Biochemical Engineering Journal 128 (2017) 3344

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Biochemical Engineering Journal

journal homepage: www.elsevier.com/locate/bej

Regular article

Methods for quantication of biosorption in high-rate activated

sludge systems
Arifur Rahman a, , Harold Yapuwa b , Manel Garrido Baserba c,d , Diego Rosso c,d ,
Jose A. Jimenez e , Charles Bott f , Ahmed Al-Omari b , Sudhir Murthy b , Rumana Riffat a ,
Hayde De Clippeleir b
a
Department of Civil & Environmental Engineering, The George Washington University, 800 22nd Street, NW, Washington, DC 20052, USA
b
DC Water, 5000 Overlook Avenue, SW, Washington, DC, 20032, USA
c
Department of Civil & Environmental Engineering, University of California, Irvine, CA 92697-2175, USA
d
Water-Energy Nexus Center, University of California, Irvine, CA 92697-2175, USA
e
Brown and Caldwell, 2301 Lucien Way, Suite 250, Maitland, FL 32751, USA
f
Hampton Roads Sanitation District, 1436 Air Rail Avenue, Virginia Beach, VA 23455, USA

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

Article history: The fundamental mechanism behind biosorption to recover carbon from wastewater through high-
Received 17 May 2017 rate activated sludge (HRAS) system is insufciently studied. The most accepted method of quantifying
Received in revised form 5 September 2017 biosorption capacity of activated sludge depends on higher settling velocity solids. As HRAS had different
Accepted 7 September 2017
settling characteristics and in addition, raw wastewater fed to the HRAS system contained approximately
Available online 8 September 2017
55% settleable organics, the conventional biosorption capacity test was not capable of accurately den-
ing biosorption. Thus, we studied ex-situ aerobic yield as an alternative approach for determination of
Keywords:
biosorption in batch systems. However this approach similarly to biosorption capacity was not sensitive
Adsorption
Carbon recovery enough to quantify differences between HRAS types, potentially due to enhanced storage mechanisms
Contact-stabilization under the feast-famine approach. A viable method to determine biosorption in the batch system was
Energy based on measurement of ex-situ oxygen uptake rate (OUR) of HRAS over 40 h and the biosorption yield
Municipal wastewater was considered as the area underneath the OUR curve normalized for solids concentration. In addition,
High-rate activated sludge in-situ based biosorption quantication was proposed in this study based on detailed measurement of
carbon mass balances including contribution of extracellular polymeric substances. A clear relationship
between biosorption yield and in-situ biosorption with carbon mass-balance, carbon redirection and
solids retention time of HRAS systems was observed suggesting two viable methods for quantication of
biosorption.
2017 Elsevier B.V. All rights reserved.

1. Introduction WWTPs. A-stage intends to minimize carbon oxidation and max-

Recently, wastewater treatment plants (WWTPs) are shifting
away from energy-intensive wastewater treatment processes to
more sustainable processes that have a net low energy demand
and higher energy recovery. In addition, to achieve energy efcient
nitrogen removal via anaerobic ammonium oxidation (anam-
mox) or autotrophic short-cut mechanisms, the implementation
of carbon removal technologies such as A-stage of AB (adsorption
bio-oxidation) process and contact-stabilization (CS) as a form of
high-rate activated sludge (HRAS) systems are gaining interests in
Corresponding author.
E-mail address: arifur@gwmail.gwu.edu (A. Rahman).
imizes organic carbon recovery through biosorption at very low
solids retention time (SRT of 0.1-1 d) and hydraulic retention time
(HRT of 0.51 h) [14]. Few full-scale A-stage plants have shown
to display a high degree of biochemical oxygen demand (BOD)
removal (up to 82%) and energy-rich sludge production (0.81.4 kg
sludge/kg BOD removed) [5,1,2]. In addition to AB systems, high-
rate CS process is a variation of high-rate conventional activated
sludge systems which signicantly improve transformation of
organic carbon from wastewater to sludge matrix through biosorp-
tion [69]. Thus, to achieve maximum organic carbon recovery
through high-rate carbon removal technologies, the quantication
of biosorption in HRAS systems is crucial for turning the WWTP
into a water resource recovery facility.

