
101.6
Fresh

70.9P

wastes. After obtaining the anaerobic sludge in 20 L plastic buck-

7.75
7.72

66.0
11

ets, it was acclimated and stabilized in an incubator at 55 ± 2 °C

for 1 week, to ensure depletion of readily available remaining
394.6IJ
566.8C

69.4PQ
568.3

organic matter in the sludge. This sample is referred to as fresh Ori-

8.34

39.4
7.70
70.6

16
4

ginal Sludge (OS) in this manuscript. To prepare a Centrifuged

Sludge (CS), about 380 mL of the original sludge was placed into
679.0BE
425.1IJ

74.8PQ
568.3

400 mL centrifugation bottles and centrifuged for 20 min at

7.81
7.83
83.4
58.1
12

5000 rpm using a laboratory centrifuge (Thermo Scientific, USA),

2

and the supernatant discarded. The centrifuged solids obtained

OS + FW

753.3AF
498.3H

were harvested and further concentrated for two hours at 55 °C

113.5

568.3
87.7O
Fresh
8.17

76.9
8.01

by rotary vacuum evaporation (Buchi, Switzerland) operated at

11

3.6 Pa to produce a Centrifuged and Evaporated Sludge (CES) sam-

DMYmax (mL g1 VSadded)

ple. Prior to evaporation, the headspace in the sludge bottle was

DBYmax (mL g1 VSadded)
Storage time (Months)

EMY (mL g1 VSadded)

TMY (mL g1 VSadded)

purged with argon gas to ensure an anaerobic condition.

EBY (mL g1 VSadded)

2.2. Storage of original sludge and condensed sludge

Initial (pH)

TM90 (day)
Final (pH)
Sample

BD (%)

After stabilization, OS was placed in 15 L plastic buckets with

Table 2

minimal headspace and tight fitting lids. CS and CES were placed
into rubber-capped 250 mL glass bottles with a working volume
204 J. Li et al. / Bioresource Technology 166 (2014) 201–210
of 130 mL. The headspaces of the bottles were purged with argon
gas for 5 min to ensure anaerobic conditions. All three types of
sludge were stored at room temperature (20 ± 2 °C) for 2 and
4 months.
2.3. Substrates
Batch anaerobic digestion tests were conducted to evaluate the
methanogenic activity of fresh and stored sludge samples using FW
and NFDM as substrates. The FW was provided by a waste manage-
ment company in northern California. It was stored at 20 °C after
screening and grinding. The NFDM solution was freshly prepared
using the Non-fat Dry Milk powder, purchased from a local grocery
store, mixed with distilled water to achieve the desired 10% vola-
tile solids (VS) content. All batch anaerobic digestion tests had no
additional minerals or nutrients added. The characteristics of FW
and NFDM are shown in Table 1.
2.4. Methanogenic activity assay
Methanogenic activity measurements of sludge samples were
carried out in triplicate using 1-L glass reactors at thermophilic
temperature (55 ± 2 °C). The substrate (food) organic loading of
reactors was 2 g-VS L1 and food to microorganism ratio (F/M)
for all the batch digestion tests was 0.5 based on VS of substrate
and sludge. Fresh and stored sludge samples without substrate
addition were tested as inoculum blanks at 4 g-VS L1 and
daily biogas or methane production subtracted from sample
Fig. 2. Methane content of biogas produced from FW and NFDM using fresh and stored sludge as inoculum. 0, fresh sludge; 2, 2-month storage; 4, 4-month storage; FW, food
waste; NFDM, non-fat dry milk; OS, original sludge; CS, centrifuged original sludge; CES, centrifuged and further evaporated original sludge.
measurements. Tap water was used to make the working volume
be 500 mL. The reactors were tightly closed with rubber septa
and screw caps and the headspaces of the reactors were purged
with argon gas to create an initial anaerobic environment. All the
reactors were mixed once a day for about 5 min by manually
shaking reactors prior to measurement of biogas volume. For each
anaerobic digestion test, the total digestion time was 21 days.
2.5. Analytical methods
The TS, VS, and ash contents were measured by using standard
methods described in APHA, 2005. Elemental compositions of FW
and NFDM were determined with an elemental analyzer (Vario
EL cube, Germany) and a 2400 II oxygen analyzer (PerkinElmer
Instrument, USA). The pH value was measured with an AR50 pH
electrode (Fisher, USA).
The pressure in the headspace of each reactor was used as an
indicator to calculate the amount of biogas produced in the reactor,
and it was measured using a 3150 WAL-BMP-Test system pressure
gauge with accuracy of 0.1% (WAL Mess-und Regelsysteme GmbH,
Germany). The biogas in the headspace was released under water
to prevent gas exchange between the reactor and the ambient
air. Then the pressure in the headspace was measured again as
an initial condition for the next-day measurement. Daily pressure
differences were converted into biogas volume using the following
Eq. (1) (El-Mashad and Zhang, 2010):
DP  Vheadspace  C
Vbiogas ¼ ð1Þ
RT
 J. Li et al. / Bioresource Technology 166 (2014) 201–210 205

