10
Development and examination of a granular nitrogen-fixing wastewater treatment system Steven Pratt a, * , Michael Tan a , Daniel Gapes b , Andy Shilton a a Centre for Environmental Technology and Engineering, Massey University, Palmerston North, New Zealand b Scion, Rotorua, New Zealand Received 16 November 2006; received in revised form 12 February 2007; accepted 25 February 2007 Abstract This work presents the first success at aerobic granulation in a nitrogen deficient system. Two sequencing batch reactors (SBRs) were used to treat nitrogen deficient (the N-fix system) or nitrogen-sufficient (containing NH 4 Cl) synthetic wastewater (acetic acid as the sole carbon source). Granulation was observed in both systems, with particularly large granules (average diameter: 7 mm) grown in the N-fix system. We propose that the unique morphology of nitrogen-fixing granules is a consequence of the response of oxygen-sensitive diazotrophs to elevated oxygen concentrations. Both the nitrogen-fixing and nitrogen-supplemented systems were shown to be capable of removing all of the influent substrate carbon. Excellent biomass settleability characteristics were obtained, with the N-fix system having a final sludge volume index (SVI) of less than 100 mL g 1 and its granules having settling velocities of over 1.4 cm s 1 . However, moderately high solids discharges were recorded for both systems, which revealed a potential limitation of granular sludge processes that is not widely discussed in the literature. # 2007 Elsevier Ltd. All rights reserved. Keywords: Nitrogen fixation; Nitrogen deficient; Aerobic granules; Sludge settleability; Sequencing batch reactor 1. Introduction All wastewater treatment processes reliant on biological activity require the supply of adequate nitrogen for main- tenance of microbiological population growth. Often, the ratio of nitrogen to carbon in influent waste streams is more than sufficient to support adequate microbial growth. However, some industrial waste streams, such as discharges from pulp and paper mills, have an extremely low ratio of nitrogen to carbon. For biological treatment of these nitrogen deficient waste streams, novel processes that utilise nitrogen-fixing bacteria may be employed [1,2]. The diazotrophic organisms in these systems are able to directly fix nitrogen from the atmosphere, thus satisfying their cellular nitrogen require- ments, while maintaining extremely low nitrogen discharges in the final effluent [3]. Biological nitrogen fixation involves the reduction of atmospheric dinitrogen to ammonia, catalysed by the nitrogenase enzyme. Nitrogenase is an important cellular component of diazotrophs, comprising up to 20% of their total protein [4]. The component proteins of nitrogenase are extremely oxygen sensitive and thus aerobic bacteria have been found to possess varying mechanisms to survive oxygen inhibition via reducing the ambient oxygen environment with such means as respiratory protection and slime formation, or engaging in enzyme conformational protection [5–7]. Such mechanisms allow two apparently paradoxical processes to occur together within the same cell, namely aerobic respiratory metabolism, coupled with anaerobic nitrogenase activity. Separation of biomass from treated wastewater via gravity settling is a key requirement for full-scale implementation of activated sludge-type wastewater treatment systems. The suspended floc processes that are employed in activated sludge systems are known to be susceptible to bulking problems under conditions of nitrogen deficiency, attributable to proliferation of filamentous organisms and overproduction of extracellular polymeric substances [8]. While nitrogen-fixing (defined here as N-fix) activated sludge systems have been demonstrated to be highly effective in reducing carbon concentrations, biomass settleability has been identified as a potential problem [3]. www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 863–872 * Corresponding author. Tel.: +64 6 350 5085; fax: +64 6 350 3604. E-mail address: [email protected] (S. Pratt). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.02.009

Pratt et al 2007

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www.elsevier.com/locate/procbio

Process Biochemistry 42 (2007) 863–872

Development and examination of a granular nitrogen-fixing

wastewater treatment system

Steven Pratt a,*, Michael Tan a, Daniel Gapes b, Andy Shilton a

a Centre for Environmental Technology and Engineering, Massey University, Palmerston North, New Zealandb Scion, Rotorua, New Zealand

Received 16 November 2006; received in revised form 12 February 2007; accepted 25 February 2007

Abstract

This work presents the first success at aerobic granulation in a nitrogen deficient system. Two sequencing batch reactors (SBRs) were used to

treat nitrogen deficient (the N-fix system) or nitrogen-sufficient (containing NH4Cl) synthetic wastewater (acetic acid as the sole carbon source).

Granulation was observed in both systems, with particularly large granules (average diameter: 7 mm) grown in the N-fix system. We propose that

the unique morphology of nitrogen-fixing granules is a consequence of the response of oxygen-sensitive diazotrophs to elevated oxygen

concentrations.

Both the nitrogen-fixing and nitrogen-supplemented systems were shown to be capable of removing all of the influent substrate carbon.

