Accepted Manuscript

Respiration adaptation of activated sludge under dissolved oxygen and hypo-

chlorite stressed conditions

Zhi-Hua Li, Zhi-Bo Ma, Han-Qing Yu

PII: S0960-8524(17)31070-2
DOI: http://dx.doi.org/10.1016/j.biortech.2017.06.166
Reference: BITE 18403

To appear in: Bioresource Technology

Received Date: 6 May 2017

Revised Date: 28 June 2017
Accepted Date: 29 June 2017

Please cite this article as: Li, Z-H., Ma, Z-B., Yu, H-Q., Respiration adaptation of activated sludge under dissolved
oxygen and hypochlorite stressed conditions, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/
j.biortech.2017.06.166

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 Respiration adaptation of activated sludge under dissolved oxygen and

hypochlorite stressed conditions

Zhi-Hua Li1, Zhi-Bo Ma1, Han-Qing Yu2

1
Key Laboratory of Northwest Water Resource, Environment and Ecology, Ministry

of Education, School of Environmental and Municipal Engineering, Xi'an University

of Architecture and Technology, Xi'an 710055, China

2
CAS Key Laboratory of Urban Pollutant Conversion, Department of Chemistry,

University of Science & Technology of China, Hefei, 230026 China

* Corresponding author:

Prof. Han-Qing Yu, Fax: +86 551-3607592; E-mail: hqyu@ustc.edu.cn

1
Abstract:

In this work, the relationship between environmental disturbance and the

recovery of activated sludge at a low dissolved oxygen of 0.5 mg/l or in the presence

of sodium hypochlorite of 1~15 mg Cl2/g SS/d was examined. When microorganisms

entered their physiological adaptation period, a sharp increase in endogenous

respiration rate was observed. The activity recovery potential of sludge depended on

the ratio of the endogenous respiration rate to the maximum respiration rate. A

subsequent decrease in this ratio after a sharp increase indicates that the disturbance

was recoverable. An increase in this ratio to a certain value, e.g., 0.35, suggests that

the sludge system could not adapt to the new environments and thus was

unrecoverable. In addition, the recoveries of sludge respiration and effluent quality

were asynchronous, which was impacted by both sludge activity and operating

conditions. These results provide a useful approach for the operation of activated

sludge systems.

Keywords: Activated sludge; Dissolved oxygen; Endogenous respiration rate;

Recoverability; Respiration; Stressed conditions

2
1. Introduction

Activated sludge systems have been used for wastewater treatment for over a decade

(Jenkins and Wanner, 2014; Orhon, 2015). The main advantages of the activated

sludge system are ease of operation and management. In its long-term operation, a

series of problems including the change of influent water flow, seasonal climate

variation, fluctuation of dissolved oxygen (DO), and the change of sludge

characteristics is inevitably encountered. These turbulences will undoubtedly cause

the instability of activated sludge systems, as they affect the microbial metabolism of

activated sludge and result in the deterioration of treatment plant effluent, sludge loss,

sludge bulking and other problems. Many shocking failure cases have been reported

in the past (Dai et al., 2016; Liu et al., 2015; Wang et al., 2016a), and various

countermeasures have been undertaken to address such fluctuations (Wang et al.,

2016c; Wang et al. 2016d).

The concentration and composition of effluent are a major concern for the

operation of wastewater treatment plants (WWTPs) under stressed conditions. The

fluctuating characteristics of activated sludge, including respiratory characteristics,

microbial activity, cell integrity, intracellular reactive oxygen species, and microbial

community variations, have been investigated (Di Trapani et al., 2015; Foladori et al.,

2014; Gao et al., 2015; Ju and Zhang, 2015). DO concentration plays an important

role in the performance of activated sludge system (mand et al., 2013; Campos et

3
al., 2007; Mosquera-Corral et al., 2005). Limitation of DO frequently results in poor

sludge performance in the form of poor settleability (Martins et al., 2004) and

deteriorated nitrifications (Campos et al., 2007). On one hand, the limitation of DO

caused by shocking loads of influent or temporary failure of aeration equipment is

frequently encountered in the operation of WWTPs. On the other hand, sodium

hypochlorite (NaClO) is frequently applied in WWTPs for sludge bulking treatment

(Kocerba-Soroka et al., 2013), and membrane fouling recovery (Navarro et al., 2016;

Wang et al., 2014). The effect of NaClO on sludge characteristics has been widely

examined. It has been reported that microbial cells remained intact under a low

NaClO concentration, while a high concentration could adversely affect autotrophic

and heterotrophic bacterial metabolisms (Meng et al., 2016). With a contact time of 10

min and a dosage of NaClO at 7.9 mg/g MLSS (mixed liquor suspended solids),

almost all of the filamentous microorganisms became inactivated, and the respiration

rate of the microorganism in flocs was also reduced by 45% to 65% (Caravelli et al.,

2004).

