wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 6

Avai lab le a t www.sc iencedi rec t .com

journa l homepage : www.e lsev ie r . com/ loca te /wat res

Temperature phased anaerobic digestion increases apparenthydrolysis rate for waste activated sludge

Huoqing Ge, Paul D. Jensen, Damien J. Batstone*

Advanced Water Management Centre (AWMC), Environmental Biotechnology CRC, The University of Queensland, St Lucia,

QLD 4072, Australia

a r t i c l e i n f o

Article history:

Received 28 September 2010

Received in revised form

26 November 2010

Accepted 28 November 2010

Available online 4 December 2010

Keywords:

Temperature phased anaerobic

digestion

Thermophilic pre-treatment

Mesophilic pre-treatment

Waste activated sludge

Hydrolysis rate

* Corresponding author. Tel.: þ61 7 3346 905E-mail address: damienb@awmc.uq.edu.a

0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.11.042

a b s t r a c t

It is well established that waste activated sludge with an extended sludge age is inherently

slow to degrade with a low extent of degradation. Pre-treatment methods can be used prior

to anaerobic digestion to improve the efficiency of activated sludge digestion. Among these

pre-treatment methods, temperature phased anaerobic digestion (TPAD) is one promising

method with a relatively low energy input and capital cost. In this study, an experimental

thermophilic (50e70 �C)emesophilic system was compared against a control meso-

philicemesophilic system. The thermophilicemesophilic system achieved 41% and 48%

volatile solids (VS) destruction during pre-treatment of 60 �C and 65 �C (or 70 �C) respec-

tively, compared to 37% in the mesophilicemesophilic TPAD system. Solubilisation in the

first stage was enhanced during thermophilic pre-treatment (15% at 50 �C and 27% at 60 �C,

65 �C and 70 �C) over mesophilic pre-treatment (7%) according to a COD balance. This was

supported by ammoniaenitrogen measurements. Model based analysis indicated that the

mechanism for increased performance was due to an increase in hydrolysis coefficient

under thermophilic pre-treatment of 60 �C (0.5 � 0.1 d�1), 65 �C (0.7 � 0.2 d�1) and 70 �C

(0.8 � 0.2 d�1) over mesophilic pre-treatment (0.2 � 0.1 d�1), and thermophilic pre-treat-

ment at 50 �C (0.12 � 0.06 d�1).

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction inherently low degradability, as inertmaterials in the influent,

Anaerobic digestion is a biological decomposition process

used to treat, stabilise, and reduce the quantities of organic

wastes prior to disposal or beneficial re-use. Anaerobic

digestion has been used extensively in municipal wastewater

treatment to stabilise primary sludge and activated sludge.

Over the past decade, municipal wastewater treatment

processes have adapted to meet reduced discharge limits on

the effluent nitrogen concentration. Process adaptations

include the removal of primary settlings and primary sludge

streams; and increased retention times for biological nutrient

removal (BNR) processes, resulting in increased sludge age.

Increased sludge age results in waste activated sludge with

1; fax: þ61 7 3365 4726.u (D.J. Batstone).ier Ltd. All rights reserved

as well inert decay products accumulate in the activated

sludge (Gossett and Belser, 1982). Adaptations of modern

wastewater treatment processes have introduced new chal-

lenges for anaerobic digestion, as poor degradability of acti-

vated sludge requires long digester retention times, higher

mixing costs, and also results in poor gas production.

Incorporating a pre-treatment into anaerobic treatment

may enhance the sludge digestion by accelerating hydrolysis,

which is generally accepted as the rate-limiting step in

anaerobic digestion. Pre-treatment can enhance overall

digestion, and requires a minimal capital investment in

comparison with methods such as aerobic digestion (Ro�s and

Zupan�ci�c, 2003). Temperature phased anaerobic digestion

.

Table 1 e Characteristics of the waste activated sludgeused in this study.

Measure Activated sludge

TS (g L�1) 25.4 � 0.1�1

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 61598

(TPAD), combines a short (1e3 days) thermophilic pre-treat-

ment stage (50e70 �C) applied prior to a conventional meso-

philic anaerobic digestion (35 �C, 10e20 days). TPAD is highly

scalable as the process incorporates standard digestion

vessels and low quality heat is the main energy input.

Thermophilicemesophilic TPAD has been shown to be an

effective treatment for increasing methane production and

volatile solids (VS) destruction, compared with a single-stage

mesophilic digestion. Han and Dague (1997) reported 39% VS

destruction of primary sludge achieved in a TPAD system

(55 �C, 3 days hydraulic retention time (HRT) and 35 �C), which

was higher than 32% in a single-stage control system (35 �C).A correspondingmethane production in the TPAD systemwas

also 16% higher over the control system. An improvement of

methane production with a TPAD system treating activated

sludge was also observed by Bolzonella et al. (2007). They

found the highest specificmethane production from the TPAD

(70 �C, 2e3 days HRT and 37 �C) was 370 ml gVSadded�1 , 30e50%

higher than that from a single-stage control system (37 �C).Extending pre-treatment HRT to 5 days did not improve

methane production further.