Excellent biomass settleability characteristics were obtained, with the N-fix system having a final sludge volume index (SVI) of less than

100 mL g�1 and its granules having settling velocities of over 1.4 cm s�1. However, moderately high solids discharges were recorded for both

systems, which revealed a potential limitation of granular sludge processes that is not widely discussed in the literature.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Nitrogen fixation; Nitrogen deficient; Aerobic granules; Sludge settleability; Sequencing batch reactor

1. Introduction

All wastewater treatment processes reliant on biological

activity require the supply of adequate nitrogen for main-

tenance of microbiological population growth. Often, the ratio

of nitrogen to carbon in influent waste streams is more than

sufficient to support adequate microbial growth. However,

some industrial waste streams, such as discharges from pulp

and paper mills, have an extremely low ratio of nitrogen to

carbon. For biological treatment of these nitrogen deficient

waste streams, novel processes that utilise nitrogen-fixing

bacteria may be employed [1,2]. The diazotrophic organisms in

these systems are able to directly fix nitrogen from the

atmosphere, thus satisfying their cellular nitrogen require-

ments, while maintaining extremely low nitrogen discharges in

the final effluent [3].

Biological nitrogen fixation involves the reduction of

atmospheric dinitrogen to ammonia, catalysed by the nitrogenase

* Corresponding author. Tel.: +64 6 350 5085; fax: +64 6 350 3604.

E-mail address: [email protected] (S. Pratt).

1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2007.02.009

enzyme. Nitrogenase is an important cellular component of

diazotrophs, comprising up to 20% of their total protein [4]. The

component proteins of nitrogenase are extremely oxygen

sensitive and thus aerobic bacteria have been found to possess

varying mechanisms to survive oxygen inhibition via reducing

the ambient oxygen environment with such means as respiratory

protection and slime formation, or engaging in enzyme

conformational protection [5–7]. Such mechanisms allow two

apparently paradoxical processes to occur together within the

same cell, namely aerobic respiratory metabolism, coupled with

anaerobic nitrogenase activity.

Separation of biomass from treated wastewater via gravity

settling is a key requirement for full-scale implementation of

activated sludge-type wastewater treatment systems. The

suspended floc processes that are employed in activated sludge

systems are known to be susceptible to bulking problems under

conditions of nitrogen deficiency, attributable to proliferation of

filamentous organisms and overproduction of extracellular

polymeric substances [8]. While nitrogen-fixing (defined here

as N-fix) activated sludge systems have been demonstrated to

be highly effective in reducing carbon concentrations, biomass

settleability has been identified as a potential problem [3].

Page 2: Pratt et al 2007

Table 1

Summary of operating conditions

N-fix N-supplemented

Volume (L) 4.3 4.3

Diameter (cm) 8.0 8.0

HRT (d) 0.5 0.5

Settling period (min) 1.0 1.0

Cycle length (h) 6.0 6.0

Fill time ratio (non-aerated) 0.083 0.083

Decant time ratio 0.042 0.042

Volumetric exchange rate 50% 50%

Temperature (controlled) (8C) 30 30

Organic load (mg TOC L�1) �500 �500

Nitrogen supplementation (mg NH4–N L�1) 0 55

COD:N >100:0.1 100:3.5

Superficial upflow air velocity (cm s�1)a 0.5 0.5

DO (mg L�1)b 5.9–7.8 5.0–7.8

a Superficial upflow air velocity was determined by dividing the volumetric

air flow rate (measured by a water displacement method) by the reactor cross

sectional area.b DO was not controlled. DO and temperature were measured and recorded

throughout the study.

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872864

Manipulation of system configuration can lead to an

improvement in floc structure in such nitrogen-deficient waste-

water treatment. Dennis et al. [3] compared an N-fix system run

under sequencing batch reactor (SBR) configuration with an N-

fix system run as a conventional continuous flow activated sludge

(CF-AS) reactor. While it was shown that biomass settleability in

the SBR was better than that in the CF-AS, biomass settleability

remained non-ideal, with both reactors having sludge volume

indices (SVIs) of at least 200 mL g�1.

An alternative approach for ensuring effective separation of

solids from effluent is to immobilize the biomass in defined,

self-forming entities known as microbial granules [9–12].

Granules typically have a diameter range of between 0.2 and

5 mm, and display high density, strong microbial structure and

(importantly) good settleability when compared with conven-

tional flocculent biomass [13]. The operational consequence of

good biomass settleability is the opportunity to apply relatively

high hydraulic loads without concerns regarding biomass losses

[13].

