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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].
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-fixsystem;
� 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
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 thatof the object.
� C
ircularity: [4p � (area/perimeter2)]: for a circle this identity has a valueof 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.
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
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).
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
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 forsubstantially less operator intervention and monitoring, and
(ii) im
proved environmental performance, as N2-fixationeliminates 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
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
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-fixinggranules 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 areexcellent (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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