  
where Vbiogas is daily biogas volume (mL), DP is the absolute pres- Rm  e
M ¼ M 0  exp  exp ðk  tÞ þ 1 ð2Þ
sure difference (kPa), Vheadspace is volume of the headspace (mL), C M0
is molar volume (22.41 L mol1 at 273.15 K, 101.325 kPa), R is the
where M refers the cumulative methane yield (mL g1 VSadded), M0
universal gas constant (8.314 L kPa K1 mol1), and T is absolute
is the ultimate methane yield (mL g1 VSadded), Rm represents the
temperature (K).
maximum methane production rate (mL g1 VSadded d1), k repre-
Biogas samples were taken from each reactor using a gas-tight
sents the lag phase time (day), t refers to the digestion time (day),
syringe and analyzed for the contents of hydrogen (H2), methane
and e is a mathematical constant (2.718). These parameters were
(CH4), and carbon dioxide (CO2) using a 6890 N gas chromatograph
used to characterize and to compare methanogenic activity in batch
(Agilent, USA) equipped with a thermal conductivity detector
reactors tested in this study.
(TCD), a 1.8 m  carbosphere 80/100 stainless steel column (All-
tech, USA), and argon carrier gas at a flow rate of 32 mL min1.
2.7. Theoretical methane yield and biodegradability
The temperatures of the oven, injector and detector were 100 °C,
120 °C, and 120 °C, respectively. Three biogas standards (Scott
In this study, based on elemental composition, theoretical
Specialty Gases, USA) containing CH4, H2, and CO2 were used to
methane yields (TMY) of FW and NFDM were calculated according
calibrate the GC.
to the method (Buswell and Mueller, 1952) as shown in Eqs. (3)
and (4):
2.6. Kinetic modeling
 
a b 3c
Cn Ha Ob Nc þ n   þ H2 O
The mathematical modified Gompertz model (Eq. (2)), which 4 2 4
includes parameters for ultimate methane yield and maximum    
n a b 3c n a b 3c
methane production rate, has been widely applied in modeling ! þ   CH4 þ  þ þ CO2 þ cNH3 ð3Þ
2 8 4 8 2 8 4 8
batch methane production (van Ginkel et al., 2001).

Fig. 3. Daily and cumulative biogas production from FW and NFDM using fresh and stored centrifuged sludge as inoculum. FW, food waste; NFDM, non-fat dry milk; CS,
centrifuged original sludge; C, cumulative gas yield; D, daily gas yield.
206 J. Li et al. / Bioresource Technology 166 (2014) 201–210

Fig. 4. Daily and cumulative biogas production from FW and NFDM using fresh and stored centrifuged and further evaporated sludge. FW, food waste; NFDM, non-fat dry
milk; CES, centrifuged and further evaporated original sludge; C, cumulative gas yield; D, daily gas yield.
 