http://dx.doi.org/10.1016/j.bej.2017.09.006
1369-703X/ 2017 Elsevier B.V. All rights reserved.
34 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344
The biosorption mechanism is a combination of physical-
chemical and biological phenomena in activated sludge, which
includes outer cell surface adsorption of particulate and colloidal
organic matter and storage of soluble COD (chemical oxygen
demand) inside the cell [1014]. Biosorption capacity of the
sludge has been mostly quantied in bench scale experiments to
understand the kinetics of organic matter transformation from
wastewater to sludge matrix [10,1219]. Riffat and Dague [14] rst
measured the biosorption capacity on anaerobic granular biomass
mixed with non-fat dry milk (soluble COD) for short contact time
(230 min) in which COD removal was determined by subtract-
ing the supernatant COD (after settling) from initial COD of carbon
source. The advantage of that test was that the granular sludge gen-
erally has higher settling velocity than occulent sludge and thus
the supernatant was sufciently clear to determine the removal of
COD. Tan and Chua [18] and Zhang et al. [19] adopted the simi-
lar approach for measuring the biosorption capacity of high mean
cell residence time activated sludge (5 days) mixed with settled
domestic wastewater in a batch system open to the atmosphere.
In their study, the oxidation of COD from wastewater in a short
contact time was neglected. In addition, the non-settling fraction
of biomass was also negligible due to the better settleability of
high SRT sludge. Later, Guellil et al. [10] and Lim et al. [12] applied
a modied approach of Tan and Chua [18] by centrifuging the
supernatant after settling to separate out non-settleable biomass
from wastewater. In this case, the possible soluble COD (sCOD)
oxidation or biomass growths were also neglected. However, non-
settleable particulate matter from wastewater and the adsorption
of particulate and colloidal matter to the non-settling biomass
cannot be separated out by the centrifugation, thus overestimat-
ing the biosorption capacity. Modin et al. [17] further improved
the method by separating sCOD oxidation by active and inactive
biomass (sodium azide addition) experiment for determining the
biosorption of municipal wastewater. Lim et al. [11] reported that
inactive biomass mixed with wastewater for a short contact time
determines extracellular adsorption while active biomass quanti-
es extracellular adsorption and intracellular storage. However,
they did not measure polyhydroxyalkanoates (PHA) as a stored
compound and neglected oxidation of soluble COD. Since this
conventional biosorption capacity approach is inuenced by the
possible interference of oxidation, biomass growth, non-settleable
particulate matter from wastewater and non-settling biomass, an
alternative approach is required for HRAS systems to quantify
actual biosorption of organic matter from wastewater. In addi-
tion, the HRAS systems have comparatively different settleability
characteristics (lower efuent quality) compared to the low-rate
systems [20,21], thus a proper method is required for biosorption
quantication in HRAS systems
In HRAS systems, the inuent carbon redirection is dened as
entrapment, adsorption and storage of COD from the wastewa-
ter to the sludge matrix, which depends on the SRT [22,9]. The
lower the SRT in a HRAS system, higher the carbon redirection
from wastewater was found in previous literatures [15,8,9]. More-
over, it was found that observed yield increased with decreased SRT
and increased carbon redirection via biosorption [9]. Thus, yield, a
stoichiometric coefcient was considered as an indirect parame-
ter to predict biosorption in continuous systems [9]. In high-rate
continuous systems, the observed yield is dened as sludge pro-
duction which includes the adsorption of particulate and colloidal
organic matter, intracellular storage of soluble carbon, microbial
growth, and possibly decay. All of these mechanisms contribute
to the sludge production for removal of COD from wastewater
and thus inuence the yield coefcient. Therefore, we propose an
approach to quantify biosorption on HRAS systems by determining
the ex-situ aerobic yield through the establishment of famine and
feast conditions (similarly to CS) in the batch reactor.
Since the conventional biosorption capacity approach possibly
determines the maximum or saturation carbon capture capacity
of activated sludge, knowing the in-situ biosorption contributions
in HRAS systems is necessary to understand the carbon redirec-
tion. Rahman et al. [9] rst proposed a simple approach to quantify
biosorption in a batch reactor using the oxygen uptake rate (OUR) of
the sludge which contained biodegradable organic matter as a form
of biosorption occurred in a continuous system, and the approach
was referred as biosorption yield. In this study, we evaluated this
approach to quantify biosorption for various HRAS systems at dif-
ferent SRT conditions to nd the relationship between biosorption
yield and inuent carbon balance and other operational parameters
(SRT).
Although, the conventional biosorption capacity and the
biosorption yield was focused on ex-situ measurement based
approach, the biosorption has rarely been investigated on in-situ
continuous reactor systems to consider the impact of operational
parameters (SRT, HRT), inuent, efuent, and waste activated
sludge (WAS) characteristics and most importantly the extracellu-
lar polymeric substances (EPS) in the reactor. In fact, EPS is the key
for the microbial community to grow at high-cell density by attach-
ing their surfaces [2325]. Therefore, microorganisms are required
to hold together via EPS production for better bioocculation which
caused biosorption of organic matter from wastewater [26]. A novel
approach was proposed in this study to quantify in-situ biosorption
using operational parameters, inuent carbon balance and EPS of
a pilot-scale reactor system treating chemically enhanced primary
treatment (CEPT) efuent.
The main purpose of this study was to evaluate four differ-
ent approaches of biosorption quantication on HRAS systems and
their potential applications, advantages, and limitations in HRAS
systems. The specic objectives of this study were (a) to quantify
biosorption capacity of HRAS and biological nutrient removal (BNR)
sludge in batch systems using the conventional method (sludge
mixed with wastewater) established in literature and its limitations
for applications, (b) to determine the biosorption using ex-situ aero-
bic yield of starved and non-starved biomass of HRAS systems, (c) to
quantify ex-situ biosorption yield using OUR on various SRT sludge,
(d) to estimate in-situ biosorption on pilot-scale HRAS systems
using operational parameters, inuent carbon balance and EPS con-
centration, and (e) to evaluate these four biosorption quantication
approaches, their potential relationship with SRT and carbon redi-
rection, benets and limitations for applications in wastewater
treatment processes
2. Materials and methods
2.1. Origin of sludge and wastewater location
The sludge used for this study was collected from the Blue Plains
Advanced Wastewater Treatment plant (AWWTP) (Washington,
DC) and the pilot-scale high-rate CS systems operated there by Rah-
man et al. [9] and Rahman et al. [27]. The Blue Plains AWWTP treats
raw wastewater through enhanced primary treatment (CEPT), bio-
logical step-feed HRAS reactors (23 d SRT) and is followed by
biological nutrient removal (BNR) (15 d SRT) systems based on
conventional nitrication-denitrication with methanol dosing to
meet low nitrogen levels. The total inuent ow rate for 20 CEPT
clarier (East side) is 1095607 m3 /d and each circular clarier has
diameter of 36.57 m, side water depth of 4.37 m and center water
depth of 5.89 m. Each clarier has HRT of 2 h. The range of coagu-
lant dosage in CEPT is between 6 and 9 mg Fe3+ /L. The primary and
high-rate WAS are hydrolyzed through a pretreatment step called
thermal hydrolysis (CAMBI) and then digested in conventional
mesophilic anaerobic digestion. The plant inuent (raw wastew-
 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344
Table 1
Characteristics of full-scale plant inuent raw wastewater and chemically enhanced primary treatment (CEPT) efuent measured based on 24-h composite sample analysis
(n: 6580).
Parameter Unit
Total Suspended Solids (TSS) mg TSS/L
Volatile Suspended Solids (VSS) mg VSS/L
Total COD (tCOD) mg COD/L
Particulate COD (pCOD) mg COD/L
Colloidal COD (cCOD) mg COD/L
Flocculated ltered COD (ffCOD) mg COD/L
Ammonia mg N/L
Total Phosphorous mg P/L
ater) and CEPT efuent characteristics are described in Table 1. The
sludge was collected from the HRAS (end of reactor) and BNR (end
of reactor) processes. In bench-scale experiments, the raw wastew-
ater and CEPT efuent were used as a carbon source. Sludge samples
were also collected from different SRT conditions of pilot-scale
high-rate CS reactors and are described in the following section.
2.2. Pilot-scale reactor operation
To evaluate the different approaches of biosorption quanti-
cation on HRAS systems and their potential relationship with
continuous reactor systems, a pilot-scale CS systems treating CEPT
efuent were performed in this study. The description of the pilot
scale reactor plant are described in detail by Rahman et al. [9]
and Rahman et al. [27]. In this study, ve different high-rate CS
congurations were conducted and are abbreviated based on the
average total SRT in steady-state conditions: CS 1.7 d, CS 1.5 d,
CS 1 d, CS 0.8 d and CS 0.7 d. The average full-scale plant inu-
ent (raw wastewater) and pilot-scale reactor inuent wastewater
(CEPT efuent) composition are described in Table 1. The oper-
ational parameters (volume of reactor, SRT, HRT, and volumetric
loading rate), measured parameters of dissolved oxygen (DO) and
mixed liquor suspended solids concentration, COD fractions [total
COD (tCOD), particulate COD (pCOD), colloidal COD (cCOD) and
occulated ltered COD (ffCOD)] removal efciency and efuent
solids concentration of different CS congurations are illustrated
in Table 2. A COD mass balance was performed on each scenario
dividing the inuent COD into four different fractions as waste
activated sludge (WAS), efuent pCOD, efuent cCOD plus efuent
ffCOD and oxidation which was calculated by closing the mass-
balance of 100% [9,27]. The carbon harvesting efciency as% was
calculated by dividing the% WAS by the summation of% efuent
pCOD and % WAS from the carbon mass-balance. Since the main
focus of this study was to quantify biosorption, the detailed reactor
performance was not discussed. The CS 1.7 d, CS 1 d, CS 0.8 d and
CS 0.7 congurations detailed performance are described by Rah-
man et al. [9] and Rahman et al. [27]. Additional data of these four
congurations are reported in this study to evaluate the biosorp-
tion quantication. The pilot-scale high-rate continuously stirred
tank reactor (CSTR) and plug-ow (PF) congurations studied by
Rahman et al. [9] are also reported in this study for evaluation of
biosorption quantication.
2.3. Conventional approach to quantify biosorption capacity
The biosorption capacity on HRAS in a batch reactor was
quantied using the most acceptable methods established in the
literature [14,18,19]. Biosorption capacity was dened by the dif-
ference between the initial wastewater COD addition to the mixture
(sludge and wastewater) and the supernatant COD after settling
and it was determined using the equation adopted from Tan and
Chua [18].
35
Raw wastewater Chemically enhanced primary treatment efuent
243 39 30 20
209 33 26 15
419 23 151 36
317 17 67 36
19 6 22 10
84 12 63 12
26 2.5 23 3.6
4.2 0.7 1.9 0.3
Biosorption capacity (mgCOD/gVSS)
CODinitial (mgCOD/L) CODfinal (mgCOD/L)
= (1)
Initial sludge VSS concentration (g/L)
where, CODinitial denotes initial total COD concentration of
wastewater added to the mixture (wastewater and sludge) plus
supernatant COD concentration of a sludge control (food to
microorganism ratio, F/M = 0), CODfinal denotes supernatant total
COD concentration after settling in the mixture (sludge and
wastewater). It is to be noted here that the biosorption capacity
is expressed as mgCOD/gVSS (volatile suspended solids) instead
of mgCOD/gTSS (total suspended solids). This is because the
biosorption capacity should be calculated based on the biomass
concentration. The use of TSS concentration includes inorganic car-
bon and inert carbon which will impact the biosorption capacity
measurement.
The biosorption tests were performed on HRAS (2 d SRT) col-
lected from the full-scale secondary reactor and biological nutrient
removal (BNR) sludge (SRT >10 d) collected from full-scale nitri-
cation/denitrication reactor of Blue Plains AWWTP, Washington,
DC. A 1 L sample of sludge was mixed with 1 L of raw wastew-
ater or CEPT efuent in a batch reactor and mixed for 10 min
which was considered as contact time and then settled for 60,
90 and 120 min. The mixing was done using a magnetic stir bar
and a magnetic stir plate and the mixing speed was 230 rpm
(approximate G value of 50 s1 ). The experiments were conducted
under both active and inactive biomass conditions at variable
food to microorganism (F/M) ratio. The biomass was inactivated
using sodium azide (0.2 gNaN3 /gTSS) addition to avoid any bio-
logical activity happened in the reactor [11,17]. The sludge was
exposed to sodium azide for at least 3 h and intermittent sludge
samples were collected to measure OUR. After 3 h of sodium
azide exposure, the constant OUR (below 0.5 mgO2 /L/h) in the
batch experiment was considered as the start of the inactivation
time.
The biosorption capacity experiments were also conducted on
HRAS (2 d SRT) for various contact time (140 min) and a xed
settling time (30 min) to understand the impact of contact time.
Table S1 to S5 describes the detailed experimental conditions of
biosorption capacity experimental approaches.
It is to be noted here that this study did not distinguish between
adsorption and intracellular storage mechanisms and thus, these
two mechanisms are expressed together as a biosorption mecha-
nism for all methods.
2.4. Ex-situ aerobic yield coefcient approach to quantify
biosorption
The well-known theory of feast-famine condition for enhancing
biosorption in CS technology was applied to quantify biosorption
36 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344

