Formation and Stability of Nitrifying Granules under High Loading

Rates

Yan-ping Wang1, Fang-yuan Chen

1, Jing-lu Nie1, Ping Ning

1

1.College of Environmental Science and Engineering, Kunming University of Science and Technology, 282

Xuefu Road, Kunming 650093, PR China

Abstract. Nitrifying granules are generally believed to have high nitrification ability due to the

immobilization of large quantities of nitrifying bacteria with low growth rate. However, high loading rate is

normally not recommended for nitrifying granules due to the high inhibition statue. In this study, nitrifying

granules, able to treat ammonia nitrogen as high as 1000 mg N/L (2 kg N/m3·d), were cultivated in the

laboratory scale sequencing batch reactor. During around 200-day operation, the granules exhibited good

performance with 99% ammonia removal efficiency. In the meantime, MLSS increased from initial 6 g/L to

the final 10 g/L, and the value of SVI30 decreased from around 100 to 15 ml/g. However, it was observed that

the nitrifying granules gradually disintegrated into small aggregates with mean size decreased from 286 to

138 μm from the operation day 50 to 120, after which they gradually recovered by themselves with increased

mean size of 235 μm at the end of the operation. Notably, this disintegration did not compromise

performance and characteristics of the nitrifying granules. These results demonstrated that nitrifying granules

may have the capability to undertake ammonia loading rates as high as 1000 mg N/L with good self-healing

ability, which make the process more stable and promising in practice.

Key words: nitrifying granules, ammonia oxidation, self-healing, stability, sequencing batch reactor

(SBR)

1. Introduction

Aerobic granulation has been widely reported to be effective in the field of wastewater treatment. Under

selection pressure, normally believed as settling time [1], [2] and exchange ratio [3]-[5], activated sludge

could further aggregate and form compact granules with a certain size, dense structure, diverse microbial

species, good settling ability and high tolerance to shock load and toxins. So far, aerobic granules have been

proved effective in the treatment of wastewater with carbon, nutrients and many toxic materials. In

traditional biological nitrogen removal system, nitrifying bacteria was characterized by slow growth rate and

high sensitivity to both toxic compounds and unfavorable environmental conditions. However, nitrifying

granules, as aggregates of nitrifying bacteria, have been proved effective in enhancing nitrification rate and

system stability by the high retention of nitrifying bacteria [6]-[9].

Information on the nitrifying granules cultivated with inorganic wastewater rich in ammonium nitrogen

has been reported. Tsuneda proved that nitrifying bacteria able to treat ammonia nitrogen as high as 500 mg

N/L could be self-immobilized in an aerobic upflow fluidized reactor, with nitrifying granules of 350 μm in

diameter produced after 300 days of operation [6]. Liu cultivated nitrifying granules with size of 240 μm

and SVI of 40 mg/L on day 21 in SBR, which was able to treat 450 mg N/L of ammonia [8]. Shi reported

that the compact nitrifying granules were formed after 120 days of operation, and an NH4+-N of 100 to 250

mg/L was found to be appropriate for the operation of the nitrifying granules [9]. It could be seen that

ammonia nitrogen concentration in the reported literature is generally below 500 mg N/L for nitrifying

granular system. However, in practice, high ammonia concentration, e.g. as high as 1000 mg N/L, is

Corresponding author: Fang-yuan Chen ; Tel: +8613708897074,

E-mail address: chenfy1220@hotmail.com

International Proceedings of Chemical, Biological and Environmental Engineering, V0l. 96 (2016)

DOI: 10.7763/IPCBEE. 2016. V96. 3

12

common in high strength nitrogen-containing wastewater, such as leachate and feedlot wastewater

etc.[10]-[13]. Therefore, granules with high nitrogen removal efficiency are still highly desired.

High loading rate has been reported to be one of the main causes of the failure of granular system during

long-term operation [14], [15]. The cultivated aerobic granules could keep their structural integrity and high

COD degradation rates at certain COD level and started to disintegrate at a high loading pressure [14]. Jin

reported that the nitrifying granules began to appear on day 30 and matured in 75 days in continuous-flow

airlift reactor (ALR), which went worse upon increasing of the influent ammonia concentration up to 1120

mg N/L [16]. The proposed breakdown mechanism of the granules includes over growth of filamentous

microorganisms [17], intracellular protein hydrolysis and degradation at the anaerobic granule core and the

reduced protein quantity secreted by cells [18]. For nitrifying granules, free ammonia (FA) and free nitrous

acid (FNA), which may become tenser with the increment of loading rate in influent, exert extra inhibition to

the nitrifying granules. FA has been reported to hinder the formation of aerobic granules seriously through its

inhibition to energy metabolism of microorganisms [19]. In the meantime, FNA has been proved to be more

toxic and has a much lower inhibition threshold for nitrifying bacteria [20]. Therefore, high loading rate

could exert severer inhibition on nitrifying granules, which could cause bigger perturbations to nitrifying

granules and readily failure of the system. However, the acclimation of nitrifying granules to high FA and

FNA conditions at high nitrogen loading rates has also been reported, which may facilitate nitrifying

granules to withstand higher inhibition conditions [21]. In the meantime, nitrifying granules observed in the

present studies mostly have smaller size and higher density and settling ability than heterotrophic granules,

which make them more stable in structure. Therefore, there is possibility that nitrifying granules could

undertake higher loading rate, at least higher than the applied loading rates in current studies.