Hailei et al. [14] and Wang et al. [15] concluded that

granules should not be expected to form in a nutrient deficient

environment, due to over-proliferation of filamentous micro-

organisms. The current paper presents a challenge to this

negative paradigm, with the first successful investigation of

aerobic granulation in a nitrogen deficient system. The

experimental focus of the work was on describing:

� tr

eatment performance characteristics of the aerobic N-fix

system;

� s

Table 2

Feed composition

Compound Concentration (mg L�1)

NaCH3COO 2055.0

MgSO4�7H2O 30.0

CaCl2�2H2O 15.0

NaHCO3 6.9

KH2PO4 35.1

Nitrilotriacetic acid 15.0

NH4Cl [N-supplemented only] 213.8

FeSO4�7H2O 0.4005

H3BO3 0.4650

CoSO4�7H2O 0.1920

CuSO4�5H2O 0.0165

MnCl2�4H2O 0.0144

Na2MoO4�2H2O 0.1950

ludge quality of the N-fix system, quantified in terms of

granule characteristics and sludge settleability.

In an aerobic environment granulation can be encouraged by

maintaining adequate turbulence through aeration [11]. Due to

the oxygen sensitivity of the nitrogenase activity as described

above, moderate aeration could be perceived as being

incompatible with effective nitrogen fixation activity. However,

it is important to recognize that high levels of aeration do not

necessarily preclude the existence of low oxygen environments.

Ivanov et al. [16] and Tay et al. [18] have shown that the

complex spatial structure in aerobic granules enables the

coexistence of aerobic and anaerobic bacteria. In this work, the

potential for aerobic granules to facilitate the oxygen-sensitive

process of nitrogen fixation is discussed.

2. Materials and methods

2.1. Reactor operation and seed sludge

Two sequencing batch reactors were used for the study: one operated with

severe nitrogen limitation (the nitrogen-fixing or N-fix system) to which sodium

acetate was fed as the sole carbon source; the other operated as a control (the

nitrogen-supplemented system) to which acetate and ammonia sufficient for

balanced growth (COD:N of 100:3.5) was fed. The organic load to both systems

was representative of a moderate strength pulp and paper mill effluent [2]. The

temperature of the reactors was controlled at 30 8C, elevated above that used in

most granular work, but representative of conditions in industrial effluents

emanating from processes such as pulp and paper mill operations. A short

settling period was employed in order to select for denser, faster settling

particles [17]. The operating conditions are outlined in Table 1 and the

wastewater compositions are outlined in Table 2.

Both systems were seeded from the same mixed liquor, so the only

difference between the N-fix system and the control was the nitrogen content

of the feed. The seed mixed liquor was sourced from two non-granular SBR

reactors, one of which was running under nitrogen-sufficient conditions and the

other, to ensure a capacity for nitrogen fixation, under nitrogen-deficient

conditions. The two biomass samples were mixed together, homogenized to

remove any preexisting structures, and inoculated to an initial concentration of

1.8 g L�1 within each reactor.

The period of study was 75 days; less time than is typically allowed for

granular systems to reach true steady-state in terms of mixed liquor concentra-

tion, but considered sufficient for defining the characteristics of the novel

nitrogen-fixing granular sludge system and allowing distinction between its

performance and that of a conventional nitrogen-supplemented granular system.

2.2. Image analysis

A Nikon CoolPix990 digital camera with 3� Zoom and 3.34 Mega Pixel

resolution attached to a Leica MZ125 stereomicroscope was used for micro-

scopy. Digital camera pictures were acquired for granule characterization using

a SONY DSC-F707 digital still camera with 10� Zoom and 5.0 Mega Pixel

Page 3: Pratt et al 2007

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872 865

resolution (entities smaller than 200 mm [equivalent to 4 pixels] were not

captured). The pictures taken were edited using ImageJ V1.33 [19], with the

‘analyse particle’ function being used to determine the following parameters:

� A

rea: calculated based on the number of square pixels within an object.

� P

erimeter: the length of the outside boundary of an object.

� E

quivalent diameter: the diameter of a circle with the same area as that

of the object.

� C

ircularity: [4p � (area/perimeter2)]: for a circle this identity has a value

of 1, while as an object becomes increasingly elongated this value

approaches 0.

2.3. Analytical methods

The carbon concentration was measured using a total organic carbon

analyzer (highTOC II, Elementar Analysensysteme GmbH, Germany). The

soluble organic carbon content was used to track system performance.

Samples of the feed and effluent from the reactors were externally analysed

to determine phosphorus and nitrogen content (as per Method 4500 outlined in

APHA Standard Methods [20]). The phosphorus data was used to confirm that

the systems were not phosphorus limited. For nitrogen the analyses were for

nitrates and nitrites, ammonia and dissolved and total Kjeldahl nitrogen, and

these data were used to track nitrogen transformations in the reactors.