Note: HRT-Hydraulic retention time, SRT-Solids retention time, DO-Dissolved oxygen, tCOD-Total COD, pCOD-Particulate COD, cCOD-Colloidal COD, ffCOD-Flocculated ltered COD, WAS-Waste activated sludge, CS-Contact-
in batch experiments. In famine conditions, biomass is gener-

5.43 1.03
508 194
ally aerated for a longer period (> 2 h) to oxidize biosorbed COD
(extracellular adsorbed and intracellular stored COD) to create a

227
86
starvation phase. This creates more extra and intra cellular space

S
for adsorption and storage of organic carbon from wastewater in
feast condition. For this approach, it was assumed that the starved

2.18 0.23
0.69 0.17
0.51 0.46
CS 0.69 d

253 86
biomass expected to have higher aerobic yield coefcient under

76 36
31 15
52 8
56 9

21 4
58 5
pulse feed condition (feast regime) in the batch reactor. In conven-
227
30
C

tional high-rate systems (full-scale step-feed or A-stage), biomass is

not exposed to famine conditions like CS systems, thus, freshly col-
5.71 1.36

lected HRAS is expected to exhibit a lower aerobic yield under pulse

571 81

feed conditions in the batch reactor. This is because the sludge with-
227
86

out experiencing the starvation condition, are already saturated

S2

(or partially saturated) with biosorbed COD from high-rate full-

scale systems. Hence, the addition of pulse feed in the batch reactor
1.44 1.22
564 193

will not create feast response to biomass due to their pre-occupied

biosorption (adsorption and storage) space. The difference in aer-
18
50
S1

obic yield coefcients between starved biomass and non-starved

biomass under feast conditions provides an indication of biosorp-
2.25 0.24
0.75 0.11
0.46 0.26

tion capacity of the sludge.

CS 0.75 d

239 39

41 13
38 28
51 32
35 12

49 14
24 9

The ex-situ aerobic yield coefcient was estimated based on the

227

most acceptable formula [28,29] using oxygen consumption rate

30
C

(rO2 ) and soluble COD removal rate (rCOD) in a batch experiment

at high F/M ratio.
1129 188
3.57 0.61
Operational parameters, COD fractions removal efciency and efuent solids concentration in high-rate contact stabilization (CS) congurations.

rO2
227

Y =1 (2)
86
S

rCOD
1.09 0.11
0.66 0.33

2.30 0.26

Where, Y denotes the aerobic yield coefcient (gCOD/gCOD), rO2

CS 1.09 d

479 39

75 16
56 34

73 12
35 10

denotes the oxygen consumption rate (mgO2 /L/h) due to COD oxi-
59 8

25 4

dation and rCOD denotes the soluble COD (ltered through 1.5 m
227
30
C

glass-ber lter) removal rate in mgCOD/L/h.

In this study, the aerobic yield for starved biomass and non-
1033 322
3.61 1.62

starved biomass exposed to feast condition in the batch test are

expressed as Yield with Pretreatment (YP ) and Yield with No-
227
86

Pretreatment (YNP ), respectively. The assumption for biosorption

S

calculation was that removal of soluble COD in no-pretreatment (no

1.52 0.47

2.05 0.53
0.28 0.20

starvation) sludge was equal to pretreatment (starvation) sludge

449 142
CS 1.52 d

47 14
46 32
51 45

26 13
46 24
40 17

test. In this case, the adsorption of particulate and colloidal COD

and storage of soluble COD from wastewater in no-pretreatment
227
30
C

sludge is assumed negligible, and oxidation is the only process that

can contribute to COD removal. In pretreatment sludge, biosorp-
1279 293
3.63 1.03

tion (adsorption, and storage), and oxidation are two mechanisms

which contributed to the removal of COD from wastewater. The
227
86

difference between these two yields provides an indication of

S

biosorption of the sludge. A conceptual diagram of the biosorption

quantication using pretreatment and no-pretreatment approach
1.73 0.54

2.18 0.26
0.36 0.25
618 139
CS 1.73 d

is illustrated in Fig. 1.
61 11
62 25
77 29

67 14
48 8
20 7

To calculate the biosorption rate, the aerobic yield coefcient

227
30
C

for both sludge can be computed using Eq. (2), producing:

kgCOD/m3 /d

For no pretreated sludge :

mgVSS/L

mgVSS/L
mgO2 /L

(3)
Unit

rO2,NP
min

1 YNP =
%
%
%
%

%
d
L

rCODNP = rsCODNP = rCODP

Stabilization, C-Contactor, S-Stabilizer.
Biomass concentration in reactor

rO2,P
For pretreated sludge : 1 YP =
Efuent solids concentration
Carbon harvesting efciency

rCODP
ffCOD removal efciency
pCOD removal efciency
cCOD removal efciency
tCOD removal efciency
Volumetric loading rate

rO2,P rO2,P (4)