Asynchronous changes of sludge respiration and effluent quality under stressed

conditions have also been reported. For instance, when nitrifying sludge was exposed

to copper, the reactor effluent did not simultaneously deteriorate with the decrease in

respiration rate. Only when the respiration rate decreased by up to 52%, could the

deterioration of effluent quality be observed (Ouyang et al., 2016). On the contrary, an

increase in respiration rate does not necessarily indicate good performance of

4
activated sludge. For example, an increase in ATP could be used as an index for an

early warning signal of sludge bulking (Brault et al., 2011).

Thus far, the response and subsequent recovery of the microorganisms in

activated sludge under stressed conditions, i.e., whether the system will fluctuate and

recover, or fail completely under certain stressed conditions, remain unclear.

Therefore, in order to solve such a problem and provide a simple and effective tool for

the management of activated sludge systems under stressed conditions, experiments

were conducted to examine the respiration characteristics of activated sludge under

two types of typical stressed conditions: low oxygen or low dose of NaClO.

Additionally, a new evaluating index, recovery index (RI), which is defined as the

ratio of the endogenous respiration rate to the maximum respiration rate, was

proposed to quantitatively assess the activated sludge recovery status.

2. Materials and Methods

2.1 Reactor, wastewater and seeding sludge

A laboratory-scale sequencing batch reactor (SBR) used in this work had a

working volume of 10 L and an inner diameter of 15 cm and a height of 70 cm. The

cycle time of the SBR was 6 h, consisting of 4 min feeding reaction, 294 min aerobic

reaction (aeration mixing together), 58 min settling and 4 min discharge. A

programmable logic controller was used to control the sequence operation. During the

5
feeding period, 4 L of wastewater was added into the reactor with a hydraulic

retention time (HRT) of 15 h. Temperature of the bioreactor was maintained at

201 with a thermostatic heater and the pH level was maintained at 7.0-8.0 by

dosing 0.1 mol/l HCl to avoid chemical phosphorus precipitation. The solids retention

time (SRT) of the SBR was maintained at 15-18 days by controlling sludge wastage,

resulting in a MLSS concentration in a range of 2000-4000 mg/l. DO concentration

was controlled by adjusting the aeration intensity.

A synthetic wastewater was fed to the SBR and composed of chemical oxygen

demand (COD) 600-750 mg/l using sodium acetate as the carbon source, NH4+-N

60-70 mg/l, PO43--P 6-8 mg/l and trace elements 0.4 ml/l. The trace elements

consisted of 0.15 g/l H3BO3, 0.03 g/l CuSO45H2O, 0.18 g/l KI, 0.12 g/l MnCl24H2O,

0.06 g/l Na2MoO42H2O, 0.12 g/l ZnSO47H2O, 0.15 g/l CoCl26H2O, 1.54 g/l

FeSO47H2O and 12.74 g/l EDTA. The seeding sludge was taken from a WWTP in

Xian City, China.

2.2 Experimental designs and operational conditions

Three types of operating conditions were examined independently. In Run 1, two

types of stressed conditions, i.e., low oxygen (DO 0.5 mg/l) and introduction of

NaClO, were adopted to examine the fluctuation and recoverability of activated

sludge. Three periods was divided into 3 phases: DO concentration of 2-4 mg/l was

provided in the period P1 (Days 0-7), then, the low DO operation (DO 0.5 mg/l)

6
was applied in the period P2 (Days 7-15), and finally NaClO stressing was applied

and the DO level was recovered to 2-4 mg/l in the period P3 (Days 15-21). The

dosage of NaClO was increased from 10 to 30 mg/l with a step of 10 mg/l/day, and

from Day 17 a dosage of 30 mg/l had been maintained, resulting in the final available

chlorine loading of 15 mgCl2/g SS; In Run 2, experiments were carried out to further

confirm the recoverability of the nitrification strains at a low DO level. In this case,

the following series of parameters were monitored: OURs, ammonia uptake rate

(AUR), nitrate accumulation rate (NAR), and ammonia nitrogen affinity coefficient

(Kn). In Run 3, the activated sludge samples respectively collected from a

laboratory-scale reactor and a WWTP were used to further verify the effects of

different dosages of NaClO on activated sludge.