Nges and Liu (2009) evaluated the effect of pre-treatment

temperature, and reported that the digestion performance of

mixed primary and activated sludge was not influenced by

thermophilic temperature, as the same VS destruction of 42%

was achieved at pre-treatment temperatures of both 50 and

70 �C (2 days HRT). However, this result was still greater than

the VS destruction achieved in the single-stage mesophilic

control (39%). Watts et al. (2006) reported a substantial

improvement of activated sludge digestion with increased

thermophilic temperature. They found that a TPAD system

(47 �C, 2 days HRT and 37 �C) achieved the similar VS

destruction of 24% as a single-stage mesophilic digester

(37 �C). VS destruction was not improved with the thermo-

philic temperature increased to 54 �C, but was significantly

enhanced to 34% at 60 �C.The majority of research has focused on achieving

improved performance by varying pre-treatment conditions

during TPAD, with performance comparisons against single-

stage thermophilic or mesophilic anaerobic digestion.

However, there is little analysis to determine the nature of the

pre-treatment process; whether it improves the rate or extent

of subsequent sludge degradation, or both properties. As

a result, optimal pre-treatment conditions (temperature, pH

and HRT) have not been established. Additionally, thermo-

philicemesophilic TPAD rarely has been evaluated in

a parallel comparison of mesophilicemesophilic with the

same retention times. This study is based on our previous

investigation of primary sludge (Ge et al., 2010), and further

investigates pre-treatment mechanisms of TPAD on waste

activated sludge, with a direct comparison against a control

mesophilicemesophilic process.

VS (g L ) 17.5 � 0.1

pH 6.5e7.5

COD (g L�1) 27.4 � 3.5

VFA (g COD L�1) 0.2 � 0.1

TKN (g N L�1) 1.9 � 0.5

NH4þeN (g L�1) 0.06 � 0.04

Error margins indicate standard deviation across 14 different feed

collections used in the study over 15 months.

2. Materials and methods

2.1. Substrate

Substrate was waste activated sludge, collected from a bio-

logical nutrient removal (BNR) process with 10 days sludge age

and water temperature of approximately 20 �C in the Elanora

wastewater treatment plant, located at Gold Coast, Australia.

The feed was prepared monthly by centrifuging the sludge to

a total solids (TS) concentration of 2e3%, and subsequently

stored below 4 �C. Regular analysis was performed to deter-

mine the characteristics and consistency of the feed material.

Table 1 shows the average characteristics of the activated

sludge feed based on 14 feed collections over 15 months.

2.2. Start-up and operation

Two identical two-stage systems were used throughout.

These consisted of thermophilic pre-treatment (TP) and

mesophilic pre-treatment (MP) pre-treatment stages (0.6 L, 2

days HRT), andmesophilicmethanogenic stages (4.2 L, 14 days

HRT), as shown in Fig. 1. The basic set-up and operation of

thermophilic (TP1)emesophilic (TP2) TPAD and mesophilic

(MP1)emesophilic (MP2) TPAD systems were described in

Ge et al. (2010). Approximately 0.3 L per day of substrate was

fed simultaneously by pumping 0.05 L through the pre-treat-

ment stage andmethanogenic stage at intervals of 4 h per day

(6 times daily, also weighed daily). Gas production was

measured daily from each reactor using tipping bucket gas

meters, and continuously logged. Reactor pH was also recor-

ded online from each reactor continuously. Each reactor was

inoculated using methanogenic inoculum from the meth-

anogenic second stage (35 � 1 �C, 14 days HRT) of a lab-scale

thermophilicemesophilic TPAD system (Ge et al., 2010). This

provided a diverse microbial community and a common

starting point for each reactor.

The systems were operated in parallel for over 15 months.

During this time the temperature of TP1 was altered to create

different operating periods:

� Period 1: 50 �C (186 days), including two periods of pH 5 by

dosing 1 M HCL (Day 39e48, and Day 55e77)

� Period 2: 60 �C (100 days)

� Period 3: 65 �C (67 days), including a period of HRT reduced

to 12 days in TP2 (Day 330e356)

� Period 4: 70 �C (68 days).

The temperature of TP2, MP1 andMP2was held constant at

35 �C during all periods. During periods where the pH of TP1

was reduced, the pH of MP1 was also reduced, and periods

Pretreatment 0.6LTP1 = 50-70°C

MP1 = 35°C

Main Digester 4LTP2=35°CMP2=35°C

PLCF

Gas meterF

Gas meter

Feed reservior Effluent drum

Feedpump

EffluentpumpDigester

pump

Heatingcoil

Water jacket temperature

control

Gas to exhaust

Fig. 1 e Schematic diagram of thermophilic pre-treatment TPAD system and mesophilic pre-treatment TPAD system.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 6 1599

where the loading of TP2 was increased, the loading of MP2

was also increased, as a result of HRT shortened to 12 days.

This was done in steps of 20% around the average in an

attempt to provide better model-parameter identifiability.

After each acid dosing period, the pH of TP1 and MP1 returned

to their natural levels of 6.6 and 6.8, respectively.

2.3. Chemical analysis

Gas production and composition (H2, CH4, CO2) were analysed

by GCeTCD as described previously (Tait et al., 2009). Liquid

samples were collected from each reactor three times per

week. Analysis was performed for TS, VS, volatile fatty acid

(VFA), chemical oxygen demand (COD), total Kjeldahl nitrogen

(TKN) and ammoniumenitrogen (NH4þeN). Analytical

methods were based on Standard Methods (APHA, 1998). The

preparation and measurement of VFA, soluble COD (COD(S))

and NH4þeN were as described previously (Ge et al., 2010).