= =
rsCODP + Biosorption rO2,NP
Volume of reactor

+ Biosorption
1 YNP
Parameter

rO2, P rO2, NP
Biosorption = (5)
Table 2

1 YP 1 YNP
HRT
SRT
DO
 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344
Fig. 1. Conceptual diagram of biosorption yield determination, ex-situ aerobic yield coefcient determination using aerobic pretreatment and no-pretreatment approach in
a batch experiment.
where, biosorption computed in mgCOD/L/h unit, YNP , YP = Yield
coefcient (gCOD/gCOD) in no-pretreatment (NP) and pretreat-
ment (P) batch test, respectively; rO2,NP and rO2,P = oxygen
consumption rate (mgO2 /L/h) in NP and P batch test, respectively;
rCODNP and rCODP = removal rate of COD (mgCOD/L/h) ltered
through 1.5 m glass ber lter in NP and P batch test, respectively,
rsCODNP and rsCODP = removal rate of soluble COD ltered through
1.5 m glass ber lter in NP and P batch test, respectively.
In pretreatment sludge experiment, a 2 L of freshly collected
HRAS from the full-scale step-feed reactor (SRT of 2 d) was col-
lected and aerated (DO > 4 mgO2 /L) for 24 h to create starvation
phase. After that, a 30 mL of aerated sludge were mixed with 1.9 L
of CEPT efuent as a carbon source and continuously aerated (DO
of 45 mgO2 /L) for 2 h to measure oxygen consumption rate (rO2 )
using a respirometry setup of DO controller. The respirometry
setup is described in Section 2.5. The samples were taken for
every 1030 min to measure COD ltered through 1.5 m. In no-
pretreatment sludge experiment, above procedures were followed
except pre-aerating the sludge. The aerobic yield coefcient for
both types of sludges was measured using Eq. (3) and (4). The both
pretreatment and no-pretreatment sludge experiments were also
conducted to determine yield coefcient for ltered (1.5 m) CEPT
efuent as a carbon source to avoid the impact of active biomass
from CEPT efuent. Then, the biosorption was calculated using Eq.
(5).
2.5. Quantication of biosorption yield
An oxygen uptake rate (OUR) technique was employed to quan-
tify biosorption yield on HRAS in a batch reactor. The biosorption
37
yield is dened here as the amount of oxygen consumed from
biodegradable organic matter contained in the sludge as a form
of adsorbed COD and stored COD for any SRT condition systems.
This denition was based on the ASM3 model which assumed
that the readily biodegradable COD from wastewater can be stored
as a form of intracellular products under aerobic conditions [30].
In biosorption yield experiments, the biodegradation happens
through biological oxidation and hydrolysis processes in the batch
reactor. The oxidation of biodegradable COD from the sludge matrix
for certain aeration period provides the measurement of total oxy-
gen consumption after subtraction of oxygen consumption due to
endogenous respiration.
A volume of 2 L of HRAS (mixed liquor) was collected from high-
rate full-scale (2.5 d and 3.5 d) and pilot scale systems (CS 1.7 d, CS
1.5 d, CS 1 d, CS 0.8 d, CS 0.7 d) at different SRT conditions and then
settled for approximately 30 min to remove supernatant. The set-
tled sludge was then diluted with nal efuent (inert soluble COD
of 1220 mgCOD/L) of the full-scale plant to make the total volume
of 2 L. The sludge was aerated without adding any external sub-
strate for certain time in which the decreased OUR response with
the time indicated the oxidation of biodegradable COD. After com-
plete degradation of biodegradable COD, OUR followed a constant
response showing the endogenous respiration phase. The whole
area under the OUR vs time curve (excluding endogenous respira-
tion) was considered as total oxygen consumed by biodegradation
of biosorbed COD. A conceptual diagram of biosorption capacity
determination is illustrated in Fig. 1. The biosorption yield was
calculated by the following formula:
38 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344

Biosorption Yield (mgO2 /gVSS)

Total O2 consumption due to biosorbed COD oxidation (mgO2 /L)
= (6)
Initial sludge VSS concentration (gVSS/L)
Cumulative O2 consumption (mgO2 /L) O2 consumption from endogenous respiration (mgO2 /L)
=
Initial sludge VSS concetration (gVSS/L)
It is to be noted here that, the pCOD was not measured in this
test, thus the unit of biosorption yield as mgO2 /gVSS cannot be The oxidized COD redirected for new biomass growth, and EPS
corrected to gCOD/gCOD. The OUR was measured using a respirom- production can be calculated as below.
etry setup consisting of a 2 L batch reactor and a dissolved oxygen
(DO) membrane probe attached to a DO controller (6309PDTF, Jenco CODcell growth + EPS (gCOD/d) = Qin tCODin CODoxidation
Instruments, Inc. San Diego, CA). The reactor was covered by a plas-
Y
tic lid to prevent any oxygen intrusions from the atmosphere. In (8)
1Y
addition the head space (approximately 2.5 cm) between top of
liquid surface and plastic lid was lled with styrofoam to decrease Where CODcell growth + EPS denotes inuent oxidized COD redirected
chance of oxygen intrusion. A magnetic stir plate and a stir bar were to biomass growth and EPS productions in gCOD/d, Y denotes ordi-
used to mix the mixed liquor suspended solids in the reactor. The nary heterotrophic organism yield of 0.67 gCOD/gCOD.
DO controller was used to record and maintain the DO between The percentage of inuent oxidized COD shunted for EPS pro-
45 mgO2 /L. A sh air pump supplied air to the reactor until the duction (CODEPS ) with respect to particulate COD of the reactor
DO reached to 5 mgO2 /L and was turned off by the DO con- sludge per gram VSS concentration was determined as below.
troller. A linear reduction of DO from 5 to 4 mgO2 /L was observed
CODEPS or EPS production (%)
because of biological activity. As the DO reached to 4 mgO2 /L, the
air was turned on to raise the DO to 5 mgO2 /L. This cycle was Total EPS concentration
=   100 (9)
repeated until a constant OUR was achieved which considered as pCOD/VSS
reactor
an endogenous respiration state [31]. The recording of DO between
4 and 5 mgO2 /L conrmed that there was no oxygen intrusion from Where total EPS measured in contactor reactor as gCOD/gVSS and
the atmosphere into the reactor. A data recorder (DataSuite data pCOD to VSS ratio of the contactor reactor measured as gCOD/gVSS.
recorder) linked with the DO controller recorded the DO data and The percentage of inuent oxidized COD redirected to new
transferred to a computer using the DataSuite software. The slope biomass growth only (excluding EPS production) was computed
was calculated between DO of 5 and 4 mgO2 /L to compute OUR. as below.
 