The suspended solids in the mixed liquor and supernatant were determined

using two methods: (i) a dry-weight method similar to Method 2540D outlined

in APHA Standard Methods [20], and in order to expand the data set (ii) by

relating the concentration of non-soluble organic carbon to the solids content by

assuming that mixed liquor organic carbon content in nitrogen-supplemented

and nitrogen fixed systems is 45% and 39% respectively. Suspended solids were

subsequently reported as the total suspended solids (TSS).

Biological nitrogen fixation was confirmed on day 75 via the acetylene

reduction assay, as per the procedure presented in Sprent and Sprent [21] and

used by Dennis et al. [3]. An ethylene peak 10� greater than that of the blank

was taken as indicative of nitrogenase activity.

Terminal restriction fragment length polymorphism (T-RFLP) was used to

provide a comparison of the microbial diversity within the samples. The

procedure (following Hiraishi et al. [22]) involves PCR amplification and

fluorescent labelling of the environmental 16S rRNA genes, digestion of the

PCR product with restriction enzymes, and T-RF separation by automated

electrophoresis. The restriction fragment profiles then provide a basis for

comparison of the diversity in the measured microbial population.

2.4. Settling velocity and specific gravity

The settling velocity was determined by placing individual granules in a

column of feed medium and recording the time taken for them to fall 15 cm. The

specific gravity of the granules was measured by placing the granules in column

of water and then titrating with a sucrose solution until buoyancy was observed.

A total of 15 granules (selected from the sample that was used for image

analysis) from each system were analysed.

2.5. Calculation of sludge volume index (SVI)

The sludge volume index was determined in situ by recording the volume of

the sludge blanket as a fraction of the total volume after a 15 min settling period

and then dividing the result by the mixed liquor suspended solids concentration.

2.6. Modelling substrate penetration and biomass growth rates in

granules

Numerical integration of a generic equation for substrate diffusion and

consumption (Eq. (1)) was used to predict substrate (Si [g m�3]) profiles in

granules of various radii (R [m]). The substrates of interest were acetic acid and

oxygen; nitrogen was assumed to be non-limiting as (a) the diffusion of nitrogen

is relatively high (the nitrogen content of the aeration gas is over three times

greater than the oxygen content, and the diffusion coefficient for nitrogen in

water is higher than the coefficients for both oxygen and acetic acid) and (b) the

consumption of nitrogen is relatively low (the nitrogen requirement for growth

is over four times less than the carbon requirement). The granules were assumed

to be spherical and made up of a number of shells; external mass transfer

limitations were not considered. A series of steady-state mass balances over the

shells revealed the concentrations of acetic acid (SAc) and oxygen ðSO2Þ

throughout the granules:

Di

�d2Si

dr2þ 2

r

dSi

dr

�¼ qi max

Si

KSi þ SiX (1)

where r is the distance from the centre of the granule to the shell. The maximum

acetic acid consumption rate (qAc max) of 5.9 � 10�5 gAc gVSS�1 s�1, effective

diffusion coefficient (DAc) of 4.4 � 10�10 m2 s�1 and half-saturation constant

(KSAc) of 26 g m�3 were selected from data presented in Wu and Hickey [23].

The maximum oxygen consumption rate ðqO2 ;maxÞ was calculated from the

stoichiometry of carbon oxidation. The effective diffusion coefficient ðDO2Þ of

1.58 � 10�9 m2 s�1, half-saturation constant ðKSO2Þ of 1.9 g m�3 and biomass

concentration (X) of 4750 gVSS m�3 were selected from data presented in Su

and Yu [24].

For nitrogen-supplemented carbon conversion, the growth yield (Y) can be

assumed to be 0.47 gVSS gO2�1 [25]. However, for diazotrophs the growth yield

has been shown to vary as a function of dissolved oxygen (DO). At elevated DO

diazotrophs exhibit extremely high maintenance rates, which result in signifi-

cant yield reductions [26,27]. For example, the biomass yield of Azotobacter

vinelandii, a representative diazotroph, is severely reduced at elevated oxygen

concentration [27]. In this work, a relationship for the yield of diazotrophs

(Eq. (2)) was developed from data presented in Kuhla and Oelze [27].

nitrogen fixing system : Y

�gVSS

gO2

�¼ 0:47� 0:47� SO2

SO2þ y

(2)

where y is an empirical constant: 0.7 gO2 m�3 (from data presented in Nagai

and Aiba [28]).

The rate of biomass growth (f [gVSS s�1]) through the granules was

calculated as a function of biomass yield and substrate utilisation at various

distances from the centre of the granule (r):

fr ¼ Y � qO2max

SO2

KSO2þ SO2

SAc

KSAcþ SAc

X (3)

For the purpose of comparing the biomass activity between the nitrogen-

supplemented and N-fix systems, the effective growth rates (hg) of granules of

various volume (VS [m3]) were determined:

hg ¼PR

r¼0 rgrð1=VSXÞmmax

(4)

MATLAB (Mathworks Inc.) was used to solve the model presented in (1)

along with the algebraic relationships presented in (2)–(4). The modelling

experiments were designed to represent the system shortly after carbon addi-

tion: bulk acetic acid concentration was assumed to be 500 gAc m�3 and bulk

oxygen concentration was assumed to be 6 gO2 m�3.

