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Performance of high-loaded ANAMMOX UASB reactorscontaining granular sludge

Chong-Jian Tang a, Ping Zheng a,*, Cai-Hua Wang a, Qaisar Mahmood b, Ji-Qiang Zhang a,Xiao-Guang Chen a, Lei Zhang a, Jian-Wei Chen a

aDepartment of Environmental Engineering, Zhejiang University, Hangzhou 310029, ChinabDepartment of Environmental Sciences, COMSATS University, Abbottabad, Pakistan

a r t i c l e i n f o

Article history:

Received 1 November 2009

Received in revised form

28 May 2010

Accepted 10 August 2010

Available online 17 August 2010

Keywords:

ANAMMOX

Granular characteristics

Process performance

UASB reactor

* Corresponding author. Tel./fax: þ86 571 86E-mail addresses: chjtangzju@yahoo.com

0043-1354/$ e see front matter ª 2010 Elsevdoi:10.1016/j.watres.2010.08.018

a b s t r a c t

The performance of high-loaded anaerobic ammonium oxidizing (ANAMMOX) upflow

anaerobic sludge bed (UASB) reactors was investigated. Two ANAMMOX reactors (R1 with

and R2 without effluent recycling, respectively) were fed with relatively low nitrite

concentration of 240 mg-N L�1 with subsequent progressive increase in the nitrogen

loading rate (NLR) by shortening the hydraulic retention time (HRT) till the end of the

experiment. A super high-rate performance with nitrogen removal rate (NRR) of

74.3e76.7 kg-N m�3 day�1 was accomplished in the lab-scale ANAMMOX UASB reactors,

which was 3 times of the highest reported value. The biomass concentrations in the

reactors were as high as 42.0e57.7 g-VSS L�1 with the specific ANAMMOX activity (SAA)

approaching to 5.6 kg-N kg-VSS�1 day�1. The high SAA and high biomass concentration

were regarded as the key factors for the super high-rate performance. ANAMMOX granules

were observed in the reactors with settling velocities of 73e88 m h�1. The ANAMMOX

granules were found to contain a plenty of extracellular polymers (ECPs) such as

71.8e112.1 mg g-VSS�1 of polysaccharides (PS) and 164.4e298.2 mg g-VSS�1 of proteins (PN).

High content of hemachrome (6.8e10.3 mmol g-VSS�1) was detected in the ANAMMOX

granules, which is supposed to be attributed to their unique carmine color.

ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction process inwhich half of ammonium is converted to nitrite, the

Anaerobic ammonium oxidation (ANAMMOX) is a promising

biotechnology for the treatment of ammonium-rich waste-

water (van der Star et al., 2007; Joss et al., 2009). Under anoxic

conditions, the ANAMMOX bacteria accomplish autotrophic

ammoniumoxidation todinitrogengas employingnitrite as an

electron acceptor (Strous et al., 1998). It offers several advan-

tages over conventional nitrification-denitrification systems

including higher nitrogen removal rate, lower operational cost

and less space requirement (Jetten et al., 2005; van der Star

et al., 2007; Joss et al., 2009). Combined with single reactor

high activity ammonium removal over nitrite (SHARON)

971709..cn (C.-J. Tang), pzheng@ier Ltd. All rights reserved

first full-scale ANAMMOX process (70 m3) was applied to treat

sludge dewatering effluents in Rotterdam, The Netherlands in

2002 (van Dongen et al., 2001; van der Star et al., 2007). It stably

operated achieving nitrogen removal rate (NRR) up to

9.5 kg-N m�3 day�1 (van der Star et al., 2007).

High-rate is one of the prime objectives for ANAMMOX

process. The NRR of conventional nitrogen removal biotech-

nologies was less than 0.5 kg-N m�3 day�1 (Jin et al., 2008);

while for ANAMMOX process, it was higher than 5 kg-

N m�3 day�1 as obtained by a number of researchers using

different reactors such as upflow biofilter, upflow anaerobic

sludge blanket (UASB) reactor and gas-lift reactor (Sliekers

zju.edu.cn (P. Zheng)..

