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Dissolved oxygen as a key parameter to aerobic granule formation

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					  781                                                                            Q IWA Publishing 2008 Water Science & Technology—WST | 58.4 | 2008




Dissolved oxygen as a key parameter to aerobic granule
formation
B. S. McSwain Sturm and R. L. Irvine



ABSTRACT

Much research has asserted that high shear forces are necessary for the formation of aerobic                B. S. McSwain Sturm
                                                                                                            Department of Civil, Environmental & Architectural
granular sludge in Sequencing Batch Reactors (SBRs). In order to distinguish the role of shear and             Engineering,
                                                                                                            University of Kansas,
dissolved oxygen on granule formation, two separate experiments were conducted with three                   Lawrence, KS 66045,
bench-scale SBRs. In the first experiment, an SBR was operated with five sequentially decreasing              USA
                                                                                                            E-mail: bmcswain@ku.edu
superficial upflow gas velocities ranging from 1.2 to 0.4 cm s21. When less than 1 cm s21 shear
                                                                                                            R. L. Irvine
was applied to the reactor, aerobic granules disintegrated into flocs, with corresponding                    SBR Technologies, Inc.,
                                                                                                            Chicago, IL,
increases in SVI and effluent suspended solids. However, the dissolved oxygen also decreased                 USA
from 8 mg L21 to 5 mg L21, affecting the Feast/Famine regime in the SBR and the substrate                   E-mail: Robert.Irvine@sbrtechnologies.com

removal kinetics. A second experiment operated two SBRs with an identical shear force of
1.2 cm s21, but two dissolved oxygen concentrations. Even when supplied a high shear force,
aerobic granules could not form at a dissolved oxygen less than 5 mg L21, with a Static Fill. These
results indicate that the substrate removal kinetics and dissolved oxygen are more significant to
granule formation than shear force.
Key words     | aerobic granules, dissolved oxygen, feast and famine, shear force, SBR




INTRODUCTION

In recent years, the Sequencing Batch Reactor (SBR) has                  loading rate on the formation and structure of granules.
been used to form compact sludge structures, termed                      A high shear force has been shown to be an important
aerobic granules. Granules can be described as a collection              factor in the formation of aerobic granular sludge, and an
of self-immobilized cells into a spherical form. This special            extensive literature review presented the effect of superficial
case of biofilm growth occurs without the addition of carrier             gas velocity in a variety of reactor systems including UASB,
material. Granular sludge have a wide range of beneficial                 fluidized-bed reactors, and aerobic granular reactors (Liu &
properties compared to activated sludge flocs, most notably               Tay 2002).
their strong structure and good settling property. A brief                   Shear caused by upflow aeration is the main detach-
review of both anaerobic and aerobic granulation shows                   ment force in column-type SBRs during the formation of
that a variety of microbial species are able to form granules,           granules. Once granules are formed, interaction between
leading researchers to hypothesize that granulation is not a             granules may also serve as a detachment force. Most papers
function of microbiological groups but of reactor operating              published for aerobic granulation describe the shear force in
conditions.                                                              terms of the superficial upflow gas velocity, which is the
     Research on the necessary factors for aerobic granule               aeration rate applied over the surface area of the reactor.
formation has focused on operational parameters, such as                 Although the superficial upflow gas velocity ignores shear
the effect of settling time, shear force, and volumetric                 force caused by particle interaction, it is a constant and
doi: 10.2166/wst.2008.393
 782   B. S. McSwain Sturm and R. L. Irvine | Dissolved oxygen in aerobic granule formation                                     Water Science & Technology—WST | 58.4 | 2008