3. Results

3.1. Confirmation of nitrogen fixation

As expected, only the N-fix system yielded a positive

response to the acetylene reduction assay (data not shown),

confirming the presence of nitrogen fixation within this reactor.

A nitrogen balance, which consistently showed elevated total

nitrogen in the effluent from the system with the nitrogen

deficient feed, was further evidence of nitrogen fixation in the

N-fix system.

Page 4: Pratt et al 2007

Fig. 1. Digital camera images of (A) the reactor set-up and (B) samples from the N-fix and nitrogen-supplemented reactors.

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872866

3.2. General observations

The physical characteristics of the granules in the N-fix

system were markedly different to those in the nitrogen-

supplemented system (Fig. 1), with the former producing very

large granular structures, up to approximately 10 mm in

diameter. Clearly, the severe nitrogen deficiency in the feed,

and subsequent modification of the bacterial microbiota to

Fig. 2. Population diversity in reactors based on terminal restriction fragments, norm

total (A) N-fix system and (B) nitrogen-supplemented system.

allow metabolism of atmospheric nitrogen, resulted in a

different granule morphology.

The differences in measured microbial community between

the two reactors are highlighted by the T-RFLP profiles (Fig. 2).

The T-RFLP profile for the nitrogen-supplemented system

indicates the dominance of one microbial group in that system,

while the more varied array of terminal fragments in the

measured microbial population associated with the nitrogen

alised to the sum of fragments present at abundances greater than 0.5% of the

Page 5: Pratt et al 2007

Fig. 3. Organic carbon removal (^ = N-fix system and � = nitrogen-supple-

mented system).Fig. 5. Nitrogen in effluent (^ = N-fix system and� = nitrogen-supplemented

system) (solid line shows TKN; dotted line shows dissolved KN).

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872 867

deficient system implies a greater diversity in microbial

population in that system. The T-RFLP profiles also show that

the communities in the two systems did not change markedly

with time, while the observation of equal-length fragments in

the two reactors does indicate the presence of some similar

bacterial populations capable of proliferating under the two

nutrient environments.

3.3. Reactor performance

As shown in Fig. 3 both reactors demonstrated excellent

treatment performance, reducing the incoming organic carbon

from 500 mg L�1 to less than 25 mg L�1.

The solids concentrations within both reactors showed some

significant fluctuations (Fig. 4A), but despite the short-settling

times, even in the early development stages critical loss of

biomass was not observed. The extent of fluctuation was most

pronounced in the N-fix system, in which the biomass

concentration dropped during the early development stages

(days 7–20) and then significantly increased during the later

development stages (post day 20). Although the amount of

solids within the nitrogen-supplemented reactor was at some

stages higher than that within the N-fix system, at the end of the

study there was approximately twice as much biomass within

the N-fix system.

The concentration of suspended solids in the effluent is

shown in Fig. 4B. It can be seen that this discharge from both

systems was relatively stable throughout the study. The solids

concentration in the effluent from the N-fix system was

approximately half of that observed in the effluent of the

nitrogen-supplemented reactor. This reduced washout from the

Fig. 4. Solids concentration in (A) the mixed liquor and (B) the efflu

N-fix system contributed to this system’s relatively higher

solids concentration.

Fig. 5A shows that during the later development stages (post

day 20) the dissolved nitrogen content in the effluent of the N-

fix system (average 5 mg L�1) was consistently lower than that

in the nitrogen-supplemented system (average 11 mg L�1).

During these stages the TKN of the effluent of both systems was

high (averaging 27 mg L�1 for the N-fix system and 45 mg L�1

for the nitrogen-supplemented system), directly attributed to

the presence of biomass (as shown in Fig. 4B). The TKN in the

effluent of the nitrogen-supplemented system was only

marginally different to the feed nitrogen concentration

(56 mg L�1). However, the TKN of the effluent from the N-

fix system was considerably higher that the feed concentration

(1 mg L�1), providing further confirmation of nitrogen fixation

in the N-fix system. Negligible oxidized nitrogen (NOx) was

observed in either system with the maximum observed NOx

being 0.04 mgN L�1.

3.4. Granule morphology

Stereomicroscope images (Fig. 6) of the individual entities

show that the granules in the N-fix system were spherical with

small fibrous surface features. The granules in the nitrogen-

supplemented system appeared less regular in shape, and were

devoid of any obvious surface features.