Nomenclature

ANAMMOX Anaerobic ammonium oxidation

ECP(s) Extracellular polymer(s)

HLR Hydraulic loading rate

HRT Hydraulic retention time

NLR Nitrogen loading rate

NRR Nitrogen removal rate

PN Protein

PS Polysaccharide

SAA Specific ANAMMOX activity

SEM Scanning electron microscopy

SHARONSingle reactor high activity ammonium removal

over nitrite

SVI Sludge volume index

TEM Transmission electron microscopy

TSS Total suspended solids

UASB Upflow anaerobic sludge bed

VSS Volatile suspended solids

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4136

et al., 2003; Imajo et al., 2004; Isaka et al., 2007; van der Star

et al., 2007; Tang et al., 2009a). To date, the highest NRR

reported was 26.0 kg-N m�3 day�1 at hydraulic retention time

(HRT) of 0.24 h (Tsushima et al., 2007). Previous works on

anaerobic processes including anaerobic digestion (Thiele

et al., 1990) and denitrifying process (Franco et al., 2006)

attributed high volumetric removal rates to three main

aspects. Firstly, the reactors should have high-quality sludge

retention for sufficient biomass accumulation. Secondly, the

microbial communities should aggregate as granular sludge or

biofilms for optimummetabolic activity. Finally, the substrate

requirements of ANAMMOX bacteria should be satisfied

simultaneously avoiding substrate inhibition, especially

nitrite inhibition (Strous et al., 1999; Isaka et al., 2007;

Tsushima et al., 2007).

The granular sludge characterized by good settling prop-

erty and high activity plays a pivotal role in the performance

of high-rate bioreactors (Thiele et al., 1990; Franco et al., 2006;

Zhang et al., 2008). The characteristics of granular sludge such

as heterotrophic aerobic granules (Beun et al., 1999; Beun

et al., 2002; Zheng and Yu, 2007; Adav et al., 2008), anaerobic

granules (Hulshoff Pol et al., 2004; Show et al., 2004; Wu et al.,

2009), hydrogen-producing granules (Mu and Yu, 2006; Zhang

et al., 2008), denitrifying granules (Franco et al., 2006) and

autotrophic nitrifying granules (Tsuneda et al., 2003; Liu et al.,

2008; Belmonte et al., 2009) have been extensively studied. In

case of ANAMMOX granules, the settling property, diameter

Table 1 e Operational parameters of the ANAMMOXsludge and the two UASB reactors before the start of theexperiment.

Characteristic R1 R2

A: Characteristics of the sludge

Diameter (mm) 1.9 2.1

TSS/VSS (%) 82 85

SAA (kg-N kg-VSS�1 day�1) 0.3 0.2

B: Operational characteristics of the two UASB reactors before the

start of the experiment

Influent ammonium concentration (mg-N L�1) 300 200

Influent nitrite concentration (mg-N L�1) 360 240

Effluent recycling ratio 0.5 e

HRT (h) 6.90 11.7

Sludge concentration (g-VSS L�1) 18.7 26.8

NRR (kg-N m�3 day�1) 6.0 2.9

distribution and substrate diffusion have been reported

(Arrojo et al., 2006; Ni et al., 2009). The characteristics of

carmine color of ANAMMOX granules and their associated

extracellular polymers (ECPs) have also drawn considerable

attention for the process optimization. The hydroxylamine

oxidoreductase and hydrazine oxidoreductase are two

important enzymes of the ANAMMOX pathway. Both of these

enzymes are rich in heme c (Klotz et al., 2008; Schmid et al.,

2008), which endows the granular sludge with the carmine

color. The extracellular polymers are assumed to be a key

factor in the formation of granular sludge, which can be

secreted by ANAMMOX bacteria (Cirpus et al., 2006).

In the present study, two ANAMMOX UASB reactors were

operated to investigate the performance of high-loaded reac-

tors possessing carmine granular sludge.