2.3 Respirogram and kinetic parameters

The respirogram was composed of a series of respiration rates. The respiration

rates were measured offline using an automatic respirogram equipment (WBM400,

Xian Lvbiao Water Environmental Technology Co., China) that composed of a DO

probe (VisiFerm DO 120, Hamilton, Switzerland) and a pH probe (Polilyte Plus H

Arc 120, Hamilton, Switzerland) and a programmable logic controller (PLC), and the

following procedures were taken: 1) 0.3 L of activated sludge and 0.9 L of wastewater

were pumped into a built-in Plexiglas sleeve vessel of the equipment with a water

bath; 2) online monitoring of pH and temperature variations of the built-in vessel of

7
the equipment, followed by the dose of HCl or NaOH solution to maintain the pH at

7.20.5. The equipment was operated at a constant temperature of 200.5 ; 3) a

predetermined amount of nitrogen and carbon was added into the vessel (resulting in a

final increase in COD and NH4+-N concentrations in the vessel by 300 mg/l and 50

mg/l, respectively) to ensure the maximum microbial respiration without any stressed

conditions; and 4) aeration was provided until the oxygen concentration reached 7

mg/l, and then stopped while mixing was carried out. The oxygen uptake rate (OUR)

was measured with the decline of oxygen concentration during the mixing without

aeration.

The detailed offline determination procedure of the respirogram is shown in Fig.

1. OURe represents the endogenous respiration of activated sludge, OURen indicates

the maximum ammonia nitrogen removal capacity of activated sludge with sufficient

substrate and oxygen under unstressed conditions, and similarly, OURt shows the

maximum ammonia nitrogen and COD removal capacity of activated sludge.

Additionally, OURh and OURa could also be calculated (Fig. 1b). In order to

eliminate the effect of sludge concentration on the respiration rate, the specific

oxygen uptake rate (SOUR) was also used (SOUR=OUR/MLSS). Thus, the recovery

index RI could be calculated as RI=OURe/OURt. The ammonia nitrogen affinity

coefficient (Kn) was measured using a respirometer-based method (Liwarska-Bizukojc,

E. and Bizukojc, M.,2012). Briefly, after measuring the respiration rate OURi under

8
different initial concentrations of ammonia Si, the KN value could be estimated with

the equitation OURi=vmax*Si/(KN+Si).

2.4 Analysis

COD, ammonium (NH4+-N), nitrate (NO3--N), nitrite (NO2--N), total phosphate

(TP), MLSS, MLVSS and sludge volume index (SVI) were measured according to the

Standard Methods (APHA 1995). The total organic carbon (TOC) and total inorganic

carbon (TIC) were analyzed using a TOC/TNb analyzer (VarioTOC, Elementar Co.,

Germany). The pH level and DO in the SBR reactors for Runs 1-3 were determined

by using a HQ40d portable DO meter (HACH Co., USA).

3. Results and discussion

3.1 Recovery of activated sludge under stressed conditions

As shown in Fig. 2, three periods could be discerned based on to the DO level

and the dosage of NaClO. DO was at a high level in periods P1 and P3 of Run 1, and

was at a low level in period P2. When the DO level was low, the sludge settling ability

became deteriorated and the SVI value increased (Fig. 2A, P2). In order to inhibit the

growth of filamentous bacteria, dosage of 15 mg/gSS/d NaClO was applied in period

P3, and the SVI slightly decreased (Fig. 2A, P3).

9
 With regard to the reactor effluent, the concentrations of NH4+-N and TIN

increased during the whole period P2 and the COD increased 3 days later after the

decrease in DO (Fig. 2B). Interestingly, the TP increased from 0.20 mg/l to a

maximum of 13.2 mg/l after 8 hours from the beginning of period P2, and then

decreased to 5.8 mg/l, which was almost the same level as that in the influent (Fig.

2C). After resuming the DO level and the dosage of NaClO (Fig. 2B and C, P3), the

removal efficiencies of COD, TP and NH4+-N appeared to recover. However, with the

continuous exposure to NaClO, these removal efficiencies all decreased (Fig. 2B and

C, P3).