In this study, ammonia concentration as high as 1000 mg N/L was loaded in the feed in a stepwise

manner to investigate the capability of the nitrifying granules to withstand high loading conditions. In the

meantime, long-term stability of the system under the high loading rate was investigated.

2. Material and Methods

2.1. Experiment Set up and Operation Bubble column with a diameter of 5 cm and H/D (height/diameter) ratio of 20 was employed in this

study. It was operated sequentially with a cycle time of 6 hr, which included 3 min of influent filling, 323 to

346 min of aeration, 5 to 30 min of settling and 4 min of effluent discharging. Effluent was discharged from

the middle port of the reactor with a volumetric exchange ratio of 50%. Fine air bubbles for aeration were

supplied through an air sparger at the reactor bottom with an airflow rate of 1.66 cm s-1

.

2.2. Media A synthetic inorganic wastewater with the following compositions was used as influent for the

cultivation of nitrifying granules: (NH4)2SO4, KH2PO4, NaHCO3, and micronutrients. (NH4)2SO4 was

nitrogen source and NaHCO3was inorganic carbon source as well as pH control material. The NH4+-N

concentration in the influent was adjusted during the operation period, but the NaHCO3: NH4+-N : P ratio

was always kept at about 65: 5: 1 (w: w: w). The micronutrients in the influent contained CaCl2∙2H2O 25 mg

L-1

, MgSO4∙7H2O 20 mg L-1

, FeSO4∙7H2O 10 mg L-1

, EDTA-2Na 10 mg L-1

, MnCl2∙4H2O 0.12 mg L-1

,

ZnSO4∙7H2O 0.12 mg L-1

, CuSO4∙5H2O 0.03 mg L-1

, (NH4)6Mo7O24∙4H2O 0.05 mg L-1

, NiCl2∙6H2O 0.1 mg

L-1

, CoCl2∙6H2O 0.1 mg L-1

, AlCl3∙6H2O 0.05 mg L-1

, H3BO3 0.05 mg L-1

.

2.3. Analytical Methods

Commercial photochemical test kits (Hach Lamge GmbH, Dusseldorf, Germany, Test LCK303, LCK304,

LCK339, LCK340, LCK341, LCK342; spectrophotometer type LANGE Xion500) were used for the

measurement of NH4+-N, NO2

--N and NO3

--N in the reactor. Sludge volume index (SVI), biomass dry weight

(MLSS) and mixed liquor volatile suspended solids (MLVSS) were analyzed in accordance to the standard

methods (APHA 1998). Average particle size was determined by laser particle size analysis system with a

measuring range from 0 to 2000 μm (Malvern MasterSizer Series 2600, Malvern Instruments Ltd, Malvern,

UK). Samples for DNA extraction were preserved every one or two weeks. Both polymerase chain reaction

13

(PCR) amplification of extracted bacterial 16S rRNA gene and denaturing gradient gel electrophoresis

(DGGE) were conducted based on the methods described by Liu et al [22].

3. Results

3.1. The Cultivation of the Nitrifying Granules Nitrifying sludge with size of 109 μm and SVI30 of 92 ml/g, which had been stored in 4℃ fridge for

about 2 months, were seeded in the reactor for the cultivation of nitrifying granules. At the beginning of the

reactor operation, the ammonia nitrogen concentration in the feed was set at 200 mg N/L. However, it was

found that the nitrifying sludge consumed all the ammonia nitrogen in the influent during one batch cycle. So

influent ammonia nitrogen concentration was increased gradually after 2-day operation, till the 56th day when

it was increased to 1000 mg N/L. Since then, influent nitrogen concentration was maintained at this level till

the end of the operation. Meanwhile, settling time of the reactor was shortened gradually from 30 min to 7

min within 40 days to stimulate the formation of nitrifying granules.