2. Material and methods

2.1. Synthetic wastewater

Ammonium and nitrite were supplemented to mineral

medium as required in the form of (NH4)2SO4 and NaNO2,

respectively. The composition of the mineral medium was

(g L�1 except for trace element solution) (Trigo et al., 2006):

KH2PO4 0.01, CaCl2$2H2O 0.00565, MgSO4$7H2O 0.3, KHCO3

1.25, FeSO4 0.00625, EDTA 0.00625 and 1.25 mL L�1 of trace

elements solution. The trace element solution contained

(g L�1): EDTA 15, H3BO4 0.014, MnCl2$4H2O 0.99, CuSO4$5H2O

0.25, ZnSO4$7H2O 0.43, NiCl2$6H2O 0.19, NaSeO4$10H2O 0.21,

NaMoO4$2H2O 0.22 and NaWO4$2H2O 0.050 (adapted from van

de Graaf et al. (1996)).

2.2. ANAMMOX bioreactors

The experimental work was carried out in two glass-made

UASB reactors of 1.1 L capacity having internal diameter of

50 mm. Both reactors were completely covered with black

cloth to avoid the growth of phototrophic organisms and the

related oxygen production (van der Star et al., 2008). The

reactors were fed with synthetic wastewater which was

flushed with 95%Ar-5%CO2 continuously to maintain anoxic

conditions. The temperature was set at 35 � 1 �C according to

Tsushima et al. (2007) and the influent pH was controlled in

the range of 6.8e7.0 (Tang et al., 2009b). The produced gas was

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4 137

initially absorbed by NaOH solution and then recorded by

a wet gas meter.

The two reactors (designated as R1 and R2) were initially

inoculated with anaerobic granular sludge taken from a paper

mill wastewater treatment plant (100 m3, located in

Zhejiang Province, China). The average diameter of the

anaerobic granular sludgewas about 2.2mm; and the VSS/TSS

contentwas about 85%. The reactors were successfully started

up and operated stably for 214 days before the experiments.

The operational characteristics of R1 and R2 are listed in

Table 1.

2.3. Reactor operation

In order to avoid the nitrite inhibition, both reactors were

operated at low influent nitrite concentration. R2 was

constantly fed with 240 mg-NO2�-N L�1 without effluent recy-

cling;while, R1was constantly fedwith 360mg-NO2�-N L�1with

effluent recycling ratio (recycling flow to inflow ratio) about 0.5.

Thus, the influentnitrite concentrationwasabout 240mg-NL�1

after dilution. Ammonium was supplemented relatively in

excess and was progressively increased during the shortening

of HRT in order to gain better nitrite removal efficiency and

performance stability (Tsushima et al., 2007). The HRT was

progressively shortened after 5 days at each step when the

reactor performance was stable. Throughout the operation, no

sludge was deliberately removed from the reactors.

Fig. 1 e Profile of nitrogen removal rate and influent fl

2.4. Analytical procedures

The influent and effluent samples were collected on daily

basis and analyzed immediately. The determination of pH,

ammonium, nitrite, nitrate, 5-min and 30-min sludge volume

indices (SVI5 and SVI30), total suspended solids (TSS) and

volatile suspended solids (VSS) concentrations were carried

out following the Standard Methods (APHA, 1998). The size of

granular sludge was measured by an image analysis system

(QCOLite) with a Leica DM2LBmicroscope and a digital camera

(Canon S30).

ECPswereextractedfromsludgebyEDTA(Shengetal., 2005),

then the extracellular proteins (PN) were determined by the

Lowry method using egg albumin as standard and the poly-

saccharide (PS) content was analyzed by the anthrone method

with glucose as standard (Wu et al., 2009). Heme c content was

determined according to Berry and Trumpower (1987) and

Sinclair et al. (1999). The nitrogen ligands from protein-bound

heme were replaced by pyridine in alkali, and the resultant

heme cwas quantified through the difference between spectra

of the reduced (sodium dithionite crystals) and oxidized

(potassium ferricyanide) compounds. The heme c concentra-

tion was calculated based on the millimolar extinction coeffi-

cient of 23.97 (mM cm�1) for the difference in absorption

between the peak at 550 nm and the trough at 535 nm.

Specific ANAMMOX activity (SAA) was determined

following the procedures described by Tang et al. (2009b). The

initial substrate concentration was maintained at

ow rate of the two ANAMMOX R1 (A) and R2 (B).