CODcell growth + EPS
2.6. Biosorption quantication using inuent COD mass-balance, Cell growth (%) = 100 CODEPS (%)
Qin tCODin
EPS and operational parameters of a pilot-scale system (10)

A novel approach was proposed in this study to quantify the

percentage of inuent COD redirected through biosorption mecha- The percentage of inuent total COD redirected to biosorption
nism. This approach hypothesized that the inuent redirected COD pathways was calculated by closing the mass-balance from cell
through efuent pCOD (assumed as biomass) and WAS can only be growth (%) and EPS production (%).
transformed into three different pathways: cell growth, EPS pro-
Biosorption (%) = 100 Eq.9 Eq.10 (11)
ductions and biosorption (adsorption and storage). The assumption
was that the inuent COD is oxidized and a fraction of that oxidized The actual percentage of biomass growth, EPS productions and
COD directs for biomass growth and EPS productions and remaining biosorption were then computed by closing the inuent COD mass-
used for energy productions. The percentage of inuent COD redi- balance for a treatment process congurations.
rected to biosorption pathway was calculated to close the mass Inuent Carbon redirection (%) = % efuent pCOD + % WAS
balance from biomass growth and EPS productions. The method
(Qin QWAS ) pCODeff QWAS tCODWAS
quanties the biosorption based on reactor operational parameters = 100 + 100 (12)
(SRT, inuent ow rate, and wasting rate), the measured parame- Qin tCODin Qin tCODin
ter (inuent, efuent, reactor sludge and WAS COD fractions) and Eq.10
reactor sludge EPS concentration in the system. The detailed calcu- Actual biomass growth (%) = Eq.12 (13)
100
lation procedures are described below. A schematic of wastewater
Eq.9
ow diagram of a pilot-scale CS conguration is shown in Fig. S1. Actual EPS productiom (%) = Eq.12 (14)
100
The inuent COD oxidation was calculated based on inuent,
efuent and WAS ow rate and COD fractions. Eq.11
Acutal biosorption (%) = Eq.12 (15)
100
CODoxidation (%) =

Qin tCODin (Qin  QWAS ) 2.7. Analytical measurements

 (7)
pCODeff + cCODeff + ffCODeff QWAS tCODWAS
100
Qin tCODin
where, CODoxidation denotes% of inuent COD oxidation, Qin denotes
inuent ow rate (L/d), tCODin denotes inuent total COD concen-
tration (gCOD/L), QWAS denotes wasting rate (L/d), pCODeff denotes
efuent pCOD concentration (gCOD/L), cCODeff denotes efuent
colloidal COD concentration (gCOD/L), ffCODeff denotes efuent
occulated ltered COD concentration (gCOD/L), tCODWAS denotes
WAS total COD concentration (gCOD/L).
The COD concentrations were divided into total (tCOD), par-
ticulate (pCOD), colloidal (cCOD) and occulated-ltered (ffCOD)
COD fractions. The total COD (tCOD) was measured on raw sample
(unltered). The occulated-ltered COD (ffCOD) was measured by
following the methods of Mamais et al. [32] and the ffCOD is consid-
ered as true soluble COD. The colloidal COD (cCOD) was measured
by subtracting the ffCOD from COD ltered through 1.5 m glass
ber lter. The particulate COD was determined by subtracting
COD ltered through 1.5 m from tCOD. The COD, ammonia and
 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344
Fig. 2. Inuence of COD loading (mass of substrate added per mass of biomass)
on biosorption capacity (and corrected biosorption capacity) using conventional
approach for (a) biological nutrient removal sludge (full-scale nitrication stage of
SRT >10 days) and (b) high-rate activated sludge (full-scale system of SRT 2 days)
under active and inactive conditions.
total phosphorous were measured and analyzed using HACH test
kits and HACH spectrophotometer (HACH, Loveland, CO, USA). The
total suspended solids (TSS) and volatile suspended solids (VSS)
were measured by following the Standard methods [33]. The EPS
on pilot-reactor sludge sample (contactor) was measured using the
modied protocol of base extraction method adopted from Rahman
et al. [9].
3. Results and discussion
3.1. Conventional biosorption capacity approach
HRAS sludge (2 day SRT) was used to evaluate the biosorp-
tion method as described by Riffat and Dague [14], Tan and
Chua [18] and Zhang et al. [19]. However, no COD removal was
observed under different contact times (140 min), various F/M
ratios (0.031.27 gCODwastewateradded /gCODbiomass ), and 30 min of
settling time (Table S1) thus resulted in negative biosorption
capacity for all conditions (n = 12). The main reason for the neg-
ative biosorption capacity was due to the poor settleablity of the
sludge which did not allow enough separation between activated
sludge and the wastewater matrix. Therefore, settling times were
increased to 60 min in subsequent tests. In addition, to avoid the
interference of heterotrophic growth resulting in the increase of
particulate COD and decrease of soluble COD, biomass was inacti-
vated as suggested by Modiun et al. [17] and Lim et al. [11].
Fig. 2 illustrates the biosorption capacity measured on both
active and inactive HRAS (SRT 2 d) and BNR sludge (SRT > 10 d)
at xed contact time of 10 min, xed settling time of 60 min and at
different F/M ratios. It was observed that as F/M ratio increased, the
biosorption capacity increased for both types of active and inactive
sludges. At similar range of F/M ratio, both type of sludge sam-
ples had similar range of biosorption capacity under active (HRAS:
30 8 to 101 5 mgCOD/gVSS, BNR: 18 1 to 93 2 mgCOD/gVSS)
and inactive (HRAS: 14 2 to 105 4 mgCOD/gVSS, BNR: 19 2 to
39
103 2 mgCOD/gVSS) conditions (Fig. 2). This suggested that the
approach was independent of SRT and origin of sludge character-
istics. The BNR sludge was able to adsorb (extracellular) carbon
from wastewater even though the sludge was not acclimated to
raw wastewater and the capacity was slightly lower than HRAS.
This might have been because the BNR sludge had comparatively
higher occulation ability than HRAS at Blue Plains AWWTP [20].
The predominance of heterotrophic population of methylotrophs in
BNR sludge might have not been able to store carbon which might
explain the lower biosorption capacity of BNR sludge in comparison
with HRAS.
In addition, the biosorption capacity under active and inactive
conditions was similar which indicated that carbon oxidation must
not have played a role. However, in active conditions, intracellu-
lar storage may also play a role for COD removal from wastewater
and investigation of storage determination can be a future research
aspects. Results also showed that the biosorption capacity under
active and inactive conditions had a positive relationship with the
amount of wastewater added (F/M) (Fig. 2). Thus, it was hypothe-
sized that the impact of wastewater matrix had a signicant impact
on the results.
The biosorption capacity was corrected for settleable organic
matter present in the wastewater matrix to separate settling from
adsorption. The biosorption capacity corrected for settleable solids
in the wastewater matrix is referred in this study as corrected
biosorption capacity. It was observed that about 55% of the total
COD in the raw wastewater settled out within the 60 min time
frame in the control experiment. Fig. 2 describes the corrected
biosorption capacity for both HRAS and BNR sludge under active
and inactive conditions. Results showed that corrected biosorption
capacity of HRAS and BNR sludge were in the range of 26 8 to
46 6 mgCOD/gVSS and 15 1 to 34 2 mgCOD/gVSS under active
conditions, respectively. Thus, the corrected biosorption capac-
ity were signicantly lower than the initial observed biosorption
capacity. In addition, the corrected biosorption capacity did not
change with the increase of F/M ratio for both sludges. Table S2
to S5 describes the biosorption and corrected biosorption capacity
measurements on HRAS and BNR sludge under active and inac-
tive conditions at settling time of 60, 90 and 120 min. It was
observed that the biosorption capacity of inactive HRAS decreased
with settling time (Table S5). This might be due to deocculation
of the sludge at longer settling time (90120 min) which caused
non-settleable fractions of biomass in the supernatant. However,
under deocculation condition, the sludge can be resuspended and
biosorption capacity can be measured on that conditions. For future
research aspects, the biosorption capacity needs to be evaluated for
mixing, settling and deocculated sludge conditions.
The results obtained in this study when corrected for settleable
organics in the wastewater matrix are within the range of previ-
ous literature studies. Tan and Chua [18] and Guellil et al. [10]
reported the biosorption capacity for activated sludge (SRT 5 d)
was between 2780 mgCOD/gVSS (assuming that the VSS to TSS
ratio was 0.80) (34100 mgCOD/gTSS) for domestic wastewater.
Zhang et al. [19] found that the biosorption capacity increased
from 88 mgCOD/gVSS to 136 mg COD/gVSS (assuming that the
VSS to TSS ratio was 0.80) (110170 mgCOD/gTSS) when substrate
concentration increased from 400 to 700 mgCOD/L and then the
biosorption capacity did not increase any more with the increase
of substrate concentration (> 700 mgCOD/L), suggesting the sludge
was saturated. However, they also observed that the biosorption
capacity was maximized at 2500 mgTSS/L concentration and then
started to decrease with an increase of biomass concentration sug-
gesting that the substrate was limited and thus inuenced the
biosorption. Lim et al. [12] examined the biosorption capacity in
a sequencing batch reactor (SBR) as a feast-famine regime to treat
synthetic wastewater (glucose, acetate, soluble starch, and urea).
40 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344