The granule characteristics are summarized in Table 3, along

with data from a review of biogranulation [13] and from a study

of granulation for the treatment of industrial wastes [29]. The

image analysis confirms the significant difference in granule

ent (^ = N-fix system and � = nitrogen-supplemented system).

Page 6: Pratt et al 2007

Fig. 6. Stereomicroscope images of individual granules in (A) N-fix system and (B) nitrogen-supplemented system (day 75).

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872868

size between the two systems. The average equivalent diameter

of the granules in the nitrogen-supplemented system was 2 mm,

similar to the dimensions typically reported in the literature.

However, the average equivalent diameter of the granules in the

N-fix system (7 mm) was significantly higher than those

reported in the literature.

Interestingly, the results of the image analysis suggest that

the granules in the nitrogen-supplemented reactor are actually

more circular than those found in the nitrogen-fixing system.

This contradicts the visual observations made based on the

stereomicroscope images. The discrepancy was possibly

caused by the fibrous surface features of the granules in the

N-fix system, which, when processed, resulted in a reduced

area-to-perimeter ratio and consequently a reduced circularity.

The particle size distributions were also determined, using

both a ‘number’ and ‘equivalent volume’ basis. Fig. 7 shows

that, on a number basis, the majority of particles in both systems

were small (88%<1 mm diameter in the N-fix system and 70%

<1 mm diameter in the nitrogen-supplemented system), whilst

most of the material (volumetric basis) was associated with the

large distinct granules (over 90% of the volume of material in

both systems was associated with granules with >1 mm

diameter). The major difference between the two systems was

that the size distribution of the N-fix system was wider than that

of the nitrogen-supplemented system.

Table 3

Granular sludge characteristics (standard deviation in brackets)

Day N-fix Nitrogen-sup

Individual granules

61

Average diameter (mm) 7 (1.1) 2 (0.3)

Area (mm2) 39 (12) 3 (1)

Circularity 0.21 (0.8) 0.61 (0.12

Aspect ratio – –

75

Settling velocity (cm s�1) 1.4 (0.2) 0.9 (0.2)

Specific gravity 1.002 (1) 1.019 (10

Sludge

75

SVI (mL g�1) 55 (15) 106 (43)

a Review paper.

3.5. Settleability

A feature of granular systems is the efficiency with which

sludge can be separated from the treated liquid effluent. This is

an important aspect of sludge quality, and can be quantified in

terms of the sludge volume index (SVI), e.g. [30]. With a SVI of

less than 100 mL g�1 being considered desirable [31], Fig. 8

shows that this aspect of sludge quality of both the systems was

good. Importantly, during the later stages of the study (post day

20) the average SVI (55 mL g�1) of the N-fix system was

significantly lower than that of flocculent N-fix systems, which

have been reported as displaying SVIs of greater than

200 mL g�1 [3].

As well as a low SVI, the nitrogen-fixing granular sludge had

granules that exhibited very high settling velocities (average

1.4 cm s�1); higher than those from the nitrogen-supplemented

reactor (average 0.88 cm s�1, a similar level to velocities

reported by Su and Yu [29]) and much higher than that of

activated sludge flocs (0.17–0.42 cm s�1, Li and Yuan [33]).

The specific gravity of the N-fix system was found to be

lower than that of the nitrogen-supplemented system, with both

values being within the range observed from other work

(Table 3). From Stokes law, both the density and diameter

impact on the settling velocity, and clearly the elevated size of

the N-fix granules compensated for the lowered density levels.

plemented Liu and Tay (2004)a Su and Yu (2005)

0.6–1.3 0.9–1.1

– –

) – �0.55–0.63

0.73–0.79 0.74

Up to 0.8–1.9 1.02 � 0.24

) 1.004–1.065 1.017

30.8 � 5.3

Page 7: Pratt et al 2007

Fig. 7. Cumulative particle size distribution for (A) the N-fix and (B) the nitrogen-supplemented systems (� = number of entities and & = equivalent volume of

entities).

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872 869

3.6. Substrate penetration and biomass growth rates in

granules

The simulations shown in Fig. 9 provide descriptions of

acetic acid and oxygen penetration and biomass growth rates in

granules. For the representative conditions tested (see Wu and

Hickey [23] and Su and Yu [24] for details) the simulations

showed that substrate penetration would be very similar in both

systems (penetration for the nitrogen-supplemented system is

shown). Acetic acid can be expected to fully penetrate all

granules and oxygen can be expected to penetrate to the centre

of granules with radii of less than 1.0 mm. For larger granules,

significant environments with low/no dissolved oxygen are

predicted. So, for large-size aerobic granules, acetic acid

(organic substrate) is not a limiting factor, rather the whole

microbial process would be dominated by the availability of

DO [32].