Fig. 2 e Effluent nitrite concentration and nitrite removal

efficiency of R1 (A) and R2 (B) at different HRTs.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4138

100 mg-N L�1 for both ammonium and nitrite and the sludge

concentration was kept about 0.6 g-VSS L�1. The cellular yield

and the specific growth rate were calculated based on the

procedures of Chen et al. (2010). Sludge granules settleability

(50 in number) was measured during the last 200 mm through

a 300 mm water column (Franco et al., 2006). Specific density

was measured according to Beun et al. (2002). Scanning elec-

tron microscopy (SEM) and transmission electron microscopy

(TEM) were performed according to Tang et al. (2009a) and

Tang et al. (2009b), respectively. Nitrogen removal rate was

calculated as the sum of ammonium and nitrite consumption.

3. Results and discussion

3.1. Nitrogen removal performance

3.1.1. Volumetric capacityThroughout the reactors’ operation, the influent nitrite

concentration was maintained constant whereby the nitrogen

loading rate (NLR) was progressively increased by shortening

HRT. The nitrogen removal performance of R1 and R2 is

depicted in Fig. 1A and B, respectively. During the first 250

days, the hydraulic loading rate (HLR) of R1was increased from

3.5 L L�1 day�1 to 114.3e123.8 L L�1 day�1, that corresponded to

HRT of 0.21e0.19 h; the NRR was enhanced to 72.5 � 2.8

(70.3e78.5) kg-Nm�3 day�1 with the NLR of 89.1e99.0 kg-Nm�3

day�1 (Fig. 1A). The nitrogen removal performance of R2 also

showed a similar trend when the influent flow rate was

progressively increased during the first 290 days. During that

period, the NRR was 65.4 � 3.0 (60.9e69.8) kg-N m�3 day�1

(HRT, 0.18e15 h; NLR, 78.9e97.9 kg-N m�3 day�1). Further

increasing the influent flow rate caused the nitrogen removal

performance of both reactors to decline (Fig. 1A and B).

Subsequent attempts made to increase influent flow rate of

both reactors did not significantly improve NRR. At the end of

the operation, the NRR of R1 was recorded to be 74.3 � 6.7

(66.8e82.8) kg-N m�3 day�1 (HRT, 0.16 h; influent flow rate,

152.4 L L�1 day�1; NLR, 125.0 kg-N m�3 day�1; days 399e412);

while for R2, the NRR was up to 76.7 � 4.5 (69.8e84.6) kg-Nm�3

day�1 (HRT, 0.11 h; influent flow rate, 221.0 L L�1 day�1; NLR,

137.1 kg-N m�3 day�1; days 410e417). The NRRs observed for

both reactors were three times of the highest reported values

(Tsushima et al., 2007).

3.1.2. Removal efficiencyThe influent ammonium concentration was gradually

increased for both reactors as described in Materials and

Methods section. The ammoniumremoval efficiencywasup to

90% when the HRT of R1 was longer than 1.58 h (influent

ammonium concentration, 330 mg-N L�1; NLR, 10.5 kg-N m�3

day�1); while it decreased to 70% when the HRT was further

shortened to 0.21 h (influent ammonium concentration,

420 mg-N L�1; NLR, 89.1 kg-N m�3 day�1). For R2, the

ammonium removal efficiencywas around 90%and 70%at the

corresponding HRT of 1.29 h (influent ammonium concentra-

tion, 220mg-NL�1;NLR, 8.6kg-Nm�3day�1) and0.30h (influent

ammonium concentration, 250 mg-N L�1; NLR, 39.7 kg-N m�3

day�1), respectively.

Nitrite removal efficiency of both the reactors at different

HRTs is presented in Fig. 2. For R1, the nitrite depletedwith the

removal efficiency higher than 96.8% and the average effluent

nitrite concentration was about 11 mg-N L�1 when working at

HRTs longer than 0.39 h. But it increased to 124.4 mg-N L�1

when HRT was shortened to 0.16 h with a sharp decrease in

nitrite removal efficiency to 65.4% (Fig. 2A). Fig. 2B shows the

nitrite removal efficiency of R2 at HRT range of 0.21 he0.10 h.

The average effluent nitrite concentration increased to

90.8 mg-N L�1 with nitrite removal efficiency of 62.2% when

HRT was shortened to 0.10 h.