mL CH4 22:4  1000  ðn2 þ 8a  b4  3c
8
Þ sludge samples, 82.0% for centrifuged sludge, and 71.5% for centri-
TMY ¼ ð4Þ
g VS 12n þ a þ 16b þ 14c fuged and further evaporated sludge samples. It is notable that
centrifugation resulted in a 14-fold reduction in sludge volume,
In addition, the biodegradability could be estimated from while the combination of centrifugation and evaporation resulted
experimental methane yield (EMY) and theoretical methane yield in a 22-fold volume reduction. The characteristics of sludge and
(TMY) according to Eq. (5) (Elbeshbishy et al., 2012) substrates are shown in Table 1.
BD ð%Þ ¼ EMY=TMY  100 ð5Þ The FW had TS and VS contents of 23.4% and 22.0%, respectively,
and NFDM had TS and VS contents of 95.9% and 88.0%, respectively.
The VS/TS ratio was 94.0% and 91.8% for FW and NFDM, respec-
2.8. Statistical analysis tively. The C/N ratio of FW and NFDM was determined to be 18.3
and 24.4, respectively, either of which was within a range typically
Experimental data for different pretreated and stored condi- considered appropriate for anaerobic microorganisms (Mshandete
tions were compared with one-way ANOVA using the MATLAB et al., 2004).
software (MathWorks, USA). The levels of a = 0.05 and a = 0.01
were used as the significant probability for all statistical tests. 3.2. Effect of storage time on the methanogenic activity of original
Intervals above and below average values represent standard sludge
deviations.
Biogas and methane yields with fresh, 2-month, and 4-month
3. Results and discussion stored OS are depicted in Fig. 1 and show similar trends indicating
reduced methane production rates and amounts with increased
3.1. Characteristics of anaerobic sludge and substrates storage time. Biogas and methane production started within the
first day after inoculation. As can be seen from the biogas
Anaerobic sludge processed to three different MCs were inves- production profile using FW as feedstock (Fig. 1A and B), the
tigated in this research; the MC averaged 98.7% for the original reactors inoculated with fresh OS produced the most biogas and
 J. Li et al. / Bioresource Technology 166 (2014) 201–210
Fig. 5. Ratio of EMY or Rm between stored sludge and fresh original sludge. FW, food waste; NFDM, non-fat dry milk; OS, original sludge; CS, centrifuged original sludge; CES,
centrifuged and further evaporated original sludge; EMYFOS, EMY of fresh OS; Rm -FOS, Rm of fresh OS.
methane in the first week, followed by a rapid decrease in produc-
tion. The daily biogas and methane yields with fresh OS peaked at
113.5 and 76.9 mL g1 VSadded, respectively, on day 6, while the
2-month stored OS peaked at 83.4 and 58.1 mL g1 VSadded, respec-
tively, on day 8, and the 4-month stored OS sample was at a
maximum of 70.6 mL g1 VSadded and 39.4 mL g1 VSadded, respec-
tively seen on day 3. Over 90% of the final methane production dur-
ing 21 days of digestion was produced in 11 days with fresh OS,
12 days with 2-month stored OS, and 16 days for 4-month stored
OS (Table 2). The cumulative 21-day biogas and methane
yields with fresh OS were the highest for FW at 753.3 and
498.3 mL g1 VSadded, respectively. The biogas and methane yields
were 679.0 and 425.1 mL g1 VSadded for 2-month stored sludge
and 566.8 and 394.6 mL g1 VSadded for 4-month stored sludge,
respectively. Based on the contents of C, H, O, and N, the
theoretical methane yield (TMY) of FW was calculated to be
568.3 mL g1 VSadded (Table 2). Thus, the 21-day anaerobic
biodegradability (BD) of FW tested with three types of OS was