easily calculated description of shear force caused by                                    a municipal wastewater treatment plant. All SBRs were fed
aeration.                                                                                 with a synthetic wastewater of glucose and peptone with
    In one study of the impact of shear force on granule                                  nutrients (similar to that used by (Moy et al. 2002)) at a
formation, Beun et al. utilized three different superficial gas                            volumetric loading rate of 2.4 kg COD m23 day21 over 90
velocities in the operation of one column-type SBR. At the                                minutes of FILL for 4 cycles per day (30 min Static Fill,
                                                                  21
highest superficial gas velocity of 4.1 cm s                            , smooth           180 min React, 2 min Settle, 15 min Draw, 13 min Idle).
granules formed. At lower superficial gas velocities of 1.4                                      For the first experiment, granules were formed in an
               21
and 2.0 cm s        , stable granules did not form (Beun et al.                           open SBR (Reactor 1) under a high superficial upflow gas
1999). Tay et al. then studied the effect of aeration rate using                          velocity of 1.2 cm s21. After steady-state operation was
four different column-type SBRs with superficial gas                                       reached and stable granules were formed, the aeration
velocities of 0.3, 1.2, 2.4, and 3.6 cm s21, respectively.                                rate was systematically decreased to test the effect of shear
Granules did not form in the reactor with the lowest                                      force on the maintenance of granular sludge. After each
superficial gas velocity of 0.3 cm s21. The authors also                                   change, the reactor was allowed to stabilize for at least two
showed that SVI decreased as the superficial gas velocity                                  weeks before final measurements were taken. Table 1
increased (Tay et al. 2001).                                                              presents the days of operation and aeration rates used,
    Although these papers suggest that a shear force is                                   together with the corresponding superficial upflow gas
necessary for aerobic granulation to occur, the shear force is                            velocities.
calculated from the aeration rate to the reactor. In these                                      Except for the source of compressed air, the two
experiments, the superficial upflow gas velocity is decreased                               reactors used for the second experiment were operated
by decreasing the aeration rate, which inherently changes                                 identically: both an open SBR (Reactor 2) and a closed
the dissolved oxygen supply and concentration in the                                      SBR           (Reactor       2_lowDO)          had         a   superficial   upflow
                                                                                                                                21
reactors. However, the dissolved oxygen concentration is                                  gas velocity of 1.2 cm s                   . Compressed air was supplied
often times not reported, so it is hard to discern the                                    constantly to Reactor 2 with the DO always near saturation
respective effects of a decreased shear force versus a                                    (8– 8.5 mg L21) during React. Reactor 2_lowDO received a
decreased DO on granule formation.                                                        mixture of recycled air, nitrogen gas, and compressed air,
    The purpose of this study is to distinguish the effect of                             with the DO always controlled to be less than 5 mg L21.
shear force and dissolved oxygen concentration on granule                                       Standard wastewater measurements were taken regu-
structure and formation in SBRs with a Static Fill. To                                    larly according to Standard Methods (Clesceri et al. 1998).
accomplish this objective, two reactor experiments were                                   The development of flocs and granules were observed using
conducted. In the first, one SBR was operated with                                         a stereomicroscope (Leica Wild MPS 46/52), and images
sequentially reduced aeration rates, and the aeration rate                                were taken of mixed liquor. Image analysis was performed
and DO were coupled. In a second experiment, two SBRs                                     to measure the average diameter and aspect ratio of all
were operated with identical aeration rates, but one reactor                              particles greater than 300 mm in diameter. For each sample,
was fed a nitrogen/air mixture to maintain a lower DO than                                300 –500 particles were measured using image analysis.
the SBR receiving air only.
                                                                                          Table 1   |   SBR Aeration rates applied in experiment 1


                                                                                                                         Aeration rate           Superficial upflow gas velocity
METHODS                                                                                   Days of operation              (L hr21)                (cm s21)

                                                                                          114                            275                     1.2
Experiments were performed in two open and one closed
                                                                                          14                             230                     1.0
column-type SBRs (Figure 1). All SBRs were aerated at a
                                                                                          22                             185                     0.8
rate of 275 L h21 unless otherwise noted, with a 50%
                                                                                          38                             140                     0.6
volumetric exchange ratio and 4 L total volume. The
                                                                                          21                               95                    0.4
reactors were inoculated with 4 L of activated sludge from
 783       B. S. McSwain Sturm and R. L. Irvine | Dissolved oxygen in aerobic granule formation                                    Water Science & Technology—WST | 58.4 | 2008




Figure 1   |   (Left) Schematic of reactor 1 and reactor 2 with compressed air supplied to create shear force; (right) schematic of reactor 2_lowDO with compressed air and nitrogen gas
               supplied to maintain a high shear force with a reduced dissolved oxygen concentration.


The aspect ratio (min diameter/max diameter of a given                                          SVI30 measurement (height of sludge bed after 30 minutes)
particle; 0 ¼ line, 1 ¼ circle) reflects the roundness of a                                      does not adequately describe the settling property of
particle, with an aspect ratio of one being a perfect circle.                                   granular sludge, which settles in reactors within a few
                                                                                                minutes. In order for the SVI measurement to reflect
                                                                                                settling velocity as well as sludge bed compactness, the
                                                                                                SVI should be measured after shorter settling times
RESULTS
                                                                                                (Schwarzenbeck et al. 2004). In Figure 3, the SVI is
Experiment 1—different shear forces in one reactor                                              reported after 2 and 30 minutes settling; two minutes

At the initial superficial gas velocity of 1.2 cm s                      21
                                                                             , Reactor 1        was chosen since it is the time of the Settle phase in the

had completely granular sludge with an average MLSS of                                          SBR. Except for the SVI at the lowest aeration rate, there