SOURe was monitored under two stressed conditions in Run 1 (Fig. 3A). When

the DO level was reduced below 0.5 mg/l, SOURe soon increased from 0.4960.098

mgO2/gSS/h to 1.488 mg mgO2/gSS/h, and then stabilized on the whole period (Fig.

3A, P1 and P2). In period P3, SOURe increased again. With continuous exposure to

NaClO, SOURe continuously increased (Fig. 3A, P3).

In Run 1, after the decrease in DO, SOURa, which indicates the capacity of

nitrification, decreased immediately (Fig. 3B, P1 and P2). However, after continuous

operation at a low DO level, it gradually recovered (Fig. 3B, P2). Meanwhile, SOURh,

which represents the capacity of COD removal, kept increasing after reducing DO and

peaked on the 3rd day after DO reduction. Both SOURa and SOURh decreased rapidly

after resuming DO to 2-4 mg/l and dosing NaClO. SOURa decreased to zero on the 3rd

day (Fig. 3B, P3), implying the deterioration of nitrification ability. SOURh decreased

10
by 50% at 2-4 mg/l of DO on the 3rd day. When the dosage of NaClO was increased to

15mg/gSS/d, the respiration rate and the total respiration rate of the activated sludge

decreased rapidly, suggesting that the activated sludge could not recover.

3.2 Recovery of nitrifying bacteria at a low DO level

In Run 2, the effect of DO concentration on nitrifying bacteria was examined in

the absence of NaClO (Fig. 4). The similar recovery tendency of the nitrification

respiration rate was also observed after the operation at a low DO level for several

days as in Run 1 (Fig. 3B). SOURa immediately decreased and then gradually

returned to its original level, whereas after a long-time exposure to a low DO level, it

decreased again (Fig. 4, P2). Both SOURt and SOURh shared the same trend where

they decreased in a very short time and then increased soon, remaining relatively

stable, even though their values were higher than those in period P1 (Fig. 4). It should

be noticed that the decrease in SOURh after the DO reduction was different from that

in Run 1, as the latter exhibited no decrease (Fig. 3B).

Table 1 lists the changing profiles of AUR, NAR and Kn at DO of 2-4 mg/l and a

low DO of 0.5 mg/l, respectively. The maximum of both AUR and NAR at a high DO

level were higher than those at a low DO level. In addition, the Kn value was

0.75280.0306 and 0.1550.0216 mg/l for these two cases, respectively.

3.3 Effect of NaClO on microbial respiration

11
 Figure 5 shows the effect of NaClO dosage on microbial respiration of the two

types of sludge: one collected from the lab-scale SBR and another from WWTP

respectively. Generally, SOURe and SOURa of the two sludge samples exhibited the

same performance. At a NaClO dosage of 1-2 mg/gSS/d (Fig. 5B, C, E and F, P1),

SOURe slightly increased soon after the dose was administered and then gradually

recovered after a few days. Nitrification was inhibited as SOURa decreased during the

whole exposure time. However, the variation of SOURh and SOURt of heterotrophic

bacteria depended on the sludge source (Fig. 5B, C, E and F, P1). These values of the

sludge from the lab-scale SBR slightly increased (Fig. 5B and C, P1), while those of

the sludge from the WWTP decreased (Fig. 5E and F, P1).

When the NaClO dosage increased to 4 mg/gSS/d and further 8 mg/gSS/d,

SOURe slightly decreased in a short time (3 days), but then increased to a high level.

The nitrification capacity was inhibited and finally compressed (Fig. 5B and E, P2).

The sludge from the SBR deteriorated more rapidly than the sludge from the WWTP.

Both SOURh and SOURt of the heterotrophic bacteria could recover after a short

inhibition time (Fig. 5B, C, E and F, P2).

After the dose of NaClO stopped, the respirogram of the two sludge samples,

with the exception of SOURa, decreased in a very short time and gradually recovered

to the same level of the control group at a dosage of NaClO less than 8 mg/gSS/d.

Meanwhile, SOURa of the nitrifying sludge from the WWTP recovered after 7 days

(Fig. 5F, P3).

12
3.4 Comparison of the endogenous respiration rate ratio of recoverable and

unrecoverable activated sludge

As mentioned above, the endogenous respiration rate normally increased when

sludge was exposed to stressed conditions, and the RI values could be calculated

based on the above results. In the recoverable cases, e.g., low DO level in Runs 1-3,

RI initially increased, and then decreased. For instance, when the DO was reduced in

Run 1, RI sharply increased from 0.0420.008 to 0.100 and then decreased to

0.0650.012 (Fig. 6A, P1 and P2). Similar results were also found in Run 2 (Fig. 6B).