87.7%, 74.8%, and 69.4%, respectively.
As described in Fig. 1C and D, NFDM also exhibited a trend with
lower relative cumulative biogas and methane totals seen with
increased sludge storage time. The NFDM has a slightly lower
TMY of 424.6 mL g1 VS as compared to FW, which explains the
slightly lower biogas yield (EBY) and methane yield (EMY) values
obtained during testing (Table 2). The daily biogas and methane
yields from fresh OS peaked at 162.6 and 69.1 mL g1 VSadded on
day 2, whereas the peak with 2-month stored OS appeared on
day 1, at 147.7 mL and 59.3 mL g1 VSadded, respectively. Four-
month stored sludge still demonstrated low daily gas generation
rate after day 3, however, from day 11 to day 18, recovered modest
gas production capability before decreasing substantially. Cumula-
tive biogas and methane yields tested with fresh OS were 662.7
and 414.8 mL g1 VSadded, 596.5 and 351.6 mL g1 VSadded, with
2-month stored OS, and 431.2 and 273.3 mL g1 VSadded with
4-month stored sludge, respectively. The corresponding BD of
NFDM with these three OS were 97.7%, 82.8% and 64.4%,
respectively, which compared similarly to the FW range of 87.7%
for fresh, decreasing to 69.4% with four month stored OS.
207
The methane contents of the biogas produced from stored OS
are described in Fig. 2A. Although gas production from sludge-only
inoculum blanks were used to correct sample biogas production
values for the effect of residual sludge VS, it was informative to
note that the final methane content for the reactors without any
substrate addition decreased from 51.2% to 34.1% after 4 month
storage indicating reduction of methanogenic activity in the
sludge. Fresh and 2-month stored OS with FW and NFDM reached
stable methane content in the biogas of approximately 68% by vol-
ume and exceeded 60% within 5–8 days. However, 4-month stored
OS, while exceeding 60% methane in 8 days with FW, took 16 days
to exceed this value with NFDM. Additionally, while the stabilized
methane content with NFDM was similar for fresh, 2-month, and
4-month stored sludge samples, the methane content of biogas
produced from FW with 4-month stored OS was almost 5% higher
than that of the corresponding fresh and 2-month stored sludge.
3.3. Effect of storage time on methanogenic activity with centrifuged
sludge
Figs. 2B and 3 show the methane content and biogas yields for
three types of CS, including fresh CS, 2-month stored CS, and
4-month stored CS. All the reactors show a quick start-up after
inoculation. Fig. 3A and B represents the cumulative and daily biogas
yields from FW. In the first 6 days of digestion, maximum daily bio-
gas yields were 101.6, 70.6, and 115.3 mL g1 VSadded, for fresh CS,
2-month stored CS, and 4-month stored CS, respectively, with cor-
responding methane yields of 66.0, 49.5, and 80.6 mL g1 VSadded,
respectively. There was no difference on the cumulative biogas
yields with fresh CS and 2-month stored sludge on day 21, how-
ever, as depicted in Fig. 2B, 2-month stored CS had a consistently
higher methane content resulting in 431.9 mL g1 VSadded of meth-
ane yield, which is 7% more than that for fresh CS and a significant
increase (p < 0.05). Compared with fresh CS and 2-month stored CS,
4-month stored CS reached 695.9 and 495.7 mL g1 VSadded of bio-
gas and methane yields, respectively, which was interestingly 11%
and 15% higher, respectively, than the yields of 2-month stored
sludge, even though cumulative gas production during the first five
208 J. Li et al. / Bioresource Technology 166 (2014) 201–210