Fig. 9 shows the effective biomass growth in granules

predicted in the nitrogen-fixing (Fig. 9C) and nitrogen-

supplemented (Fig. 9D) systems. The predictions are derived

by considering microbial kinetics in granules of various sizes. It

can be seen that growth in a nitrogen-supplemented system is

greatest in small granules as the substrates (acetic acid and

Fig. 8. SVIs of the aerobic granular systems (^ = N-fix system and � = nitro-

n-supplemented system) [3pt median average included].

oxygen) are most readily available. However, the simulations

indicate that for a nitrogen-fixing system, effective biomass

growth is actually retarded in small granules, as the presence of

oxygen affects the growth yield.

4. Discussion

This work has demonstrated for the first time an effective

aerobic granular sludge system for the treatment of severely

nitrogen-limited influent. The system described in this work

functioned as a result of the establishment of a stable population

of nitrogen-fixing microorganisms, capable of abstracting their

nitrogen requirement from atmospheric dinitrogen. Dennis

et al. [3] summarized that the significant advantages of

nitrogen-fixing systems are:

(i) s

elf-regulation of nitrogen requirements, allowing for

substantially less operator intervention and monitoring, and

(ii) im

proved environmental performance, as N2-fixation

eliminates the potential for excess supplementation and

consequent discharge of nitrogen with the effluent.

This paper demonstrates that these advantages are also

relevant for granular N-fix systems. Indeed, as shown in

Fig. 5A, a granular N-fix system can result in discharges of

dissolved nitrogen approximately half of those released from a

nitrogen-supplemented granular system loaded at conventional

COD to nitrogen ratios.

With regard to carbon removal, the granular N-fix system

performed extremely well. Fig. 3 confirms that the granular N-

fix system is suitable for effective oxidation of carbonaceous

inputs, displaying comparable removal with that of the

conventional granular system having supplemental nitrogen

addition, and with acetate-based granular systems reported in

the literature [12].

Importantly, the performance of the granular N-fix system

did not appear to be compromised by the high dissolved oxygen

concentration (ranging from 5.5 to 7.5 mg L�1), achieved with

Page 8: Pratt et al 2007

Fig. 9. Simulation of substrate penetration (nitrogen-supplemented system and effective biomass growth (hg) for 10 granules with various radii (R). (A) Acetic acid

penetration; (B) oxygen penetration; (C) hg: N-fix system; (D) hg: nitrogen-supplemented system.

S. Pratt et al. / Process Biochemistry 42 (2007) 863–872870

moderate superficial upflow air velocity (0.5 cm s�1). This is a

significant finding as it demonstrates that oxygen-sensitive

processes like nitrogen fixation can proceed in well aerated

granular systems. The morphology of the granules themselves

could have contributed to the effective functioning of the

system, as mass transfer resistances alter the environment

within granules from that measured in the bulk liquid. Fig. 2

shows that both systems supported a diverse microbial

community, likely a result of the varied environment within

the granules [16] and the anticipated ubiquity of microorgan-

isms capable of acetate degradation. Using microelectrode

studies, Wilen et al. [34] showed the rapid depletion of oxygen

and presence of significant fractions of low/zero oxygen

concentrations in granules observed at bulk liquid DO levels

between 2 and 8 mg L�1, and Tay et al. [18] showed that

obligate anaerobes can concentrate at depths of 0.8 mm below

granule surfaces. The predicted oxygen profiles in granules of

various radii are shown in Fig. 9B. Low oxygen, or

microaerophilic, conditions are ideal for aerobic nitrogen

fixation, and so the sizeable granules, as developed in the N-fix

system (7 mm diameter), are predicted to contain a large

volume fraction with the oxygen deficient conditions conducive

to proliferation of nitrogen fixation, even at elevated bulk liquid

oxygen concentrations.

The size, shape and well-defined edge characteristics of the

granules in the nitrogen-supplemented system were compar-

able to results of many other studies on aerobic granules.

However, the morphology of the granules in the nitrogen

deficient system was unique; the granules were particularly

large with tendril surface features. In conditions of elevated

oxygen concentration, as would be found throughout small

granules, diazotrophs have extremely high maintenance

requirements, and consequently exhibit severely reduced net

cellular growth yields [4], thus retarding granule growth. We

propose that the yield sensitivity to oxygen which is displayed

by diazotrophs provides a metabolic selection pressure for

increased granule size, over and above the hydraulic pressure

imposed by short settling times in granular reactors. This is

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S. Pratt et al. / Process Biochemistry 42 (2007) 863–872 871

supported by the results of the simulation studies. These clearly

show that mass transfer limitations are effective at reducing the

dissolved oxygen environment, and this limitation increases

with granule size. While oxygen is available throughout the

granule, effective granule growth (hg) in the N-fix system is

predicted to increase with granule size (Fig. 9C), reflecting net

increase in cellular yields as a result of lowered dissolved

oxygen levels within the granular structure. This lies in stark

contrast to the nitrogen-supplemented system, where hg is

predicted to continuously decrease with granule size, thus

providing no selective pressure for elevated granule size in such

systems.