After 45-day’s acclimation, granules ratio of the mixture of flocs and granules in the reactor increased up

to about 70%, indicating the dominance of the nitrifying granules. Liu suggested using the sludge volume

percentage with size below 200 μm (SVP-SB200) to indicate the dominance of granules when SVP-SB200 is

below 50% [22]. Considering the slow growth rate and relatively smaller size of nitrifying granules, the

sludge was assumed as granule dominant when the sludge volume percentage with size below 150 μm is

below 50% in this study. From thereon, fast size increment of the nitrifying granules was observed. The

mean size doubled within 40 days from 147 μm to 286 μm (Fig. 1 (A)). In the meantime, SVI30 in the both

reactors maintained at around 45 ml/g (Fig. 1 (B)), indicating good settling ability of the nitrifying granular

sludge. However, biomass concentration did not show the same trend as size in the reactor, which kept

decreasing from initial 6 g/L to around 3 g/L as MLSS and 5 g/L to 1.7 g/L as MLVSS (Fig. 1 (C)),

respectively. Since shorting settling time was adopted, nitrifying flocs with poor settling ability was washed

out of the reactor. Persistent washout of the slow settling nitrifying flocs and the slow growth rate of the

nitrifying bacteria resulted in a slow accumulation of biomass, which led to great decrease of the biomass

during the initial 50-day’s operation.

(A)

(B) (C)

Fig. 1: Characteristics of the nitrifying granules during the whole operation period. (A) Size; (B) SVI30; (C) MLSS and

MLVSS

Then, in the following 50 days, dramatic changes occurred in the reactor. Firstly, the value of SVI30

decreased from 45 ml/g to 10-15 ml/g in about 10 days, revealing a big increase of settling ability of the

14

nitrifying granules. Secondly, size of the nitrifying granules decreased to 138 μm. The big decrease of size of

the nitrifying granules was proved to be caused by the disintegration of the nitrifying granules into flocs and

aggregates. Fig. 2 (A) shows detailed size distribution statue during disintegration of the nitrifying granules.

It’s obvious that the size distribution was in a narrow range with little small-size flocs inside before the

disintegration of the nitrifying granules, while it turned to be a much wider range with a lot of flocs inside

during the size reduction period. However, the nitrifying granules gradually recovered in the following

operational days with the size distribution from a relatively wider range to a narrower one, which probably

resulted from the washout of small size flocs and the prevailing of even-sized granules. Fig. 2 (B) gives the

volume percentage of flocs (SVP-SB150) in the sludge on the corresponding day in Fig. 2 (A). SVP-SB150

was below 20% in the dominance of nitrifying granules (on the 55th day), while it increased up to 65%

during the disintegration period. Since there was no any external perturbation in the reactors during the

operation, the disintegration of the nitrifying granule was proposed to be of internal origin. At the same time,

MLSS and MLVSS in the reactors stopped decreasing, remaining at around 3 g/L and 2 g/L, respectively.

(A) (B)

Fig. 2: Conditions of the nitrifying granules during disintegration. (A)Size distributions of the nitrifying granules;

(B)Volume ratio of sludge with size below 150 μm

After the disintegration of the nitrifying granules, a rapid evolution of the nitrifying granules in the

reactor was observed. The size of the nitrifying granules gradually increased to 235 μm. MLSS and

MLVSS began to rise and sharply increased to around 10 g/L and 7 g/L, respectively, in two months. It

seemed that nitrifying granules finished self-adjustment and prospered with large quantities after the unstable

and critical state.

The value of SVI5/SVI30 maintained in the range of 1-1.2, indicating the dominance of the nitrifying

granules in the whole operational process.

After about 200 day’s operation, nitrifying granules was successfully cultivated with high biomass

concentration and settling ability.

3.2. Performances of the Nitrifying Granules

As can be seen in Fig. 4, ammonia removal efficiency maintained higher than 99% throughout the whole

operation period, with effluent ammonia concentration lower than 6 mg N/L. NO2--N and NO3

--N

concentration during the whole operating process were also traced, which revealed complete nitrification in

the reactor during most of the experiment period. Although nitrifying granules experienced size reduction

from day 70 to 120, the ammonia nitrogen removal was not impacted negatively during the period. In

addition, the decrease of biomass concentration during the first 50-day operation did not compromise

ammonia nitrogen removal either. Fig. 5 shows cycle profile of the nitrifying granules on the 160th day of the

operation, during which nitrifying granules recovered from the disintegration and retained stable. To reduce

the influence of the residue from last cycle, the upper liquid in the leftover was changed with tape water. In

the cycle, ammonia was totally oxidized within 3 hours, during which there were a big accumulation of

nitrite, indicating that the limiting step of the process was nitrite oxidation by nitrite oxidizing bacteria

(NOB). Upon the depletion of ammonia, nitrite concentration increased to the climax and then quickly

oxidized to nitrate since there was no oxygen limitation caused by ammonia oxidation. The end product of

the oxidation process was totally nitrate, indicating a complete nitrification of the process.