days was slightly lower than the fresh and 2-month stored CS

0.999
samples. The BD for FW was therefore calculated to be 87.2% for

1.72
37.6
355.5
the 4-month stored CS sample as compared to 70.9% and 76.0%,

4
for the fresh and 2-month stored CS samples, respectively.
The biogas production profiles from NFDM (Fig. 3C and D) gen-

0.997
0.32
326.2
28.6
erally agree with those seen with FW and notably the interesting

2
finding is replicated whereby 4-month stored CS results in sub-

CES + NFDM
stantially higher final biogas and methane yields as compared to

0.998
0.76
392.6
63.0
Fresh
the fresh and 2-month stored CS samples. The highest cumulative
methane yield produced by 4-month stored CS with NFDM, at
408.8 mL g1 VSadded, was significantly different (p < 0.01) with

0.999
1.07
that of fresh CS and represented a BD of 96.3%, which was about

412.4
41.8
20% higher than the other two kinds of CS (Table 2). One slight dif-

4
ference between the FW and NFDM tests with CS was that fresh CS

0.997
0.30
with NFDM as feedstock showed the higher initial gas production

33.5
341.4
rates in comparison with the other two kinds of CS. The maximum

2
CS + NFDM
daily methane yields with fresh CS and 2-month stored CS were

0.996
80.8 and 44.1 mL g1 VSadded on day 2, and the highest methane

0.66
310.1
59.4
Fresh
yields with 4-month CS appeared on day 5, at 45.7 mL g1 VSadded.
There is no significant difference observed between the cumulative

0.918
biogas yields with fresh CS and 2-month stored sludge.

0.74
15.5
324.0
As seen in Fig. 2A and B, CS without substrate addition results in

4
higher methane content as compared to similar OS samples,
exceeding 60% for all three types within 21 days. The methane con-

0.985
0.63
364.8
33.2
tent of biogas produced by the stored CS was slightly higher than
that of fresh sludge with both FW and NFDM. With FW, the meth-

2
OS + NFDM
ane content was 67.9% with fresh CS, and 72.1% and 71.7% with

0.996
0.70
403.5
57.3
stored CS, and similarly with NFDM, was 67.0% with fresh CS and

Fresh
70.5% and 70.9% with stored CS.

0.998
2.20

M0, ultimate methane yield; Rm, the maximum methane production rate; k, the lag phase time; R2, correlation coefficient.
489.1
50.1
3.4. Effect of storage time on methanogenic activity with centrifuged
Modified Gompertz equation parameters of methane production using FW and NFDM with stored and processed sludges.

4
and further evaporated sludge

0.999
1.39
45.4
422.1
Three kinds of CES were also evaluated with batch digestion
tests and Fig. 4 shows biogas production results for FW and NFDM
2
CES + FW

with fresh, 2-month, and 4-month stored CES samples. As shown

1.000
1.37
436.6
55.7

by comparing Fig. 4A and B to the corresponding Fig. 3A and B,

Fresh

the maximum values for daily gas production with CES were
slightly lower than those of the corresponding CS samples.
0.999
1.93

Fresh CES demonstrated the highest biogas production rates

64.3
496.9

during the first week of testing, however, as shown in Fig. 2C,

4

stored CES achieved a higher methane content from the beginning.

0.999
0.88

The FW with fresh CES obtained the highest biogas yield,

48.5
434.5

at 735.5 mL g1 VSadded, while the cumulative biogas yields

2

using 2-month and 4-month stored CES were 614.6 and

0.999
CS + FW

671.6 mL g1 VSadded, respectively.

1.18
393.7
59.0
Fresh

Fig. 4C and D shows the biogas production results for NFDM

with fresh and stored CES. As observed with the OS, fresh CES
0.960

achieved the highest biogas and methane generation rate and

2.25
409.8
26.9

cumulative totals in contrast with the stored CES samples. Cumu-

4

lative biogas and methane yields for NFDM with 4-month stored
CES were 555.9 and 349.0 mL g1 VSadded, respectively, which were
0.997
2.21
429.9
51.0

8% and 11% higher than the corresponding values for the 2-month
stored CES. This result was in accordance with the trend seen with
2

stored CS, perhaps indicating that CES methanogenic activity were

OS + FW

0.997
1.52

initially suppressed but recovered with storage time.

491.5
70.7
Fresh

Rm (mL g1 VSadded d1)

3.5. Evaluation of process and storage stability of methanogenic

Storage time (Months)

activity seeded with OS, CS and CES

M0 (mL g1 VSadded)

The pH as an important indicator was used to evaluate the

health of the anaerobic reactor and generally, a range of pH
Sample

k (day)

between 6.7 and 7.4 can provide an optimum environment for

Table 3

methane production (Bitton, 1994). In this research, pH in all the

R2

reactors was in the range from 7.05 to 8.45 (Table 2).