2.4. Calculation

2.4.1. VS destructionThe two calculation methods used to determine VS destruc-

tion were the Van Kleeck equation and the mass balance

equation. The Van Kleeck equation (1) assumes the amount of

mineral solids is conserved during digestion (Switzenbaum

et al., 2003), and uses the volatile fractions (VS/TS � VSfrac)

in the inlet and outlet as references.

VS destruction% ¼ VSfrac;in � VSfrac;out

VSfrac;in � �VSfrac;in � VSfrac;out

� (1)

where VSfrac,in ¼ volatile fraction (VS/TS) in the inlet solids;

VSfrac,out ¼ volatile fraction (VS/TS) in the outlet solids.

The mass balance equation (2) uses VS concentrations

(VSconc) in the inlet and outlet, expressed as

VS destruction% ¼ VSconc;in � VSconc;out

VSconc;in� 100 (2)

where VSconc,in ¼ VS concentration of inlet; VSconc,out ¼ VS

concentrations of outlet.

Results of the mass balance calculation are sensitive to

systematic sampling issues, which may cause dilution, while

the results of the Van Kleeck calculation are influenced by

accumulation ofmineral inerts within the reactor (under non-

steady state conditions).

2.4.2. Extent of solubilisationExtent of sludge solubilisation in each pre-treatment stage

was calculated using the ratio of total solubilised products

(methane production and COD(S)) and particulate COD

concentration in the inlet feed (Song et al., 2005). Hydrogen

was not detected in either pre-treatment stage. Extent of

solubilisation can be expressed as

Extent of solubilisation% ¼ CODCH4 þ CODðSÞo�CODðSÞiCODðTÞi�CODðSÞi

� 100

(3)

where CODCH4 ¼ methane production as mg COD during pre-

treatment; COD(S)i ¼ COD(S) concentration of inlet; COD

(S)o ¼ COD(S) concentration of outlet; COD(T)i ¼ total COD

concentration of inlet.

2.5. Mathematical analysis

Mathematical analysis was based on the IWA Anaerobic

Digestion Model No. 1 (ADM1) (Batstone et al., 2002). Imple-

mentation of ADM1 for a TPAD process is described by Ge et al.

(2010), with the input model of Nopens et al. (2009). Initial

conditions were adjusted based on measurements of organic

solids, organicacids, ammonia,TKN,etc.Therewereapprox420

input changes over 450 days used in the model. Degradability

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 61600

extent (fd) and apparent first order hydrolysis rate coefficient

(khyd) were the main parameters used to assess and compare

two TPAD systems. In each system, khyd and fd were simulta-

neously estimated to achieve the average optimal values for the

wholeTPADprocess,whichwere thenset todetermine the two-

parameter uncertainty surface for khyd and fd based on the

method of Batstone et al. (2003, 2009). In the TP system, confi-

dence regions were estimated based on system performance at

each pre-treatment temperature. For the MP system, operating

conditionswere constant throughout theexperiment; therefore

only one confidence region was estimated for comparison.

A 95% confidence limit was used, with appropriate F-value

(2.996) for 2 parameters and the number of degrees of freedom.

Van Kleeck VS destruction was used as a measured variable,

with sum of squared errors (c2) as an objective function. Mass

balance VS destruction gave similar optimal parameter esti-

mates. However, confidence regions were enlarged, and the

upper limit of the region could not generally be determined.

Therefore theanalysis isbasedontheVanKleeckdataandmass

balance based regions are not shown.

3. Results

3.1. Performance of combined TPAD systems

Fig. 2 shows VS destruction in each system calculated bymass

balance (Fig. 2A) and Van Kleeck (Fig. 2B) equations. During all

periods, VS destruction determined using the mass balance

equation (2), was consistent with VS destruction determined

using the Van Kleeck equation (1), and this applied to both

TPAD systems. Consistent results from the VS calculation

methods confirm systematic sampling errors and/or unex-

pected behaviours were minimal.

During Period 1 (50 �C pre-treatment), thermophilic pre-

treatment offered no advantage over mesophilic pre-treat-

ment. Increasing thermophilic pre-treatment temperature to

60 �C (Period 2) improved VS destruction in the TP system from

34 � 1% to 41 � 1%. A further increase of VS destruction to

48 � 2% was observed when thermophilic pre-treatment

temperature was increased to 65 �C (Period 3), but no further

enhancement at 70 �C (Period 4). Statistical analysis (student’s

t-test, a¼ 0.05) confirmed that VS destruction in the TP system

(65 �C) was significantly greater than that achieved at 60 �C,which was also a significant improvement over that achieved

at 50 �C. VS destruction in the TP system during Periods 2e4

was also significantly better as compared to the MP system

(student’s t-test, a ¼ 0.05).

Additionally, pre-treatment pHwas temporarily lowered to

pH 5 twice during 50 �C pre-treatment (Day 39e48 and Day

55e77), which did not influence VS destruction in either

system compared to previously. Similarly, VS destruction in

each system was maintained as previously described when

HRT ofmethanogenic stages was shortened from 14 to 12 days

during Period 3 (Day 330e356).