Fig. 3. A conventional approach of ex-situ aerobic yield determination of high-rate activated sludge (full-scale step-feed system of SRT 2 days) in a (a) high F/M ratio batch
test in which R2 0.89 and (b) low F/M ratio batch test in which R2 0.07 for COD fractions (tCOD, pCOD, sCOD) response and cumulative oxygen consumption during the
test where chemically enhanced primary treatment efuent added as a carbon source.

They investigated different sludge characteristics at various F/M

ratio (0.15-0.45 g COD/g TSS), SRT (330 d) and DO (0.5-6 mgO2 /L)
conditions and found the overall biosorption capacity to be about
2357 mgCOD/gTSS. They reported that SRT positively correlated
with biosorption capacity due to high EPS concentration at higher
SRT and it was negatively correlated with the oc size. The high
SRT system had higher biosorption capacity but a high fraction of
oxidized COD played role to remove COD whereas low SRT had low
biosorption capacity because the sludge was partially saturated.
The difference is that low SRT systems capture more carbon due
to lower carbon oxidation. However, too low SRT system becomes
EPS limited and carbon capture efciency decreases with high COD
leaving through the efuent. However, in that study, no clear cor-
relation between biosorption and SRT was found. Therefore, the
conventional approach does not seem useful to understand the
biosorption contribution in HRAS systems.
3.2. Ex-situ aerobic yield approach
The ex-situ aerobic yield coefcient of HRAS (SRT 2 days) was
determined based on Eq. (2) and the change of COD (tCOD, pCOD,
and sCOD) fractions and cumulative oxygen against the experimen-
tal time are illustrated in Fig. 3. Fig. 3a describes the prole of tCOD,
pCOD, sCOD concentration and cumulative oxygen concentration
with corresponding time for determining the ex-situ aerobic yield.
The increase of pCOD explained the biomass growth due to COD
oxidation as cumulative O2 also increased linearly. The regression
coefcient (R2 ) for the change of each parameter against the time
was higher than 0.89. Fig. 3b represents another experimental data
where the change of tCOD and pCOD with time had the low regres-
sion coefcient of 0.61 and 0.07, respectively indicating that pCOD
did not change during the test. This might have been caused by the
higher initial biomass concentration (initial pCOD 200 mgCOD/L)
in Fig. 3b experiment indicating lower F/M ratio compared to the
experiment shown in Fig. 3a which had higher F/M ratio (initial
pCOD 20 mgCOD/L). This suggested that correct F/M ratio is crit-
ical to observe biomass growth in batch experiments which can be
detected by increasing pCOD concentration (shown in Fig. 3a). Since
the change of sCOD and cumulative oxygen consumption against
experimental time had better regression coefcient (R2 0.80) in
both high and low F/M ratio experiments, Eq. (2) was used to deter-
mine ex-situ aerobic yield to quantify biosorption.
The biosorption capacity of the sludge was then calculated
(Eq. (5)) by differentiating the two aerobic yield between starved
biomass and non-starved biomass. The three different types of sub-
Fig. 4. Quantication of ex-situ aerobic yield coefcient with and without aerobic
pretreatment approach for non-ltered, ltered (1.5 m) wastewater (chemically
enhanced primary treatment efuent) and acetate (soluble compound) as a carbon
source in batch experiments.
strate (unltered CEPT efuent, ltered CEPT efuent, and acetate)
were used as external carbon source in the batch reactor. The main
purpose of using various types of organic carbon fractions was to
observe the impact of the substrate on yield determination and thus
biosorption (adsorption and storage) qualication.
Fig. 4 illustrates the aerobic yield determined for pretreatment
and no-pretreatment sludge feeding with non-ltered wastewa-
ter, ltered wastewater, and acetate as a soluble carbon source.
The aerobic yield was slightly higher under no-pretreatment con-
dition than pretreatment condition (Fig. 4). However, overall no
signicant difference (p-value is 0.085) between the aerobic yield
with pretreatment or no-pretreatment of the sludge was observed
(Fig. 4). Since the ex-situ yield was independent of aerobic pretreat-
ment and no-pretreatment conditions in batch experiments, the
ex-situ aerobic yield was further determined on sludge collected
from pilot-scale high-rate CSTR (2.3 d and 3.4 d SRT) and CS (0.3
d, 0.8 d, 0.9 d and 1 d SRT) continuous systems studied by Rahman
et al. [9]. The ex-situ aerobic yield determined in batch tests and
in-situ observed yield calculated from the mass balance of CSTR
and CS systems at different SRT conditions are illustrated in Fig. 5.
The in-situ observed yield of the CSTR and CS systems were lower
(0.15-0.72 gCOD/gCOD) and decreased with SRT. In contrast, the
ex-situ aerobic yield determined in the batch reactor had no corre-
lation with SRT and was always high (> 0.71 gCOD/gCOD). Overall,
 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344
Fig. 5. Ex-situ aerobic yield coefcient (in a batch test) and in-situ observed yield
(continuous system) determination on reactor sludge from high-rate pilot-scale sys-
tem of completely mixed reactor (CSTR) and high-rate contact-stabilization (CS)
systems under variable SRT conditions.
the method of ex-situ aerobic yield was independent of sludge
characteristics and operational conditions (SRT) of reactor process
congurations. This is because the yield was always higher than 0.7
gCOD/gCOD suggesting that other key factors might have played a
contribution for high yield coefcient.
Based on the batch experimental results of pretreatment and
no-pretreatment conditions, the high yield coefcient in both con-
ditions may indicated that adsorption mechanism is difcult to
distinguish from intracellular storage in batch systems. Shifting the
microbial community from a continuous reactor process conditions
to a batch reactor potentially induced feast condition for microor-
ganisms and exhibited high yield which can be explained by
possible soluble COD storage [34]. The same phenomena were also
conrmed by Chudoba et al. [35] in CSTR and PF systems feeding
glucose substrate and found the ex-situ yield of 0.71 gCOD/gCOD
and 0.88 gCOD/gCOD, respectively. High ex-situ yields have also
been reported before for acetate (0.7 gCOD/gCOD) and glucose (0.8
0.9 gCOD/gCOD) [29,36]. The high ex-situ yield was potentially
caused by changing the environmental condition from continu-
ous feeding style to a sudden rise of pulse feed. The substrate
uptake rate may increase signicantly with a higher rate under
pulse feed compared to growth limiting conditions (continuous
systems). During rapid uptake of the substrate, some of the carbon
can be converted to storage polymer as the microorganisms can-
not use excess substrate for their growth because of having a risk of
metabolism imbalance [37]. Therefore, storage might have played
a dominant role in ex-situ conditions which contributed to high
yield. However, further research is needed to evaluate this method
by measuring and analyzing the intracellular stored compounds
and sludge oc structure to conrm biosorption mechanism.
3.3. Ex-situ biosorption yield
A change of oxygen uptake rate (OUR) against time for a 2.5 d
SRT sludge (full-scale step-feed system) is shown in Fig. 6. The trend
of OUR showed that it decreased rapidly with time and reached a
steady OUR condition after 10 h. The sharp drop of the OUR was
due to oxidation of biosorbed biodegradable organic matter within
the sludge as no biodegradable soluble organics were present. The
total cumulative oxygen consumption due to organic matter oxi-
dation and endogenous respiration was 87 mgO2 /L of which 73 mg
O2 /L was due to endogenous respiration. The biosorption yield was
then calculated using Eq. (6) and found to be 56 mg O2 /g VSS. Fig.
S2 and Table S6 illustrate the typical OUR proles as a function
of time for various sludge collected from pilot-scale CSTR (3.5 d,
1.2 d, 0.3 d SRT), CS (1.7 d, 1 d, 0.8 d, 0.7 d, 0.5 d SRT) and PF
(0.45 d SRT) congurations operated under different SRT with their
respective biosorption yields. The range of biosorption yield for the
41
Fig. 6. The measurement of ex-situ oxygen uptake rate (OUR) of high-rate sludge
(full-scale step feed system of 2.5 d SRT) in a batch experiment where area under
the OUR curve was divided into two areas to quantify biosorption yield.
full-scale 2.5 d SRT sludge was found to be 1256 mgO2 /gVSS and
was most probably dependent on the variable operational condi-
tions. In CSTR systems, the biosorption yield was 85 mgO2 /gVSS at
3.5 d SRT while the range was 318373 mgO2 /gVSS when SRT 1.2
d (Table S6). In CS congurations, the biosorption yield increased
from 57 to 435 mgO2 /gVSS when SRT decreased from high SRT (1.7
d) to low SRT (0.5 d) conditions. The biosorption yield of stabi-
lizer sludge (28293 mgO2 /gVSS) was always lower than contactor
sludge (57435 mgO2 /gVSS) because biosorption happened in the
contact tank whereas starvation occurred in the stabilization tank.
In this study at SRT<1 d in HRAS systems (CSTR and CS), the range of
biosorption yield was 318435 mgO2 /gVSS which was higher than
SRT>1 d. Thus the biosorption yield approach certainly can predict
the biosorption of HRAS occurred in a continuous system.
A relationship between carbon redirection, carbon oxidation
and total SRT observed from pilot-scale reactor is shown in Fig. 7a.
This indicated that SRT had a positive correlation with carbon
oxidation and negative correlation with carbon redirection. Sim-
ilar to these observations, the relationship between biosorption
yield and SRT (Fig. 7b), in-situ observed yield (Fig. 7c) and carbon
redirection, carbon oxidation (Fig. 7d) of pilot-scale high-rate CS
congurations were observed. It was found that biosorption yield
of contactor sludge negatively correlated (R2 0.85) with total SRT
of the system (Fig. 7b). The biosorption yield was also linearly cor-
related with the observed yield of the CS system because observed
yield of a high-rate system involved sludge production through
growth, adsorption, and storage (and possibly decay) by remov-
ing COD from wastewater (Fig. 7c). In addition, biosorption yield
was positively correlated with carbon redirection indicating the
importance of biosorption to enhance carbon recovery (Fig. 7d).
Ge et al. [38], Jimenez et al. [15] and Rahman et al. [9] reported
that carbon redirection negatively correlated with SRT. In addi-
tion, the carbon oxidation was positively correlated (R2 0.79) with
SRT exhibited in this study (Fig. 7d). Thus, the biosorption yield
had a negative correlation (R2 0.75) with carbon oxidation. At low
SRT and HRT, carbon oxidation is comparatively lower than high
SRT as biosorption taken place as the dominant mechanism of
removing particulate and colloidal organic matter from wastew-
ater and suggested the higher biosorption yield. The biosorption
yield is so far the rst parameter that can be measured in ex-situ
conditions and shows a direct correlation with SRT, carbon mass
balance and potential contribution of biosorption for carbon recov-
ery. However, further research is needed to evaluate this method by
42 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344