Additionally, when the NaClO dosage increased from 2 mg/gSS/d to 8 mg/gSS/d,

RI of the sludge from the SBR increased sharply from 0.0620.006 to 0.181, and then

gradually fell back to 0.145 (Fig. 6C, P1 and P2). However, for the unrecoverable

case (NaClO dosage of 15 mg/gSS/d in Run 1), RI continuously increased to 0.35

(Fig. 6A, P3).

However, it should be mentioned the classification of recoverable and

unrecoverable cases in this work depended on the variation pattern RI after ceasing

the stressing. When the RI value continuously increased to a certain level and did not

fall back, the continuous deterioration of effluent and decrease in biomass could be

observed, thus, neither respiration rate nor effluent quality could be recovered as

occurred in Run 1.

13
3.5 Recoverability of microorganisms at low DO levels

Previous studies have shown that a low-level of DO could lead to the

deterioration of the effluent quality and sludge settling ability (Wgner et al., 2015),

variation of microbial communities (Wang et al., 2016b), and sludge foaming and

bulking (Guo et al., 2013). However, in some cases, an activated sludge system could

work well at a low DO level in an energy-saving mode (Arnaldos and Pagilla, 2014;

Kunkel et al., 2015). This study shows that both the effluent quality and sludge

settling ability deteriorated when the DO level was reduced to 0.5 mg/l (Fig. 2), but

the respiration rates changed differently. Both OURa and OURh decreased quickly and

then recovered to a considerably high level (Figs. 3B and 4), which stopped the

further deterioration of effluent quality in terms of COD and NH4+-N (Fig. 2B).

Although the respiration rates recovered to a high value, the in-situ AUR and NAR

decreased considerably (Table 1), suggesting that the sludge acclimated to a low level

of DO, and the deteriorated effluent quality was ascribed to the shortage of supplied

oxygen. This was confirmed by the increased AUR and NAR levels during the later

period of the in-situ experiment (Table 1). At a low DO lever, the AUR and NUR

values were higher during the early period compared to those during the later period.

The different changing patterns of respiration rates and effluent quality were also

observed in other studies. For instance, a considerable inhibitory effect on the

ammonia conversion was observed after the respiratory activity (i.e., SOURNH4)

decreased by 52% in an SBR experiment (Fan et al., 2015). After the DO shortage

14
was relieved, the effluent quality improved, as shown in Run 1 in our work, and the

COD removal could be well recovered despite the dose of NaClO (Fig. 2B, P3).

Therefore, with regard to the recovery of activated sludge, recovery of effluent quality

depends not only on the respiration characteristics but also the external environmental

conditions (e.g., DO level in this work). In other words, the respiration recovery

should be a prerequisite.

3.6 Endogenous respiration ratio as an index of recoverability

Endogenous respiration, as an important metabolic index of activated sludge, can

be effectively used to characterize the activity of activated sludge (Lopez et al., 2006),

physiological adaptation of growth kinetics at different sludge SRTs (Friedrich et al.

2015), and environmental adaptability or the health status of activated sludge (Li et

al., 2014). When the short-term sludge concentration remains nearly unchanged, the

endogenous respiration rate should remain nearly constant. When the activated sludge

was exposed to the stressed conditions, an increase in RI could be observed (Fig. 6).

The RI reflects the difference in the respiration rates of activated sludge in the

absence and presence of substrate. When environmental conditions favor microbial

growth and reproduction, the OURt should increase (Friedrich et al., 2015), thus the

RI value is small. In our work, it was observed that when RI rose sharply in a short

period and then decreased, the sludge respiration characteristics could be recovered

(e.g., a low-level DO operation in Runs 1-3). However, when the RI value continued

15
to increase to a certain value, e.g. 0.35, in this work, the system became unrecoverable

(e.g., dose of NaClO in Run 1). When the sludge does not acclimate to the new

environment and the bacterial growth is inhibited in a survival-optimized

physiological status (Friedrich et al., 2015), OURt value is small. At the same time,

when the sludge deteriorates, OURe increases (Figs. 3A, 4 and 5). These two factors

result in the increase in RI. However, when the RI value continuously increased,

bacterial growth stopped. In this case, it became difficult for the sludge to recover.