Thenitrateproductiondidnotsignificantlyfluctuate inboth

reactors and it correlated to the substrate removal. The stoi-

chiometric ratios of ammonium conversion, nitrite removal

andnitrateproductionwere1: (1.31�0.03):(0.23�0.01) (R1) and

1:(1.32� 0.06):(0.25� 0.02) (R2), both were close to the reported

ratios (Strous et al., 1998).

3.1.3. Biomass growthProgressive increase of the ANAMMOX granules was observed

throughout the reactors’ operation (Fig. 3). The biomass

concentration in R1 enhanced to 57.7 g-VSS L�1 after 400 days.

While, the biomass concentration in R2 gradually increased to

42.0 g-VSS L�1 during that period. The VSS/TSS ratios of the

ANAMMOX granules in both reactors were in the range of

0.82e0.90. Relatively low calcium and phosphorous concen-

trations were included in the mineral medium based on the

results of Trigo et al. (2006), which gave rise to the high volatile

Fig. 3 e Apparent characteristics of the sludge in R1 (A) and R2 (B) at different periods, and the washed-out sludge (C).

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4 139

Table 2 e Diameter, settling velocity (vs), sludgevolumetric index (SVI5), density and specific density ofthe granules in the two UASB reactors at the end of theexperiment (average value).

Parameter R1 R2

Diameter (mm) 2.5 2.4

vs (m h�1) 88 73

SVI5 (mL g-VSS�1) 25 24

Density (g mL�1) 1.0323 1.0260

Specific density

(g-VSS L-granules�1)

108 94

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4140

solids content of the enriched granules in both the UASB

reactors (Trigo et al., 2006).

The ANAMMOX biomass yield in R1 was 0.23 g-VSS g-NH4þ-

N�1 with the specific growth rate of 0.0047 h�1 corresponding

to the doubling time of 6.1 days under NRR higher than 70 kg-

N m�3 day�1. The corresponding biomass values in R2 were

0.22 g-VSS g-NH4þ-N�1, growth rate of 0.0060 h�1 with the

doubling time of 4.8 days. The cellular yields in our studywere

approximately 2e3 times of the previously reported values

(0.07 g-VSS g-NH4þ-N�1, Trigo et al., 2006; 0.088 g-VSS g-NH4

þ-N�1, Strous et al., 1998; and 0.11 g-VSS g-NH4

þ-N�1, van Dongen

et al., 2001); and the doubling times were shorter than the

value reported by Strous et al. (1998) (11 days).

3.1.4. Specific ANAMMOX activityThe specific activities of the ANAMMOX granules progres-

sively increased with the passage of time. Amazingly, high

values of 5.6 � 0.9 kg-N kg-VSS�1 day�1 were detected for the

carmine granules when the NRR was higher than 70 kg-N m�3

day�1. The high activity was an important factor leading to the

super high NRR of the UASB reactors.

3.1.5. Sludge washoutThe nitrogen gas production rate progressively increased

along the development of nitrogen removal performance,

resulting in the increased superficial gas upflow velocity. The

nitrogen gas production rates for R1 and R2 were 52.42 � 7.42

and 52.68 � 5.24 L L�1 day�1 with the accompanied gas upflow

velocity up to 1.11 and 1.17 m h�1, respectively, when super-

ficial liquid upflow velocities were gradually increased to 5.78

and 5.24 m h�1, respectively.

Fig. 4 e ECP content of the sludge in R1 (A) and

The high shear force from liquid upflow and gas upflow in

both reactors led to the severe sludge washout (Figs. 1 and 3).

The effluent VSS as high as 8.3 g day�1 (R1) and 7.7 g day�1 (R2)

were observed during days 433e435. Consequently, the NRR

declined (Fig. 1).

3.2. Characteristics of ANAMMOX granules

3.2.1. Diameter distribution and settling propertyGranulation of ANAMMOX microorganisms resulted in gran-

ular diameters of 1.0e6.4 mm in both reactors. The average

granule diameter was 2.5 mm (R1) and 2.4 mm (R2). The

percentage of granules with diameter larger than 2 mm

ranged 68%e71%.