The particularly large N-fix granules may be further

explained by the contribution of extracellular polymeric

substances (EPS) to granule development. It has been suggested

that large granules (diameter >4 mm) are difficult to maintain

as mass transport limitations restrict the supply of nutrients to

their core, causing starved bacterial cells to consume EPS, a key

binding agent, to sustain growth [35]. It is possible that the

capacity for nitrogen-fixing organisms to produce excessive

EPS as a means for providing protection from oxygen [7] may

have assisted maintenance of the large nitrogen-fixing granules.

Excess EPS production could also explain the tendril surface

features as strands of protein rich EPS adhered to the granule

structure would encourage microbial growth in an otherwise

nitrogen deficient environment. If this were the case, it would

also explain the microbial diversity in the nitrogen deficient

system, as it would offer a mechanism for supplying nitrogen to

a range of environments. The contributions of excess EPS

production, and indeed oxygen availability, to microbial

diversity and to granule size and morphology should be the

focus of future examinations of nitrogen-fixing granules.

The major draw-card of granular treatment systems is the

apparent opportunity for effective solids-liquid separation, which

is critical on two fronts: (i) to ensure adequate biomass retention

and (ii) to ensure an effluent free of polluting particulates. This

work confirms that biomass can be effectively retained in a

granular N-fix SBR system with a short settle phase (Fig. 2A).

The significant difference in solids concentrations between the

N-fix and nitrogen-supplemented systems is related to the

difference in the biomass settleability of the two systems, which

has been reported in terms of the sludge volume index and the

settling velocities of the granules within the systems. The higher

settling velocity of the granules in the N-fix system resulted in

elevated biomass retention within this reactor.

The N-fix granular sludge had a low SVI of just 55 mL g�1,

which can be attributed to the relatively high settling velocities

of the granules (1.4 cm s�1). An SVI of less than 60 mL g�1 is

normally indicative of a readily settleable sludge [31]. It was,

therefore, surprising to find that the effluent suspended solids

from this reactor was, on average, over 150 mg L�1. Such

levels of solids discharge are not unique to granular N-fix

systems; a similar effluent quality from a granular system was

reported by McSwain et al. [36], and a significantly worse

effluent was obtained from the nitrogen-supplemented reactor

reported in this work. The explanation for this contradiction

between low SVI, which is normally taken to indicate sludge

with good settling characteristics, and the poor effluent

suspended solids concentrations, lies in the fact that granular

systems can have a wide particle size distribution compared to

conventional flocculant systems (Fig. 7). While most of the

sludge is bound in large, rapid settling granules, a still

significant percentage of the sludge can be described as ‘fines’

that are washed out with the effluent: 2.8% of material in the N-

fix system (Fig. 7A) and 9.3% in the nitrogen-supplemented

system (Fig. 7B) is smaller than 1 mm in diameter. This

phenomenon raises two important considerations. Firstly,

before these systems can be scaled up to industrial application

the issue of reducing the high effluent suspended solids needs

further research. Secondly, this work highlights that the widely

accepted dogma that a low SVI implies high quality settling

does not necessarily apply to granular systems with short

settling phases.

5. Conclusions

For the first time, a nitrogen-fixing granular activated sludge

has been developed. A sequencing batch reactor using a very

short settling phase and moderate aeration regime was used for

granule development. The nitrogen-fixing granular system has

been demonstrated capable of excellent COD reduction while

maintaining an effluent with low soluble nitrogen. A number of

important features of the system were identified:

1. R

elative to their conventional counterparts, nitrogen-fixing

granules are very large, with filamentous surface features. It

is postulated that oxygen mass transfer limitations in such

large particles contribute to enhancement of the nitrogen-

fixing capability in an otherwise inhibitory oxygen-rich

environment.

2. T

he settling qualities of granular nitrogen-fixing sludge are

excellent (as determined by SVI and particle settling

velocities). While this ensures significant biomass retention,

it does not necessarily lead to low effluent solids

concentrations. This is a potential limitation of granular

systems, and can be explained by the wide particle size

distributions of these systems.

The discrepancy between the sludge quality (as measured by

SVI and single-particle settling velocities) and the effluent

solids concentration highlights a limitation in application of

tools traditionally used to determine sludge quality for

predicting sludge settleability in granular systems with short

settling time phases.

Acknowledgement

We wish to thank Technology New Zealand for supporting

this project (Grant: Technology Industry Fellowship FRIX0303).

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