6.0 ^ 0.9 g L         21
                           , an SVI of 33 ^ 5 mL g          21
                                                                 , an effluent SS of             was no difference between the SVI2 and SVI30 values,

61 ^ 20 mL g21, and an effluent COD of 27 ^ 9 mg L21. As                                         indicating that the maximum settling and compactness
shown in Figure 2, the average aspect ratio of sludge                                           in granular reactors occurs within the first few minutes of
decreased linearly below a superficial gas velocity of                                           Settle. At the lowest aeration rate, the biomass was
0.8 cm s       21
                    . The distribution of flocculent versus granular                             predominantly flocculent, and there was a difference
sludge was analyzed using the total percentage of particles                                     between SVI30 and SVI2.
with aspect ratios less than 0.5 (flocculent) and greater then                                           The overall COD removal efficiency in Reactor 1 did
0.7 (granular). Although the values of 0.5 and 0.7 are                                          not change as the aeration rate was altered and granules
arbitrary, they are useful in categorizing particles into non-                                  disappeared from the reactor. However, the slope of
spherical (,0.5) and spherical (. 0.7) categories. At                                           substrate uptake at the beginning of React decreased as
superficial gas velocities above 1.0 cm s                     21
                                                                  , over 60% of all             the shear force decreased. The COD removal curves during
particles had an aspect ratio greater than 0.7, indicating that                                 one SBR cycle at the highest and lowest aeration rates are
the majority of aggregates were granular. Alternatively, only                                   presented in Figure 4. At the highest superficial upflow gas
36% of aggregates at the lowest aeration rate were spherical.                                   velocity of 1.2 cm s21, the duration of the feast period was
     The sludge volume index (SVI) in Reactor 1 began to                                        shortest (tfeast < 45 min) with the magnitude and concomi-
                                                                 21                             tant duration of the famine period the longest (tfamine < 135
increase at gas velocities below 0.8 cm s                          . Similarly, the
effluent suspended solids (ESS) concentration began to                                           min). At the lowest superficial upflow gas velocity of
increase at gas velocities below 1.0 cm s                         21
                                                                       . The average            0.4 cm s21, the duration of the feast period was longer,
values for each aeration rate are plotted in Figure 3. For the                                  extending over the majority of React, as the COD did not
SVI measurement, it has been suggested that the standard                                        reach background levels until 120 minutes of React.
 784       B. S. McSwain Sturm and R. L. Irvine | Dissolved oxygen in aerobic granule formation                       Water Science & Technology—WST | 58.4 | 2008




                                                                                              extended period of low DO (below 5 mg/L), and the DO in
                                                                                              the SBR never reached saturation.


                                                                                              Experiment 2—same shear forces and different
                                                                                              dissolved oxygen in two reactors

                                                                                              In the first experiment, DO and shear force were coupled
                                                                                              and incrementally reduced. In the second experiment, the
                                                                                              shear force was held constant (1.2 cm s21 superficial upflow
                                                                                              gas velocity) in Reactor 2 and Reactor 2_lowDO, but the
                                                                                              latter had a DO less than 5 mg L21 throughout the React
                                                                                              phase.
                                                                                                  In this experiment, Reactor 2 formed granules in the
                                                                                              first few weeks after the initial biomass washout that is
                                                                                              typically seen with short setting times. This reactor behaved
                                                                                              similarly to Reactor 1 in the first experiment. However, in
                                                                                              Reactor 2_lowDO, the MLSS never reached 1 mg L21, and
                                                                                              only filamentous floc structures were observed in the
                                                                                              reactor. Figure 5 shows two images of sludge formed in
                                                                                              each reactor at steady-state (Day 90 Operation). Due to the
                                                                                              nature of biomass in Reactor 2_lowDO, it was impossible to
                                                                                              perform image analysis. In order to have a representative
                                                                                              sludge sample for image analysis, 300– 500 particles were
                                                                                              measured per day. Since Reactor 2_lowDO had such low
                                                                                              biomass content, a quantitatively meaningful sample could
                                                                                              not be analyzed.
                                                                                                  In terms of COD removal, both reactors had similar
                                                                                              performance, despite the differences in MLSS content and
                                                                                              sludge properties. The influent COD varied between 350 and
                                                                                              500 mg L21 at the beginning of React, and the effluent COD
                                                                                              for both reactors was consistently less than 80 mg L21. The
Figure 2   |   Aspect ratio versus the superficial upflow gas velocity in reactor 1.            observation of such a consistently low effluent COD for
               (Top) below a superficial gas velocity of 0.8 cm/sec, the average aspect        Reactor 2_lowDO was interesting since the reactor had such
               ratio decreases linearly. (middle) The % of total particles with an aspect
               ratio less than 0.5 increases, and (bottom) the % of particles with an         a low mixed liquor sludge concentration. If COD was
               aspect ratio greater than 0.7 decreases.
                                                                                              consistently being removed by the mixed liquor, this should
                                                                                              result in a growth of biomass—which was not observed in the
    To understand why substrate uptake rates changed with                                     mixed liquor. An MLSS measurement represents only the
reduced aeration rates, the DO in the mixed liquor was                                        suspended biomass in the reactor volume, and it omits any
measured during React. With a superficial upflow gas                                            biomass accumulated on the reactor walls. Biofilm wall
velocity of 1.2 cm s21, the DO dropped to 2 mg L21 during                                     growth is generally a significant problem during the first few
the first 20 minutes of React while the majority of                                            weeks of reactor operation, when the biomass has suffered
biodegradable COD was consumed. The DO in the SBR                                             significant washout. For Reactor 2 and other granule
was maintained at saturation for the remainder of React. In                                   reactors, there was very little biofilm growth on the walls of
contrast, the SBR with the lowest shear force had an                                          the reactor at steady-state. However, for Reactor 2_lowDO,
 785       B. S. McSwain Sturm and R. L. Irvine | Dissolved oxygen in aerobic granule formation                                     Water Science & Technology—WST | 58.4 | 2008