The granules in both reactors possessed a high settling

velocity (73e88mh�1, Table 2). The SVI5 range decreased from

42e51 mL g-VSS�1 to 24e25 mL g-VSS�1 (Table 2) with

a thickening process verified by an SVI5 to SVI30 ratio of 1

suggesting a fabulous sedimentation property. The density of

ANAMMOX granules in both reactors was about 1.03 g mL�1;

and the specific density of the ANAMMOX granules

(91e120 g-VSS L-granules�1) was also comparable to aerobic

granules (40e70 g-VSS L-granules�1, Beun et al., 2002) and

high-loaded denitrifying granules (128e136 g-VSS L-gran-

ules�1, Franco et al., 2006). The formation of well settling

granules resulted in accumulation of high concentrations of

sludge in both reactors in spite of working at extremely high

NLRs and short HRTs. It was another factor contributing to the

super high volumetric nitrogen removal rates.

3.2.2. Extracellular polymersThe microbial ECPs are a rich matrix of polymers, mainly

including polysaccharides and proteins (Liu et al., 2009). They

are supposed to play a central role in the formation of granules

in bioreactors (Liu and Tay, 2002; Hulshoff Pol et al., 2004; Liu

et al., 2009). In the present study, the ECP content determined

at differentnitrogen removal levels is presented in Fig. 4. Itwas

evident that both the polysaccharide and protein contents

increased with the increasing NRR. It was obvious that the

polysaccharides increased slowly as compared to the proteins.

The polysaccharide contents (mg g-VSS�1) for R1 and R2 were

71.8 � 2.3 (69.6e74.2) and 112.1 � 2.8 (109.8e115.2) at NRR

higher than 70 kg-N m�3 day�1; whereby the extracellular

protein contents (mg g-VSS�1) increased sharply to 164.4 � 9.3

R2 (B) at different nitrogen removal rates.

Table 3 e ECP content in different microbial granules.

Granular sludge Extracellular polymers (mg g-VSS�1) HRT (h) Reference

Proteins Polysaccharides PN/PS

ANAMMOX granules 164.4 � 9.3 71.8 � 2.3 2.29 0.16 This paper

Aerobic granules z40 z16 2.5 8 Zheng and Yu (2007)

Phenol-degrading granules 240 � 13 61.0 � 9.4 3.93 N.A. Adav et al. (2008)

Anaerobic granules 42.7 � 37.8 17.3 � 6.8 2.5 N.A. Wu et al. (2009)

Hydrogen-producing granules 70.9 � 4.5 115.6 � 5.2 0.6 18 Mu and Yu (2006)

Nitrifying granules 56 � 25a 18 � 1a 3.1 2.8 Martınez et al. (2004)

Denitrifying granules N.A. N.A. 2.2 N.A. Franco et al. (2006)

a mg L�1.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4 141

(153.7e170.1) (R1) and 298.2 � 8.7 (288.1e303.7) (R2), respec-

tively (Fig. 4). It was amazing to observe that the autotrophic

ANAMMOX granules possessed a high ECP content as

compared to the heterotrophic granules (Table 3). It was

previously reported that the autotrophic bacteria unable to

utilize organic compounds would secrete low ECP content

(Tsuneda et al., 2003). ECPs couldphysically bridgeneighboring

cells to each other by altering the negative charges on bacterial

surface (Liu et al., 2004). Thus, granulationmaybe facilitatedby

large secretion of ECP. As evident from Fig. 3, complete gran-

ulation of the ANAMMOX biomass occurred in both reactors

with theaveragegranulardiameterabove2mmwhenNRRwas

around 70 kg-N m�3 day�1.

The proteins to polysaccharides ratio (PN/PS) was usually

used to evaluate the granular settleability and strength

(QuarmbyandForster, 1995;Cuervo-Lopezetal., 1999;Batstone

and Keller, 2001; Martınez et al., 2004; Franco et al., 2006; Wu

et al., 2009). The PN/PS ratio of the ANAMMOX granules was

also lowwhen compared to othermicrobial granules (Table 3),

suggesting a greater granular stability (Franco et al., 2006).