Total methane produced from TP system was consistently

higher than that fromMP system except Period 1, as shown in

Fig. 3 and Table 2. This was confirmed by student’s t-test

(a ¼ 0.05) as a statistically significant improvement, and was

consistent with enhanced VS destruction in TP system over

MP system during Periods 2e4. The methane production

increase in TP system was observed when thermophilic pre-

treatment temperature was increased to 60 �C, but not at 65 �Cand 70 �C. This was not consistent with further enhancement

of VS destruction at thermophilic pre-treatment of 65�Ccompared to 60 �C. This is likely due to three reasons: (a) As

temperature increased, different portions of VS were

degraded by the microbial community resulting in different

gas yields per VS destroyed, (b) the decrease in stage 1

methane production (and increase in stage 2 methane

production) at higher temperatures is complicating analysis,

and (c) it is possible that gas leaks (approx. 10% losses overall)

were occurring despite our best efforts. Thus, gas flows have

not been used in the detailed model based analysis below, but

have been used in solubilisation analysis, and quantitatively

compared against model outputs in Section 4.3.

During Periods 1e2, approximately 50e70% of methane

generated from the TP system was produced in the thermo-

philic pre-treatment stage, with small amounts from the

subsequent mesophilic digestion stage. Increasing tempera-

ture of TP1 to 65 �C and 70 �C caused a substantial decrease in

methane production in the first stage. A correspondingly

larger amount of methane was produced from TP2, especially

during Period 4. In contrary, in MP system, the methane

production from MP1 was less compared to that from MP2 in

all periods. Moreover, acid dosing was used to lower pH to 5 in

TP1 at 50 �C, in order to reduce the activity of methanogens

and cause washout. As expected, methane production was

severely decreased in TP1, producing a corresponding increase

in production from TP2. Once the acid dosing stopped,

methane production rapidly returned to previous levels.

3.2. Model based analysis

Fig. 4 shows the 95% confidence regions for degradability (fd)

(x-axis) and apparent hydrolysis rate (khyd) ( y-axis) in MP

system and TP system at 50 �C, 60 �C, 65 �C and 70 �C,respectively. The MP system confidence regions overall at

35 �C and TP at 50 �C overlapped, indicating statistically the

same apparent properties. khyd in the TP system increased

significantly above both MP and TP at 50 �C as temperature in

TP1 was increased to 60 �C, but did not increase further to

70 �C. The confidence region moved to the right and upward

from 60 �C to 65 �C, indicating a slight improvement in prop-

erties, but with full overlap between the regions at 65 �C and

70 �C. fd in the TP system were comparable for all tested

temperatures in TP1, and statistically overlapped with the fdobserved in MP. Overall, the results indicate consistent

increases in hydrolysis coefficient with increased tempera-

ture, but with a relatively constant degradability of 30e60%

(Table 3).

3.3. Analysis of pre-treatment stage in TPAD systems

Extent of solubilisation of the activated sludge determined

using equation (3) is shown in Fig. 5. The solubilisation in TP1

was increased from 15% to 27% with the thermophilic

temperature increased from 50 �C to 60 �C, and was not

improved further at 65 �C and 70 �C (Fig. 5). Themain profile of

solubilisation at 50 �C and 60 �C was methane production,

VS

des

truct

ion

(%)

0

10

20

30

40

50

60

70% VS destruction in TP system% VS destruction in TP system

Time in operation (days)

0 50 100 150 200 250 300 350 400 450

% VS destruction in TP system% VS destruction in MP system

Period 1 Period 2 Period 3 Period 4

60

50

40

30

20

10

0

pH 5pH 5HRT 12 days A

B

Fig. 2 e VS destruction calculated by mass balance equation (A) and Van Kleeck equation (B) during each period in the

thermophilic pre-treatment (TP) and mesophilic pre-treatment (MP) systems (% VS destruction is based on the activated

sludge feed characteristics).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 6 1601

with a relatively lower level of VFA and other soluble products

presented by COD(S). However, the majority of solubilisation

profile was changed to the COD(S) with increasing thermo-

philic temperature to 65 �C and 70 �C, as the methane

production dropped at higher thermophilic temperatures. For

all periods, solubilisation in TP1 was higher than that in MP1,

and did not appear to be affected by low pH (pH 5) at 50 �C (Day

39e48 and Day 55e77).

The VFA profiles were similar in TP1 and MP1 during all

periodswith acetate as the primaryVFAproduced, followed by

propionate as the secondmajor acid. Other VFAs (iso-butyrate,

butyrate, iso-valerate, valerate and hexanoate) were also

detected at much lower levels. The acetate concentration in

TP1was lowest during thermophilic pre-treatment of 50 �Cand

60 �C, possibly due to the combination of poor solubilisation

and good methane production (Fig. 6). Increasing the thermo-

philic pre-treatment temperature to 65 �C and 70 �C resulted in

substantial increases in acetate and propionate concentra-

tions, which was consistent with measurements of COD(S),

suggesting that most material hydrolysed was converted to

organic acids. It also indicated the methanogenesis was

limited at the higher thermophilic temperatures. Methane

production was also suppressed under acidic conditions and

resulted in increased accumulation of VFA in TP1, as hydro-

lysis and fermentation processes continued to producing

intermediate products (VFAs).