Figure 3   |   SVI and effluent SS as a function of superficial upflow gas velocity in reactor 2. Error bars represent a 95% confidence interval.


biofilm wall growth was a problem over the entire operation                                       shear forces function to detach filamentous organisms
period. Wall growth in both SBRs was estimated over a week                                       from the surface of aggregates and form spherical aggregates
using inserted glass slides. For Reactor 2_lowDO, approxi-                                       (Liu & Tay 2002). This would have major implications for
mately 4 g biomass grew attached to the reactor walls per                                        the application of granular reactors at full-scale. If this
week, while Reactor 2 accumulated only 0.25 g biomass per                                        hypothesis were true, airlift reactors would be desired over
week. In the SBR with low DO, biomass grew preferentially                                        bubble-column reactors since they create more localized
attached to the wall, with only filamentous flocs accumulat-                                       shear, and the reactor would have a high energy demand
ing in the mixed liquor.                                                                         to supply mixing via aeration (Beun et al. 2002; de Bruin
                                                                                                 et al. 2004).
                                                                                                       However, when the shear force is controlled only by the
                                                                                                 aeration rate, reactors with high versus low aeration rates
DISCUSSION
                                                                                                 do not only differ in terms of shear force, and this fact has
The results from experiment 1 correlate with previous                                            been previously ignored in literature. The DO profile in the
experiments that conclude a sufficiently high shear force is                                      reactor under the lowest aeration rate shows that the DO
necessary for aerobic granule formation (Tay et al. 2001).                                       was below 5 mg L21 during the period of maximum
Using only this data, one might extend this observation to                                       substrate uptake at the beginning of React. At high
say that a high shear force is also necessary for maintenance                                    superficial gas velocities (when the DO was always near
of an established granule reactor. This would support a                                          saturation), biodegradable substrate disappeared quickly
shear-based granule formation hypothesis that states high                                        and completely. For Reactor 1 operated with a superficial




Figure 4   |   (Left) COD removal and (right) DO during the react phase of an SBR cycle with the highest and lowest aeration conditions in reactor 1.
 786       B. S. McSwain Sturm and R. L. Irvine | Dissolved oxygen in aerobic granule formation                             Water Science & Technology—WST | 58.4 | 2008




Figure 5   |   Sludge structure at day 90 operation for reactor 2_lowDO (left) and reactor 2 (right). (scale bar ¼ 5 mm).