Nevertheless, PN/PS ratios increased during the reactor oper-

ation when hydrodynamic shear force increased (Fig. 4). As

pointed out by various researchers, the higher PN/PS ratio of

microbial granules indicated lower strength and weaker set-

tleability (Quarmby and Forster, 1995; Cuervo-Lopez et al.,

1999; Batstone and Keller, 2001; Martınez et al., 2004); thus,

the sludge floating or foaming would be easily accompanied

(Franco et al., 2006; Wu et al., 2009). In this study, we deter-

mined the ECP contents of the floated granules.We found that

extracellular proteins and PN/PS ratios of the floated ANAM-

MOX granules at highNRRs andHLRswere significantly higher

than the counterparts of well-settled ones (Table 4).

Wu et al. (2009) reported that the secretion of extracellular

protein by heterotrophic anaerobic granules was stimulated

under high hydrodynamic shear force in the internal circula-

tion anaerobic reactor. On the contrary, Tay et al. (2001)

Table 4 e ECP content of the settled ANAMMOX granules and t

NRR (kg-N m�3 day�1) Sludge Polysacchari

44.87 � 2.39 Settled granules 76

Floated granules 79

76.68 � 4.46 Settled granules 112

Floated granules 134

pointed out that the hydrodynamic shear force stimulated

the production of extracellular polysaccharides in heterotro-

phic aerobic reactors. In the present study, the secretion of

extracellular proteins and polysaccharides were enhanced

when hydrodynamic shear force was increased. But extra-

cellular proteins were produced at a higher rate, leading to the

increased PN/PS ratio. Moreover, the over-production of

extracellular proteins can raise fluid viscosity in the reactor;

as a consequence, the shear force in the reactor is intensified

in turn based on Newton’s law (Wu et al., 2009). This would

increase the risk of sludge disruption due to increasing shear

force. So, in the case of violent shear conditions, the disrup-

tion of aggregates and sludge washout becomes inevitable

(Wu et al., 2009). The over-production of extracellular proteins

might be a potential cause resulting in the severe sludge

washout from the UASB reactors.

3.2.3. Heme c contentThemorphology of microbial granules is affected by a number

of factors such as seed sludge characteristics, substrate

composition, loading rate, feeding strategy, reactor design,

and hydrodynamic shear force (Liu et al., 2009). The color of

aerobic granules, nitrifying granules, denitrifying granules

and hydrogen-producing granules is usually yellow, while the

color of methanogenic granules is black because of the

precipitation ofmetal sulfides (Hulshoff Pol et al., 2004; Franco

et al., 2006; Liu et al., 2009). The color of high-load ANAMMOX

granules is uniquely carmine (Fig. 3). Heme c, which is an

important cofactor of some ANAMMOX bacterial enzymes,

was presumed to play a key role to attribute the carmine color

of ANAMMOX sludge. The present study showed that the

heme c content significantly increased with the increasing

NRR. The heme c content was 0.7e1.4 mmol g-VSS�1 when NRR

was lower than 10 kg-N m�3 day�1; and finally reached

6.8 � 0.9 (5.9e7.6) mmol g-VSS�1 (R1) and 10.3 � 0.6 (9.7e10.8)

mmol g-VSS�1 (R2) at NRR higher than 70 kg-N m�3 day�1. The

he floated granules.

des (mg g-VSS�1) Proteins (mg g-VSS�1) PN/PS

.77 � 2.29 184.39 � 9.27 2.40

.98 � 3.54 327.48 � 35.25 4.09

.14 � 2.77 298.19 � 8.72 2.65

.23 � 4.34 520.78 � 204.75 3.88

Fig. 5 e Transmission electron micrographs of the sludges taken from R1 (A and B) and from R2 (C and D) after the end of the

experiment. The innermost compartment, the anammoxosome (A), filled with material of moderate electron density and

granular texture, but devoid of ribosome-like particles, is surrounded by a single membrane. The paryphoplasm (P), in this

case relatively electron-transparent, surrounds the rim of the cell. The scale bar in A and C [ 2 mm; in B and D [ 0.2 mm.

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4142

increase of heme c content was related with the increase of

ANAMMOX bacterial numbers, resulting in high SAA.