Fig. 7. (a) Relationship between carbon redirection, carbon oxidation, and total SRT of pilot-scale CS congurations (steady-state conditions), (b) impact of total SRT on
biosorption yield determined from pilot-scale CS congurations (steady-state conditions), (c) relationship between observed yield and biosorption yield of contactor sludge
from pilot-scale CS systems (steady-state conditions), and (d) relationship between carbon redirection, oxidation and biosorption yield of contactor sludge from pilot-scale
CS systems (steady-state conditions).

measuring and analyzing the intracellular stored compounds and

sludge oc structure to conrm biosorption mechanism. In addi-
tion, the biosorption yield can be applied for modeling purposes to
determine the stoichiometric coefcients and kinetic parameters
to evaluate biosorption.

3.4. In-situ biosorption in a continuous system

The biosorption capacity was determined on pilot-scale con-

tinuous systems using inuent COD mass balance, operational
parameters, and EPS measurements (see section 2.5). The inuent
COD mass-balance for the pilot CS congurations are illustrated
in Fig. 8. As SRT decreased from 1.7 d to 0.8 d, the carbon oxi-
dation decreased from 34 to 21% and the biomass formation (or
growth) also reduced from 21 to 11%. The EPS production was in the
range of 711% and biosorption increased from 13 to 30% when SRT
decreased from 1.7 d to 0.8 d. However, when SRT decreased even
more to 0.7 d, the biosorption decreased to 24% indicating a poten- Fig. 8. Inuent COD redirection pathways of ve different high-rate pilot-scale
tial lack of biosorption efciency. This might have been caused by contact-stabilization (CS) congurations under variable SRT conditions and the COD
redirection pathway was divided into ve different categories as biosorption, EPS
EPS limitation at lower SRT (11% vs 7% EPS at 0.8 d and 0.7 d SRT, production, biomass formation, efuent colloidal and occulated ltered COD and
respectively). EPS limitation has been shown before at lower SRT COD oxidation which was calculated to close the mass-balance (% biosorption, % EPS
by Jimenez et al. [15]. production and% biomass formation equals to% efuent pCOD (as biomass) plus%
A relationship between biosorption and carbon redirection, car- WAS).
bon oxidation, observed yield and Total SRT in the CS systems
were observed and illustrated in Fig. 9. The in-situ biosorption of biosorption while the biosorption yield, performed in an ex-situ
CS systems decreased with increasing SRT as expected (Fig. 9a). test, is still less direct measure.
In addition, a positive correlation was found between in-situ
biosorption and observed yield and carbon redirection (Figs. 9b, 3.5. Evaluation of ex-situ and in-situ biosorption capacity
9c), whereas in-situ biosorption increased with decreased carbon approaches
oxidation (Fig. 9c). These observed relationships were similar to
biosorption yield approach discussed in section 3.3. The benet The four biosorption quantication approaches for HRAS sys-
of this approach was that it determined the inuent COD cap- tems were evaluated in this study. The biosorption was quantied
tured on the sludge matrix through biosorption mechanisms which in ex-situ conditions by three approaches: conventional biosorp-
left through the system via WAS and efuent streams. Thus, this tion capacity, ex-situ aerobic yield, and biosorption yield. The
approach provides a direct fraction of carbon captured through biosorption capacity approach can only quantify the maximum (or
 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344 43