Nitrogenous organic compounds contained in the sludge

(e.g. protein) is solubilised in the form of NH4þeN during pre-

treatment process, thusNH4þeN is another important indicator

of solubilisation. Solubilisation according to NH4þeN in TP1

CH

4 pro

duct

ion

(L d

ay-1

)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

CH4 production in TP systemCH4 production in MP1CH4 production in MP system

Period 1 Period 2 Period 3 Period 4

CH4 production in TP1

Fig. 3 e Average methane production during each period in the thermophilic pre-treatment (TP) and mesophilic pre-

treatment (MP) systems (Error bars are 95% confidence in mean methane production).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 61602

increased with each step increasing thermophilic pre-treat-

ment temperature (Fig. 7). Release of NH4þeNwas greater from

TP1 than from MP1 for all periods, which was consistent with

the extent of solubilisation results. The NH4þeN results indi-

cated protein fermentation was improved under thermophilic

condition, and improved with thermophilic temperature

increase. Again, low pH did not have an impact on NH4þeN

release in TP1.

k hyd

(d-1

)

0.6

0.8

1.0

1.2

TP 60 C

TP 65 C

TP 70 C

Deg

rade

s fa

ster

4. Discussion

4.1. Overall performance of TPAD systems

An increase of pre-treatment temperature from 50 �C to 65 �Csubstantially improved the overall VS destruction from 34% to

48% for activated sludge, but not for primary sludge (54% at

50e65 �C) (Ge et al., 2010). It should be noted that performance

on primary sludge is not diminished above 65 �C, and thus

mixed feed digesters should be operated at elevated tempera-

tures. This not only decreases sludge disposal costs substan-

tially, it also allows formuchbetter hygenisationandpathogen

removal. In addition, it providesperformanceofVSdestruction

Table 2eA summary ofmethane production (L gVSfedfedL1)

in the thermophilic pre-treatment (TP) system andmesophilic pre-treatment (MP) system during eachperiod.

TP system MP system

Period 1 (50 �C) 0.10 � 0.03 0.07 � 0.04

Period 2 (60 �C) 0.16 � 0.02 0.11 � 0.03

Period 3 (65 �C) 0.15 � 0.02 0.10 � 0.03

Period 4 (70 �C) 0.17 � 0.03 0.09 � 0.04

Error margins indicate standard deviation across different gas

measurements over each period.

above 38%, which is one of the legislative levels for perfor-

mance implemented by US EPA (EPA, 1994), and most Austra-

lian legislation. At higher performance levels (Period 3), VS

destruction was not strongly impacted by a change from 14 to

12 days, consistent with a hydrolysis coefficient of >0.5 d�1.

Correspondingly, the implementation of smaller digesters or

increased organic loading rates could be possible, which could

substantially reduce capital costs.

Another advantage of the TPAD system is the use of

renewable methane to produce energy, which is used to

compensate heat requirements (heat demand and heat losses)

in the TPAD system, and is conventionally produced by

cogeneration engines. The main heat demand from the TPAD

system is to heat up the sludge to the required temperature in

fd

0.2 0.3 0.4 0.5 0.6 0.70.0

0.2

0.4

TP 50 C

MP 35 C

Degrades more

Fig. 4 e 95% confidence regions for apparent hydrolysis

coefficient (khyd, dL1) and degradability (fd) using Van

Kleeck VS destruction as an objective function in the

mesophilic pre-treatment (MP) system and thermophilic

pre-treatment (TP) system at 50, 60, 65 and 70 �C,respectively.

Table 3 e A summary of apparent hydrolysis coefficient (khyd) and degradability (fd) in the mesophilic pre-treatment (MP)system and thermophilic pre-treatment (TP) system.

Parameter TP system MP system

50 �C 60 �C 65 �C 70 �C

khyd (d�1) 0.12 � 0.06 0.5 � 0.1 0.7 � 0.2 0.8 � 0.2 0.2 � 0.1

fd 0.4 � 0.1 0.41 � 0.04 0.51 � 0.04 0.53 � 0.02 0.4 � 0.1

Numbers after ‘�’ are the linear, uncorrelated 95% confidence in parameter values.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 6 1603

the thermophilic stage, as well as to the mesophilic temper-

ature in the second stage. Heat from thermophilic stage will

also be used in the mesophilic stage, and minimises overall

heating requirements in a TPAD process. A detailed evalua-

tion of the heat balance, including losses and sensitivity to

feed concentration is contained in the supplementary infor-

mation. This analysis also considers the increased perfor-

mance provided by thermophilic operation, but does not

consider a smaller main digester.

The heat balance for thermophilic and mesophilic systems

is generally positive at a 2% feed concentration, with either

supplemental heat needed, or diversion of methane from

electricity to heat energy, as shown in Fig. i (Supplementary

information). The heat balance became negative when

increasing the feed concentration to 4% in both systems, indi-

cating the potential heat production could fully offset heat

requirements in both systems.At the feed concentration of 6%,

the potential heat production was greatly in excess, especially

in the TP systemwith thermophilic pre-treatment of 65 �C and

70 �C. This emphasises the need for pre-thickening, but

importantly, indicates that the heat balance is very similar for

standard mesophilic and TPAD systems, with TPAD processes

operated above 60 �C generally producing more excess energy

than a mesophilic process. It should also be noted that we

assume only waste heat is used, from cogeneration engines

with electricity being produced as the main process.