 J. Li et al. / Bioresource Technology 166 (2014) 201–210 209

Processing of fresh sludge generally decreased the biogas and
methane generation rate as well as reduced the cumulative meth-
ane yield. Reducing the moisture content of fresh original sludge to
82% caused a 19% decrease in cumulative methane production on
average. Possible explanations for this decrease might be physical
or chemical stresses which might include cell damage from centri-
fugation, oxygen incorporation during processing, concentration of
inhibitors along with the beneficial microbial biomass or other rea-
sons not suspected here. With both the CS and CES sludges, meth-
ane yields decreased after 2-month storage time, but appeared to
recover partially (CES) or completely (CS) after 4-month storage
time, approaching that of the fresh OS sludge samples. As shown
in Fig. 5A and B, fresh original sludge had the highest methanogen-
ic activity, however, condensed and 2-month stored sludges
retained between 76% and 87% of this activity, whereas the
processed and 4-month stored sludges retained 84–99%.
3.6. Modified Gompertz model of methane production
Table 3 and Fig. 6 summarize the results of fitting the modified
Gompertz model to digestion data obtained with FW and NFDM
using processed and stored sludges. The equation describes the
rate and methane production potential for the experimental results
well with R2 values within the range of 0.960–1.000, with the
exception of 4-month stored OS + NFDM, which had a slightly
lower R2 value of 0.918 due to the gas production curve displaying
multiple apparent inflection points. Lag phase time (k), as an indi-
cator for methanogenic activity, reflects the acclimation of
microbes to the specific substrate and environmental condition
in the digestion. In this study, short lag phase times (<2.25 days)
were observed for all the digestions which supports that initial
viability was maintained for all conditions tested.
The fitted parameters M0 and Rm further inform the methane
production relationships observed and discussed thus far. For all
conditions tested, except for the 4-month OS + NFDM sample, M0
was within ±4% of the 21-day EMY measured. For the 4-month
OS + NFDM sample, the predicted ultimate methane yield (M0)
was more than 15% greater than the observed 21-day EMY
(273.3 mL g1 VSadded).
The maximum methane generation rate (Rm) gives an indication
of the biological reaction rate which is another important parame-
ter for characterizing methanogenic activity. As observed with
the EMY and M0 values, the Rm of OS also clearly decreased
after condensing and storage. Specifically, the Rm of OS reduced
from 70.7 to 26.9 mL g1 VSadded d1 with FW, and 57.3 to
15.5 mL g1 VSadded d1 with NFDM after 4-month storage, which
are even more drastic reductions than seen with EMY on a percent-
age basis (Fig. 5). Also, concentration of fresh sludge exhibits a
modest reduction in Rm for FW (79–83%) and a slight increase
in Rm with NFDM (104–110%). As seen with the EMY values,
and in contrast to the fresh sludge samples, the Rm values for con-
centrated and stored samples were higher for the 4-month samples

Fig. 6. Modified Gompertz plots of cumulative methane yield using FW and NFDM with fresh and stored sludges. FW, food waste; NFDM, non-fat dry milk; OS, original
sludge; CS, centrifuged original sludge; CES, centrifuged and further evaporated original sludge.
210 J. Li et al. / Bioresource Technology 166 (2014) 201–210
as compared to the 2-month samples, achieving between for 66%
and 91% of the original fresh sludge performance values with the
respective substrates.
4. Conclusions
The results of this study demonstrate the impact of dewatering
and storage on anaerobic digester sludge on biogas and methane
production using batch anaerobic digestion testing with different
substrates. Trends in biogas production observed from processing
and storage were not all straightforward, indicating likely depen-
dence on several factors. Despite reduced biogas production, ben-
efits derived from reduced transportation costs for sludge volume
reduction could be substantial for industrial scale digester start-
ups. Both centrifugation and vacuum evaporation are shown to
be viable methods for processing anaerobic sludge to achieve sig-
nificant moisture and volume reductions and the processed and
stored anaerobic sludges are very good inoculum for anaerobic
digesters.
Acknowledgements
This research was supported by UC Davis Agricultural Experi-
ment Station and a fellowship provided by Chinese Scholarship
Council.
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