The ex-situ aerobic yield approach quantied maximum yield

Fig. 9. (a) Impact of total SRT on in-situ biosorption determined from pilot-scale CS
congurations (steady-state conditions), (b) relationship between observed yield
and in-situ biosorption of pilot-scale CS systems (steady-state condition), (c) rela-
tionship between carbon redirection, carbon oxidation and in-situ biosorption of
pilot-scale CS systems (steady-state conditions).
saturation) capture capacity of organic matter from wastewater to
sludge matrix. The biosorption capacity did not show to be sensitive
enough to correlate with system SRT, a known factor determin-
ing biosorption contribution in high rate systems. In addition, this
study showed that the settleable organic fraction in wastewater
had a signicant effect (increased biosorption capacity by factor of
2 due to 55% settelable organic matter) on conventional biosorption
capacity measurement studied by Riffat and Dague [14], Tan and
Chua [18], Guellil et al. [10], Zhang et al. [19], Lim et al. [12], Modin
et al. [17] and Lim et al. [11]. Therefore, conventional biosorption
capacity approach does not seem to be the best method to study
biosorption contribution for HRAS systems.
(due to storage) for any type of activated sludge because of high
F/M ratio and transfer of sludge from growth limited conditions
(continuous system) to batch systems (feast). Also, in this case, the
ex-situ aerobic yield was not able to differentiate between sludge
samples with different biosorption capabilities, even when includ-
ing pretreatment of sludge. The ex-situ yield test, therefore, can
only describe the maximum yield of the heterotrophic biomass
under increased F/M ratio and not the biosorption capacity. There-
fore, the high yield coefcient in ex-situ conditions may suggested
that extracellular adsorption is difcult to differentiate from intra-
cellular storage and thus further research on evaluating sludge
composition, oc properties and measuring storage compounds are
necessary.
Unlike, biosorption capacity and ex-situ aerobic yield approach,
the biosorption yield was the only ex-situ approach that showed
a direct correlation with SRT and the carbon mass balance of the
continuous systems. The approach quantied the biodegradable
fraction present in the ocs through a measurement of the OUR,
specied for the starting solids concentration. One of the limita-
tions of this approach was that it might have interference of EPS
degradation due to the longer time stabilization of sludge and
could therefore not differentiate between sorbed solids and the EPS
matrix. The benet of this approach was that it was positively corre-
lated with carbon redirection and observed yield of the system and
had opposite correlation with carbon oxidation and SRT of the sys-
tem. Therefore, the biosorption yield seems a promising approach
that can be applied ex-situ to evaluate biosorption contribution in
HRAS systems. In addition, it could be a helpful tool to calibrate
HRAS models and kinetic parameters in addition to mass balance
data.
The biosorption quantication using COD mass-balance, EPS,
and operational parameters was the only approach among the
tested approaches, which determined in-situ biosorption of a con-
tinuous reactor system. The approach was able to quantify the COD
fraction captured through biosorption. It correlated well with SRT
and carbon redirection. The advantage of this approach was that it
directly provides the biosorption quantication that is occurring in
the reactor under the inuence of inuent, efuent and WAS char-
acteristics, operational parameters, EPS and heterotrophic yield
coefcient. The major assumption of the method is the use of the
heterotrophic yield factor, which was set as default (0.67 g COD/g
COD) to calculate the biomass growth contribution.
By considering advantages and limitations of each approach
investigated in this study (Table 3), the best selection of biosorp-
tion quantication approach for the potential application can be
ranked as 1) biosorption yield, 2) biosorption quantication using

Table 3
Comparison of different approaches evaluated in this study to quantify biosorption capacity/yield on high-rate activated sludge systems.

Approach Advantages Limitation

Biosorption capacity Direct measurement of maximum biosorption 1. Interference of biomass growth

(Conventional method) capacity 2. Interference of settling organic matter
3. Potential interference of deocculation
4. Difcult to translate the result to biosorption contribution in
continuous systems (Observed yield)
5. Non-sensitive to sludge characteristics and F/M in the test
Ex-situ aerobic yield Provides maximum yield of the activated 1. Labor intensive experiment
coefcient sludge of a treatment process 2. Non-sensitive to sludge characteristics
3. No relationship with SRT
Biosorption yield 1. Provides in-situ sorption measurement of a continuous system 1. No differentiation between storage and adsorption
2. Has relationship with observed yield of the continuous system 2. Measurement of mass of oxygen instead of mass of COD
3. Has relationship with SRT, carbon redirection and carbon oxidation 3. Interference of EPS degradation
Biosorption 1. Provides in-situ sorption measurement of a continuous system 1. Needs (more complex) EPS data
quantication using 2. Has relationship with observed yield of a continuous system 2. Uses assumptions for biomass growth yield
COD mass-balance, EPS 3. Has relationship with SRT, carbon redirection and carbon oxidation
and operational 4. No additional batch experiments needed
parameters
44 A. Rahman et al. / Biochemical Engineering Journal 128 (2017) 3344
COD mass-balance, EPS, and operational parameters, 3) conven-
tional biosorption capacity and 4) ex-situ aerobic yield.
4. Conclusion
Biosorption quantication in HRAS system is vital to evalu-
ate the efciency of organic carbon recovery from wastewater.
This study presented ex-situ and in-situ approaches of biosorp-
tion quantication and suggested that the ex-situ conventional
approach (biosorption capacity) was limited by the inuence of set-
tleable particulate matter from wastewater. The OUR based ex-situ
approach of biosorption yield can be a potential method to quantify
biosorption because the approach solely depends on sludge char-
acteristics (any SRT) of a continuous system. The in-situ biosorption
quantication approach can also be a viable method to predict
biosorption due to the inclusion of wastewater characteristics, EPS
and operational parameters (SRT) inuence in the method. The
approaches developed in this study will further advance the appli-
cation of high-rate processes for energy recovery from wastewater.
Acknowledgements
Funding for this research was provided by the DC Water, Wash-
ington, DC, USA, the Water Environment & Reuse Foundation
(WE&RF, contract number U3R14), the Water-Energy Nexus Center
at the University of California, Irvine and the Balsells Foundation.
The assistance of all the personnel at the Blue Plains AWWTP
research laboratory is gratefully acknowledged.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2017.09.006.
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