upflow gas velocities of 1.0 or 1.2 cm s21, the feast period                                              In separate studies, aerobic granulation has been
was always short (tfeast , 45 min) with a long famine period                                       reported in Mixed Anaerobic Fill SBRs with a range of
that extended the duration of React. A decreased aeration                                          DO concentrations in the subsequent React phase. In
rate resulted in a longer feast period and the absence of a                                        Mixed Fill reactors, phosphate accumulating organisms
famine period. In turn, smooth, compact granular sludge                                            (PAOs) and glycogen accumulating organisms (GAOs) are
became filamentous and increasingly flocculent.                                                      selected, and researchers have argued that granule stability
     The importance of feast and famine periods to the                                             improves with the selection of slow-growing organisms (de
formation of aerobic granules has been previously reported                                         Kreuk & van Loosdrecht 2004). The results presented in this
(McSwain et al. 2004; Liu & Tay 2006). In the study by                                             study are not comparable to granules formed under
McSwain et al. granular sludge became increasingly                                                 anaerobic or Anaerobic, Mixed Fill conditions since the
filamentous as the intensity of feast to famine in an SBR                                           microbial species and substrate removal kinetics are
decreased. As granules disappeared, the SVI and ESS values                                         different. In Static Fill reactors, biological reactions do not
increased. In experiment 1 reported herein, the limitation of                                      begin until the Aerobic, React phase, and aerobic hetero-
DO during React altered the feast and famine periods                                               trophs are expected to dominate the reactor. For Anaerobic,
within the reactor, limiting the substrate uptake at the                                           Mixed Fill SBRs, such as those used by de Kreuk et al.
beginning of React and causing granules to disappear. Liu                                          biological reactions involved in nutrient removal occur
et al. confirmed the importance of this feast to famine                                             during the Fill phase. Therefore, the selection of microbial

transition in granule reactors. In their study, Liu et al. kept                                    species depends on different environments in Static Fill and
                                                                                                   Mixed Fill SBRs.
aeration rates high during the first 110 minutes of React,
and decreased the aerations rates once the famine period
began. Since this operation did not disrupt the substrate
uptake kinetics, the granular structure was not affected (Liu                                      CONCLUSIONS
& Tay 2006).                                                                                       The first experiment confirmed previous reports that
     In experiment 2, a DO below 5 mg L21 prevented the                                            granular sludge disintegrates under low shear forces.
formation of aerobic granular sludge at high shear forces.                                               Aerobic granular sludge is not stable at superficial
The two separate experiments indicate that the dissolved                                           upflow gas velocities below 1.0 cm s21 when the shear
oxygen concentration is more important to substrate uptake                                         force is controlled only by the aeration rate. In such cases,
kinetics and granule formation than the shear force. This                                          low aeration rates create an oxygen limitation during
conclusion differs from previous reports, but it is based on a                                     React, decreasing the intensity and extending the duration
more complete data set.                                                                            of the feast period and shortening the famine period.
 787    B. S. McSwain Sturm and R. L. Irvine | Dissolved oxygen in aerobic granule formation                          Water Science & Technology—WST | 58.4 | 2008




Although a correlation can be made between the decrease                                    Clesceri, L. S., Greenberg, A. E., Eaton, A. D. (eds) 1998 Standard
of shear force and loss of granules in a reactor, this                                          Methods for the Examination of Water and Wastewater.
                                                                                                American Public Health Association/American Water
conclusion ignores the influence of shear on DO and                                              Works Association/Water Environment Federation,
substrate removal kinetics. The current results show that                                       Washington, DC, USA.
DO and substrate removal kinetics is more important to                                     de Bruin, L. M. M., de Kreuk, M. K., van der Roest, H. F., van
                                                                                                Loosdrecht, M. C. M. & Uijterlinde, C. 2004 Aerobic granular
aerobic granule formation and maintenance than the
                                                                                                sludge technology, alternative for activated sludge. Water Sci.
shear force.                                                                                    Technol. 49(11 –12), 1–7.
                                                                                           de Kreuk, M. K. & van Loosdrecht, M. C. M. 2004 Selection
                                                                                                of slow growing organisms as a means for improving
                                                                                                aerobic granular sludge stability. Water Sci. Technol.
ACKNOWLEDGEMENTS                                                                                49(11 – 12), 9 –17.
                                                                                           Liu, Y. & Tay, J. H. 2002 The essential role of hydrodynamic shear
This research was funded by the US Department of                                                force in the formation of biofilm and granular sludge. Water
Education and the German Research Foundation. The                                               Res. 36, 1653 –1665.
authors thank Dr. Peter Wilderer and the Technical                                         Liu, Y. Q. & Tay, J. H. 2006 Variable aeration in sequencing batch
                                                                                                reactor with aerobic granular sludge. J. Biotechnol. 124(2),
University of Munich Institute for Water Quality and                                            338– 346.
Waste Management for hosting this research.                                                McSwain, B. S., Irvine, R. L. & Wilderer, P. A. 2004 The effect of
                                                                                                intermittent feeding on aerobic granule structure. Water Sci.
                                                                                                Technol. 49(11 –12), 19 –25.
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Beun, J. J., van Loosdrecht, M. C. M. & Heijnen, J. J. 2002 Aerobic                        Tay, J. H., Liu, Q. S. & Liu, Y. 2001 The effects of shear force on
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