As evident from Fig. 3, the number of red ANAMMOX

granules in both reactors increased along the reactor opera-

tion period, which was in accordance with the increase of

heme c content. On the contrary, the amount of initial seed

sludge (grey particles) decreased significantly. The carmine

granules dominated in the R2 after 400 days’ operation

(Fig. 3B). However, several black zones appeared in R1 after

about 310 days’ operation (Fig. 3A). To our visual observation,

higher concentration of granular sludge was held in R1 and

the black zones may have resulted from the sludge blockage.

3.2.4. Morphology of granular sludgeThe structure of the microbial granules developed in both

reactorswasobservedbymeansofSEMandTEM.Thescanning

electron micrographs represented a sample of red-colored

matureANAMMOXgranule characterized by a cauliflower-like

shape (Arrojo et al., 2006). The granular surface mainly con-

sisted of spherical and elliptical bacteria; few or even no bacilli

and filamentous bacteria were observed in the two reactor

enrichments, suggesting that the ANAMMOX bacteria domi-

nated after enrichment in both reactors. The shape of domi-

nating cocci in R1 enrichment was like a shrunken ball, while

that in R2 enrichment it was like a gaseous ball, which were

different from each other.

TEMperformedon theenrichedbiomass taken frombottom

of the two UASB reactors revealed that the dominant cells in

both enrichments displayed typical ultrastructural features of

ANAMMOX bacteria; i.e., a single membrane bound anam-

moxsome containing tubule-like structure (Strous, 2000;

Lindsay et al., 2001; Schmid et al., 2003; Kartal et al., 2007)

(Fig. 5B andD).As evident fromFig. 5AandC, bothenrichments

were dominated by ANAMMOX cells. The morphology of the

ANAMMOX cells in the two enrichments showed some differ-

ences of the paryphoplasm (relatively electron-transparent,

as proposed by Lindsay et al. (2001)). It is evident that the

paryphoplasm in R1 cells was larger than that in R2 (Fig. 5).

The structural differences of the two reactor enrichments

observed by SEM and TEM were interesting. As shown, the

seed sludge and the mineral medium used in the study were

the same; and the hydrodynamic conditions as well as the

substrate concentrations in both reactors were also similar.

The major difference was the effluent recycling in R1 while

no recyclingwas done for R2. Thus the recycling of ANAMMOX

products (known or unknown) for R1 may be a cause lea-

ding to the ultrastructural differences of the two reactor

enrichments.

4. Conclusions

A super high-rate performance with nitrogen removal rate of

74.3e76.7 kg-N m�3 day�1 was revealed for the lab-scale

ANAMMOX UASB reactors, which was 3 times of the previ-

ously reported top value. The performance was stable until

the HRT was shortened to 0.16e0.11 h with the hydraulic

loading rate larger than 152.4e221.0 L L�1 day�1. The biomass

concentrations in the reactors were 42.0e57.7 g-VSS L�1 with

the specific ANAMMOX activity up to 5.6 kg-N kg-VSS�1 day�1,

both of which were regarded as the key factors leading to the

super high-rate performance.

The settling velocities of ANAMMOX granules ranged

73e88 m h�1 in both the reactors. The ANAMMOX granules

wat e r r e s e a r c h 4 5 ( 2 0 1 1 ) 1 3 5e1 4 4 143

were found tocontaina largeamountof extracellularpolymers

with the polysaccharides and proteins contents of

71.8e112.1 mg g-VSS�1 and 164.4e298.2 mg g-VSS�1, respec-

tively. Relatively high ECP content and low PN/PS ratios were

attributed to the ANAMMOX granulation. High hemachrome

content of 6.8e10.3 mmol g-VSS�1 were detected in the

ANAMMOX granules, which was an important cofactor of

someANAMMOXenzymesandwassupposedtobe responsible

for the unique carmine color.

Acknowledgements

Financial support of this work by the National High-

tech Research and Development (R&D) Program of China

(2009AA06Z311), the National Key Technologies R&D Program

of China (2008BADC4B05) and the Natural Science Foundation

of China (30770039) is gratefully acknowledged. We wish to

thank the anonymous reviewers and editors for their valuable

suggestions on revising and improving the work.

Appendix. Supplementary material

Supplementary data associated with this article can be found

in the on-line version, at doi:10.1016/j.watres.2010.08.018.

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