According to the endogenous respiration models (Ramdani et al., 2010), the

number of the microorganisms in activated sludge is proportional to the endogenous

respiration rate OURe, and their activity is well correlated with OURt. It was reported

that the number of ammonia oxidizing bacteria (AOB) species under sharp shocking

stressed conditions tended to increase even if no ammonium oxidizing activity was

recorded (Marzorati et al., 2013). These reports support the usage of RI in the case of

sharp shocking. It is well known that bacteria under energy-limitation conditions

experience fragmentation, resulting in a reduced cell size and cell volume (Lever et al.,

2015). This also suggests that increasing endogenous respiration could be a survival

strategy of bacteria under stressed conditions.

To further evaluate the practicability of the index RI, an investigation from six

full scale WWTPs, located in Xian, China, was carried out for verification (Table 2).

The WWTPs (Plants 2, 3 and 5) treating domestic wastewater exhibited stable RI

values, especially for the large scale WWTPs (Plants 2 and5); whereas the WWTPs

16
treating mixed domestic wastewater and leachate fluctuated in a wide range, because

these plants suffered from toxic or occasional shocking of leachate (Plants 1 and 6).

Plant 1, accepting landfill leachate completely, exhibited a wide range of RI value,

suggesting that it was hard to be readily operated. Thus, a narrow RI range suggests a

health status of the activated sludge system, which was easily operated. For Plant 6

with a capacity of 150103m3/d for the domestic wastewater showed the highest RI

value of 0.27 because it occasionally accepted the discharge of leachate. This plant

encountered the most violent fluctuation among these plants, and was thus the most

difficult case for sludge recovery.

Activated sludge systems are frequently exposed to unfavorable conditions; it is

not yet clear how to describe the recoverability of activated sludge. Usually the

operators of WWTPs judge the status of activated sludge in terms of effluent quality,

sludge settleablity and biological community. In this work, an index, RI, was

proposed to describe the respiration status of activated sludge during its adaptation

period. These approaches were compared and are listed in Table 3. This new

approach is based on the widely used OUR measurement, and thus can be easily

implemented as an online index, which could provide an early warning signal of the

status of activated sludge when exposed to unfavorable conditions. Once the warning

signal is given, one can take effective measures to adjust the operation of WWTPs.

Therefore, this approach holds a high promise for the operation and management of

WWTPs.

17
4. Conclusions

In this work, experiments were conducted to examine the respiration characteristics of

activated sludge at a low oxygen level and dosage of NaClO. It was found that the

physiological variations under the stressed conditions could be well described using

respirogram characteristics, and an index RI could be used to characterize the

recoverability of activated sludge systems. Such an index could be readily

implemented for online monitoring purposes. The results from this study provide a

simple and practical approach for the stable operation and management of activated

sludge wastewater treatment plants.

Nomenclature
AUR ammonia uptake rate (mg/l/min)
DO dissolved oxygen (mg/l)
HRT hydraulic retention time (day)
Kn ammonia nitrogen affinity coefficient (mg/l)
NAR nitrate accumulation rate (mg/l/min)
NaClO hypochlorite (mg/l)
OUR oxygen uptake rate (mgO2/l/min or mgO2/l/h)
OURa autotrophic respiration rate (mgO2/L/min or mgO2/L/h)
OURe endogenous respiration rate (mgO2/L/min or mgO2/L/h)
OURen ammonia nitrogen respiration rate (mgO2/L/min or mgO2/L/h)
OURh heterotrophic respiration rate (mgO2/L/min or mgO2/L/h)

18
OURt total respiratory rate (mgO2/L/min or mgO2/L/h)
PLC programmable logic controller
RI A defined sludge recovery index
SBR sequencing batch reactor
SOUR specific oxygen uptake rate (mgO2/gSS/min or mgO2/gSS/h)
TP total phosphorus (mg/l)
WWTP wastewater treatment plant

19
Acknowledgments

This work was supported by the National Natural Science Foundation of China

(51538011) and the West Light Foundation for the Visiting Fellow Program.

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24
Table 1 Variations of AUR, NUR, SOURa and Kn values at various DO levels
Item Normal DO level Low DO level

AURmg/L/min 0.165 0.056

NURmg/L/min 0.125 0.062
SOURamgO2/gSS/min 0.025 0.016
Knmg/L 0.75280.0306(n=2) 0.1550.0216(n=4)
n: the number of tests for Kn.