The

exte

nt o

f sol

ubilis

atio

n (%

)

0

10

20

30

40MethaneVFAsOthers

Period 1 Period 2 Period 3 Period 4

TP1

MP1

Fig. 5 e Extent of solubilisation during each period in the

thermophilic pre-treatment stage (TP1) and the mesophilic

pre-treatment stage (MP1) (% solubilisation is based on the

activated sludge feed characteristics and equation (3)).

4.2. Model based analysis

Regions measurably moved upwards and to the right with

increased temperature from 50�C to 75 �C, reflecting the

increase in performance. The regions also decreased in area as

temperature increased, likely due to the increase in VS

destruction. Because VS destruction is a product (or fractional)

term, it can be determined with better accuracy at higher

destruction levels.

There are a wide range of hydrolysis rates reported in

literature for mixed streams containing both primary sludge

and activated sludge, between 0.1 and 1 d�1 (Pfeffer, 1974;

Ghosh, 1981; Bolzonella et al., 2007). However, the determi-

nation of hydrolysis rate for digestion of WAS only has been

limited, and is now addressed in our study.

Hydrolysis coefficient was sensitive to temperature, with

no improvement at 50 �C, but significant increases at 60 �C and

higher. This may be due to emergence of true thermophiles at

the higher temperatures that do not emerge at the interme-

diate temperature of 50 �C. However, this contrasts to our

previous work on primary sludge, where hydrolysis rate was

improved above 50 �C, but not sensitive to temperature above

this. Apparent degradability was not significantly impacted,

indicating that the improved VS destruction observed in this

study was due to increase in apparent hydrolysis rate, rather

than an increase in degradable fraction. Analysis of degrad-

ability is consistent with our previous observations on

primary sludge (Ge et al., 2010).

This increase in rate rather than degradability fraction is

similar to the effects of mechanical pre-treatments, e.g.

sonication (Climent et al., 2007; Khanal et al., 2007). This is in

contrast to high impact methods such as thermal hydrolysis,

which increase rate and extent substantially (Batstone et al.,

2009). In a full-scale plant, faster degradation could be uti-

lised in either design of a smaller main digester, or intensifi-

cation of an existing process.

4.3. Pre-treatment mechanisms

Solubilisation was enhanced in TP1 at 50 �C over MP1 at 35 �C(Fig. 5), even though hydrolysis coefficient remained the same

(Fig. 4). Bothmeasureswere consistently enhancedat 60 �Cand

above.The inconsistencyat 50 �C is likelybecause theapparent

hydrolysis coefficient was acquired from the overall perfor-

mance, and was therefore dominated by the second stage

performance, whereas the information in Fig. 5 is based on the

first stage (methaneþVFAsþotherproducts). Thiswas further

tested by simulating the first stage further, and comparing

model outputs to observed results. The model simulation of

Time in operation (days)

0 50 100 150 200 250 300 350 400 450

tVFA

con

cent

ratio

n (m

g L-1

)

0

1000

2000

3000

4000tVFA in TP1tVFA in MP1

Period 1 (50 C) Period 2 (60 C) Period 3 (65 C) Period 4 (70 C)

pH 5pH 5

Fig. 6 e tVFA concentrations during each period in the thermophilic pre-treatment stage (TP1) andmesophilic pre-treatment

stage (MP1).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 61604

solubilisation followed the same trend of measured solubili-

sation in TP1, except at 50 �C, where model predictions were

conservative compared to solubilisation measurements.

Improvements at 60 �C to 65 �C (or 70 �C) were reflected in

increased solubilisation according to both model and

measurements. Additionally, fractions of solubilisation pre-

dictedbymodelandmeasurements in theTP1wereconsistent,

but the model could not predict the decrease in methane

production at 65 �C and 70 �C, indicating a model limitation.

This comparison suggests that the hydrolysis coefficient

determined at 50 �C was conservative for the first stage (but

Time in o

0 50 100 150 200

NH

4+ -N c

once

ntra

tion

(mg

L-1)

0

200

400

600

800

1000

1200NH4

+-N in TP1NH4

+-N in MP1

Period 1

pH 5pH 5

Fig. 7 e NH4D-N concentrations during each period in the therm

treatment stage (MP1).

valid overall), while hydrolysis coefficients estimated at

increased pre-treatment temperatures were valid across both

digesters.

However, solubilisation results and model analysis both

confirmed that the solubilisation or hydrolysis was improved

in TP1 at 60e70 �C compared to MP1. Therefore, improved

first stage hydrolysis is a major factor contributing to enhan-

ced performances in the TP system. The increased thermo-

philic temperature may improve production of extracellular

enzymes to hydrolyse more complex or inert substrate mate-

rials, and have selected the specialised microbial community,

peration (days)

250 300 350 400 450

Period 2 Period 3 Period 4

ophilic pre-treatment stage (TP1) and mesophilic pre-

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 6 1605

whichwill result in an optimized hydrolysis. All these possible

improvements will increase the substrate availability for

digestion in the subsequent methanogenic stage.

NH4þeN release in the pre-treatment stage was consistent

with the extent of solubilisation data and had a significant

influence on overall NH4þeN release. It was exhibited as the

NH4þeN released from TP2 (approx. 880 mg L�1) and was 30%

higher than that from MP2 (approx. 680 mg L�1). However, the

enhanced NH4þeN release in TP1 with increased thermophilic

temperature did not influence the NH4þeN release in meth-

anogenic stage. This result was different from the similar

NH4þeN release observed in both methanogenic stages with

individual thermophilic (50e65 �C) and mesophilic pre-treat-

ment (35 �C) treating primary sludge (Ge et al., 2010). It indi-

cated that overall conversion was improved further in the

methanogenic stage.

4.4. Methanogenesis during thermophilic pre-treatmentprocess

There is some uncertainty over how best to operate TPAD

systems. If the initial step is regarded purely as a hydrolytic

step, methanogenesis is not required. This is not the case for

our results, andno difference is seenwheremethanogenesis is

inhibited by low pH or high temperature compared to where

methanogenesis is allowed to occur. Particularly for activated

sludge compared to primary sludge (Ge et al., 2010), the

methane production in the pre-treatment stage is far higher,

with approx 65% of total production occurring in the whole

system. Methanogenesis in the pre-treatment stage is not

detrimental, since it allows for increasedoverallmethanogenic

retention time, improved kinetics, and provides protection to

the secondary main stage. The presence of substantial meth-

anogenesis in this pre-treatment stage, with a very short

retention time is of some interest. Methanogenesis at short

HRT may be due to change in metabolic pathways from ace-

ticlastis to acetate oxidation, under which acetate is first oxi-

dised to hydrogen and carbon dioxide, and subsequently

converted tomethane.This is enhanced thermodynamically at

higher temperatures 50 �C to 65 �C (Karakashev et al., 2006;

Zinder et al., 1984), and is supported by microbial results indi-

cating a dominance of Methanosarcinaceae, which has been

found to be the dominant methanogen in acetate oxidising

systems by isotopic carbon analyses (Karakashev et al., 2006).

While methanogenesis in the pre-treatment stage

complicates the process by the presence of two methane

producing units, the action of a two-step acetate oxidation/

aceticlastic methanogenic process can provide advantages.

This offers better resistance to process inhibition and toxins,

since aceticlastis and acetate oxidisers are influenced by

different factors. As an example, protein-rich cattle and

piggery wastes, have high ammonia, to which acetate oxi-

disers are less susceptible than aceticlasts (Karakashev et al.,

2005). In essence, the robust acetate oxidation step can act to

protect the more sensitive aceticlastic methanogenic step,

and provide a biological buffer.

A decrease of 31% and 58% methane production in TP1 at

65 �C and 70 �C compared to 60 �C suggests the activity of

methanogenesis (presumptively acetate oxidation, or both) is

decreased. Therefore, the level of methanogenesis in the pre-

treatment stage can be tuned by temperature, especially at

60e70 �C, without negative impact on overall VS destruction.

Based on our results, it is also possible to suppress meth-

anogenesis by decreasing the pH to 5, but doing so by

changing temperature is lower cost, and easier implemented.

5. Conclusions

The following conclusions can be drawn from this study:

� VS destruction in thermophilicemesophilic TPAD was

increased by thermophilic pre-treatment at 50 �C to 65 �C(34%e48%), which was 11e30% higher than that in meso-

philicemesophilic TPAD (37%), expect thermophilic pre-

treatment of 50 �C.� Model based analysis indicated the hydrolysis coefficient in

the TP system was not improved under thermophilic pre-

treatment of 50 �C (0.06e0.18 d�1) compared to the MP

system (0.1e0.3 d�1), but significantly enhanced to 0.6 d�1 at

60 �C, up to 1 d�1 at 65 �C and 70 �C. However, increasing

thermophilic pre-treatment temperature had no impact on

the overall degradability in the TP system relative to the MP

system (0.30e0.55).

� Solubilisation was improved during thermophilic pre-

treatment relative to mesophilic pre-treatment, and

reached to maximum of 27% at thermophilic pre-treatment

of 60 �C. Further thermophilic temperature increases had no

further impacts. Higher NH4þeN was released during ther-

mophilic pre-treatment over mesophilic pre-treatment, and

further increased by increasing the thermophilic pre-treat-

ment temperature from 50 �C to 70 �C.� A large amount of methane was produced from thermo-

philic pre-treatment stage between 50 �C and 60 �C, but

started to decrease with further increase of temperature to

65 �C and 70 �C. Methane production from the pre-treatment

stage was heavily inhibited at acidic conditions (pH 5).

Acknowledgements

This work was funded by the Queensland State Government,

under the Smart State Research-Industry Partnerships

Program (RIPP), Meat and Livestock Australia, and Environ-

mental Biotechnology Cooperative Research Centre (EBCRC),

Australia as P23 “Small-medium scale organic solids stabili-

zation”. Huoqing Ge and Paul Jensen are recipients of an

EBCRC postgraduate scholarship and postdoctoral award,

respectively. We thank Gold Coast City Council (Gold coast

water) for supplying samples from their Elanora Wastewater

Treatment Plant.