25
Table 2 RI values of the activated sludge from oxic unit in different full scale
WWTPs
WWTP Process and SRT Influent MLSS Influent type RI
scale COD
(103m3/d) (day) (mg/L) (mg/L)
A/A/O+NF 10000-3 28000-3 Landfill 0.04~0.1
1 77
(1.2) 0000 2000 leachate 3 (n=3)
A/A/O 4000-50 0.12~0.1
2 14-15 300-400 Municipal
(500.0) 00 6 (n=5)
A/A/O+MBR 3500-45 0.08~0.1
3 18-20 250-300 Municipal
(4.0) 00 0 (n=2)
Municipal +
Oxidation 5000-55 0.09~0.1
4 15-17 250-300 Industrial
ditch (30.0) 00 1 (n=2)
factories
A/A/O 3000-40 0.13~0.1
5 14-16 500-600 Municipal
(160.0) 00 4 (n=2)
Municipal+
Oxidation 6000-70 occasionally 0.17~0.2
6 19-21 350-450
ditch (150) 00 discharge of 7 (n=3)
leachate
n: the number of tests;
AAO: anaerobic, anoxic, oxic;
NF: nano filter
MBR: membrane bioreactor.

26
Table 3 Comparison of the different methods for describing activated sludge under
stressed conditions
Method Water quality Settle ability Molecular biology RI
Equipment required Medium Low High Medium
Reagent required Medium High Low
Time consuming Medium Short Long Medium
Skill required Medium Low High Low
Cost required Medium Cheap Expensive Medium
Effectiveness Poor Poor Good Good

27
 Figure captions

Figure 1. Procedures for the offline determination of respirogram.

Figure 2. Overall performance of activated sludge in Run 1: (A) variations of SV30,

MLSS, and SVI; (B) inflow and effluent COD and NH4+-N levels; and (C) inflow and

effluent TIN and TP levels.

Figure 3. Variations of respirogram of the activated sludge in Run 1: (A) variations of

SOURe and SOURt; and (B) variations of SOURa and SOURh.

Figure 4. Respirogram, AUR and NAR at different DO levels in Run 2: (A)

respirogram; (B) AUR and NAR at a DO of 2-4 mg/l; and (C) AUR and NAR at a DO

of 0.5 mg/l.

Figure 5. Respirogram of the activated sludge: (A) sludge from the SBR without

NaClO; (B) sludge from the SBR with 1 and 4 mg/gSS NaClO; (C) sludge from the

SBR with 2 and 8 mg/gSS NaClO; (D) sludge from the WWTP without NaClO; (E)

sludge from the WWTP with a low dosage of NaClO; (F) sludge from the WWTP

with a high dosage of NaClO. The SOURc indicates the SOUR of the same sludge in

the absence of NaClO.

Figure 6. Variations of RI index: (A) Run 1; (B) Run 2; (C) the sludge from the SBR

in Run 3; (D) the sludge from the WWTP in Run 3.

28
 Sludge from WWTP
(A) (B)
Wash in triple using PBS buffer
solution and aeration for two hours

Measurement of endogenous
respiration rate, OURe

Adding NH4Cl

Measurement of autotrophic
respiration rate, OURen

Adding NaAc

Measurement of max respiration

rate, OURt

OURh=OURt- OURa=OURen-
OURe
OURe-OURa OURe

It can take a series of OUR,i.e.,

OURe,OURa,OURh,OURt

Figure 1

29
 SVI MLSS SV30
1000 10
5000 (A) P1 P2 P3

SVI (ml/g) MLSS (mg/L)

4000 900 8

3000

SV30 (ml)

DO (mg/L)
800 6
2000
Without NaClO
400 700 4
DO: 2-4mg/L
300 DO: 2-4mg/L
200 600 2
Worsen SVI With NaClO
100
DO: 0-0.5mg/L
Low DO
0 500 0

+ +
INF-COD EFF-COD INF-NH4 -N EFF-NH4 -N
10
P1 P2 P3
(B) 70
800
60 8

NH4 -N (mg/L)
600 50
COD (mg/ l)

DO(mg/ l)
40 6
Without NaClO
With NaClO
400 + 30
+
NH4 -N Worsen
4
20
DO: 2-4mg/l DO: 2-4mg/l
200
10
COD Worsen 2
0
0 DO: 0-0.5mg/l
-10 0
INF-TIN EFF-TIN INF-TP EFF-TP
80 10
P1 P2 P3
(C)
12
8
60