Appendix. Supplementary information

Supplementary data related to this article can be found online

at doi:10.1016/j.watres.2010.11.042.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 5 9 7e1 6 0 61606

r e f e r e n c e s

APHA, 1998. Standard Methods for the Examination of Water andWastewater, 20th ed. American Public Health Association,Washington, DC, USA.

Batstone, D.J., Keller, J., Angelidaki, I., Kalyuzhnyi, S.,Pavlostathis, S.G., Rozzi, A., Sanders, W.T.M., Siegrist, H.,Vavilin, V., 2002. IWA Task Group for Mathematical Modellingof Anaerobic Digestion Processes. IWA Publishing, London, UK.

Batstone, D.J., Pind, P.F., Angelidaki, I., 2003. Kinetics ofthermophilic, anaerobic oxidation of straight and branchedchain butyrate and valerate. Biotechnology and Bioengineering84 (2), 195e204.

Batstone, D.J., Tait, S., Starrenburg, D., 2009. Estimation ofhydrolysis parameters in full-scale anaerobic digesters.Biotechnology and Bioengineering 102 (5), 1513e1520.

Bolzonella, D., Pavan, P., Zanette, M., Cecchi, F., 2007. Two-phaseanaerobic digestion of waste activated sludge: effect of anextreme thermophilic prefermentation. Industrial andEngineering Chemistry Research 46 (21), 6650e6655.

Climent, M., Ferrer, I., Baeza, M.D., Artola, A., Vazquez, F., Font, X.,2007. Effects of thermal and mechanical pretreatments ofsecondary sludge on biogas production under thermophilicconditions. Chemical Engineering Journal 133 (1e3), 335e342.

Ge, H.Q., Jensen, P.D., Batstone, D.J., 2010. Pre-treatmentmechanisms during thermophilic-mesophilic temperaturephased anaerobic digestion of primary sludge. Water Research44 (1), 123e130.

Ghosh, S., 1981. Kinetics of acid-phase fermentation in anaerobicdigestion. Biotechnology and Bioengineering 11, 301e313.

Gossett, J.M., Belser, R.L., 1982. Anaerobic-digestion of wasteractivated sludge. Journal of the Environmental EngineeringDivision-Asce 108 (6), 1101e1120.

Han, Y., Dague, R.R., 1997. Laboratory studies on the temperature-phased anaerobic digestion of domestic primary sludge. WaterEnvironment Research 69 (6), 1139e1143.

Karakashev, D., Batstone, D.J., Angelidaki, I., 2005. Influence ofenvironmental conditions on methanogenic compositions inanaerobic biogas reactors. Applied and EnvironmentalMicrobiology 71 (1), 331e338.

Karakashev, D., Batstone, D.J., Trably, E., Angelidaki, I., 2006.Acetate oxidation is the dominant methanogenic pathwayfrom acetate in the absence of Methanosaetaceae. Appliedand Environmental Microbiology 72 (7), 5138e5141.

Khanal, S.K., Grewell, D., Sung, S., Van Leeuwen, J., 2007.Ultrasound applications in wastewater sludge pretreatment:a review. Critical Reviews in Environmental Science andTechnology 37 (4), 277e313.

Nges, I.A., Liu, J., 2009. Effects of anaerobic pre-treatment on thedegradation of dewatered-sewage sludge. Renewable Energy34 (7), 1795e1800.

Nopens, I., Batstone, D.J., Copp, J.B., Jeppsson, U., Volcke, E.,Alex, J., Vanrolleghem, P.A., 2009. An ASM/ADM modelinterface for dynamic plant-wide simulation. Water Research43 (7), 1913e1923.

Pfeffer, J.T., 1974. Temperature effects on anaerobic fermentationof domestic refuse. Biotechnology and Bioengineering 16 (6),771e787.

Ro�s, M., Zupan�ci�c, G.D., 2003. Thermophilic anaerobic digestion ofwaste activated sludge. Acta Chimica Slovenica 50, 359e374.

Song, H., Clarke, W.P., Blackall, L.L., 2005. Concurrent microscopicobservations and activity measurements of cellulosehydrolyzing and methanogenic populations during the batchanaerobic digestion of crystalline cellulose. Biotechnology andBioengineering 91 (3), 369e378.

Switzenbaum, M.S., Farrell, J.B., Pincince, A.B., 2003. Relationshipbetween the Van Kleeck and mass-balance calculation ofvolatile solids loss. Water Environment Research 75 (6), 572.

Tait, S., Tamis, J., Edgerton, B., Batstone, D.J., 2009. Anaerobicdigestion of spent bedding from deep litter piggery housing.Bioresource Technology 100 (7), 2210e2218.

US Environmental Protection Agency, 1994. A plain English Guideto the EPA Part 503 Biosolids Rule. In: Environmental protectionAgency 8322/R-93-003, September Washington, DC, USA.

Watts, S., Hamilton, G., Keller, J., 2006. Two-stage thermophilic-mesophilic anaerobic digestion of waste activated sludge froma biological nutrient removal plant. Water Science andTechnology 53 (8), 149e157.

Zinder, S.H.,Anguish,T.,Cardwell, S.C., 1984.Effectsof temperatureonmethanogenesis in a thermophilic (58 �C) anaerobic digester.Applied and Environmental Microbiology 47 (4), 808e813.