9
DO(mg/ l)

6
TP (mg/ l)
TIN (mg/ l)

40
Without NaClO 6
4
TP Worsen
DO: 2-4mg/l DO: 2-4mg/l
20 3
2
TIN Worsen
With NaClO
0
0
DO: 0-0.5mg/l
0
0 4 8 12 16 20
Running Time (Day)

Figure 2
30
SOURe(mgO2 /gSS/h) SOURt(mgO2 /gSS/h)
SOURe SOURt DO
25 10
P1 P2 P3
(A)
20
8
15
With NaClO
10

DO(mg/ L)
Without NaClO 6

3 4
DO: 2-4mg/L DO: 2-4mg/L
2
2
1

0 DO: 0-0.5mg/L 0
SOURa(mgO2 /gSS/h) SOURh(mgO2 /gSS/h)

SOURa SOURh DO
10
P1 P2 P3
20 (B)
15 With NaClO 8
Without NaClO

DO(mg/ L)
10
6
5

4
1.0 DO: 2-4mg/l
DO: 2-4mg/l
0.5
2
0.0
DO: 0-0.5mg/l
-0.5 0
0 4 8 12 16 20
Running Time (Day)

Figure 3

31
 SOURe SOURa SOURh SOURt
14 10
P1 P2
12
8
10
SOUR(mgO2 /gSS /h)

8 6

DO(mg/L)
4
1.5
DO: 2-4mg/L
1.0
2
0.5
DO: 0-0.5mg/L
0.0 0
0 4 8 12 16
Running Time (Day)

Figure 4

32
 SOURe SOURa SOURh SOURt SOURe SOURa SOURh SOURt
20 20
(A) (D)
16 16

SOUR(mgO2 /g SS /h)
SOUR(mgO2 /g SS /h)

12
12
8
8

1 1

0 0

SOURe/SOURe-c SOURa/SOURa-c SOURe/SOURe-c SOURa/SOURa-c

Cl Dosage Cl Dosage
SOURh/SOURh-c SOURt/SOURt-c SOURh/SOURh-c SOURt/SOURt-c
3 10 3 P2
10
P1
(B)
P2 P3
(E) P1 P3

Avaliable chlorine (mg/g-SS)

Avaliable chlorine (mg/g-SS)

8 8
2 2
SOUR/SOURc

SOUR/SOURc
6 6

1 4 1 4

2 2
0 0

0 0

3 10 3 P2 P3
10
P1 P2 P3 P1
(C) (F)
Avaliable chlorine (mg/g-SS)

Avaliable chlorine (mg/g-SS)

8 8
2 2
SOUR/SOURc

SOUR/SOURc

6 6

1 4 1 4

2 2
0 0

0 0
0 4 8 12 16 20 24 0 4 8 12 16 20 24
Running Time (Day) Running Time (Day)

Figure 5

33
 With 2 NaClO With 8 NaClO
Normal DO Low DO Normal DO with NaClO Without NaClO high
P1 P2 P3
10 P1 P2 P3
10
0.35

Avaliable chlorine (mg/g-SS)

(A) 0.24 (C)
0.30 8 0.20 8
With NaClO
0.25

DO(mg/ L)
Without NaClO
6 0.16 6
0.20
0.12

RI
RI

4 4
0.15 DO: 2-4mg/L
DO: 2-4mg/L 0.08
0.10 2 2
0.04
0.05
DO: 0-0.5mg/L
0 0.00 0
0 4 8 12 16 20
Running Time (Day)
Normal DO Low DO
P1 P2
10 P2 P3
10
0.07 (B) (D) P1

Avaliable chlorine (mg/g-SS)

0.24
8 0.20 8
0.06

DO(mg/ L)
6 0.16 6

RI
0.12
RI

0.05 4 4
DO: 2-4mg/L 0.08
2 2
0.04 0.04
DO: 0-0.5mg/L 0 0.00 0
0 4 8 12 16 0 4 8 12 16 20 24
Running Time (Day) Running Time (Day)

Figure 6

34
 Graphical Abstract

Stressed condition Stressed condition

SOURt SOURt

RI RI
SOURe SOURe

Time
Recoverable Unrecoverable Time

35
 Research highlights

External environmental shocks caused an increase in endogenous respiration rate

of activated sludge.
The recoverability of sludge respiration and effluent under low DO conditions was
asynchronous.
A recovery index RI, describing the recovery potential of activity, was proposed to
describe the respiration adaptation of sludge under stressed conditions.

36