15

Fig. 3: Performances of the nitrifying granules

Fig. 4: Time course of NH4-N, NO2-N and NO3-N concentration in a batch cycle of reactor operation with nitrifying

granules

3.3. FA and FNA Conditions in the Reactors

FA and FNA variations in the cycle of reactor operation on the 160th day are shown in Fig. 6. It can be

seen that the concentration of FA increased fast to the climax, i.e., 108.8 mg N/L, within half an hour of the

cycle. Then it decreased gradually to zero during subsequent 2 hours of the cycle. FNA concentration

gradually increased to the climax, i.e., 0.044 mg N/L, on the depletion of FA, and then decreased to zero in

the subsequent 2 hours of the cycle.

Fig. 5: Time course of FA and FNA concentration in a batch cycle of reactor operation with nitrifying granules

3.4. Denaturing Gradient Gel Electrophoresis Analysis (DGGE) of the Nitrifying Granules Fig. 7 shows DGGE band profile of the PCR amplification products obtained from the nitrifying

granules. Samples were collected in the developing process of the nitrifying granules, including

disintegration of the nitrifying granules. The changing trend of the bands pattern shows that the number of

the bands decreased during granulation period and increased as mature bacteria community in the granules

formed. The position of the major bands did not shift during the course of granulation (band 1, 2, 4, 5, 6),

while the number of the major bands increased after the formation of mature granules. This is coincident 16

with previous research, which demonstrated that the diversity of the population may increase with the growth

of the granules [23].

1

2

3

4

5

6

7

6d 28d 54d 68d 87d 118d

1

2

3

4

5

6

7

6d 28d 54d 68d 87d 118d

Fig. 6: DGGE profiles of the communities in the nitrifying granules

4. Discussion

Nitrification is affected by many factors, such as substrate concentration, pH, dissolved oxygen (DO),

temperature [24]-[27]. Among them, the effects of substrate are strong and complicated due to its inhibition

effects when unionized, i.e., the formation of FA and FNA. They are reported to inhibit the growth and/or

energy generation of a wide range of bacteria. Different threshold values were proposed for nitrification

inhibition, which are very sensitive to bacteria adaptation. It was stated that the FA inhibition threshold is

10-120 mg/L for ammonia oxidation, and 0.1-1.0 mg/L for nitrite oxidation. While FNA inhibition threshold

is 0.1-4.0 mg N/L for ammonia oxidation and 0.01-0.83 mg N/L for nitrite oxidation.[20]-[29]. In this study,

both the FA concentration and FNA concentration increased to a climax value during one cycle, i.e., 108.8

and 0.044 mg N/L, respectively, when influent ammonia concentration increased to 1000 mg N/L under well

controlled temperature, pH and DO. The high FA and FNA concentration may thereby exert high inhibition

to the system. However, the nitrifying granules exhibited good performance (99% nitrogen removal

efficiency) and stability (SVI30 value of around 15 ml/g) during the whole operation under stepwise loading

increasing strategy in the influent. This shows that the nitrifying granules may have strong resistance to high

inhibitions with stepwise acclimation, which may facilitate their nitrification of ammonia under high loading

rates.

As a kind of aggregated cells, aerobic granules can be expected to have better protection from high

substrate loading rates and inhibitory of toxic substrates. The tolerance towards the disadvantage conditions

is not only caused by the fast removal of the substrates outside biomass, but also by the granules’ intrinsic

structural advantage as aggregated cells, which cause diffusion limitation inside granules. Though diffusion

barrier might limit oxygen and nutrient transportation to the core of the aggregates, it can protect the inner

layer of microorganisms against exposure to disadvantageous conditions. Therefore, as one of the major

producer of diffusion limitation, size of the granules should be optimally developed with enough resistance

to the disadvantage conditions but little barrier to the nutrient transportation. In this study, it was found that

the size of the nitrifying granules was increased with the fast loading increasing in the feed, which could be a

positive responds of the nitrifying granules to the disadvantageous environment, i.e., the stepwise

accumulated FA and FNA in the bulk liquid. The high growth rate of bacterial strains was widely reported to

encourage proliferation of microbes, which may cause a rapid increase in size of the granules with a loose

structure and low density [30], [31]. In the meantime, the size of the micro-colonies was reported to be able

to determine whether fragmentation took place under the shear conditions applied and that whether this shear

could erode or further fragmentation [32]. It is thus speculated that the structure of the granules could start to

be damaged when the size of the granules grew continuously to a larger one that limited mass transfer greatly.

Once the interior structure became too weak to withstand the applied shear force, the nitrifying granules were

17

broken up into small pieces. Therefore, it is possible that the fast increase of size of the nitrifying granules

was the main cause of the break-down of size of the nitrifying granules on the 60th operational day.

However, the disintegration of the nitrifying granules did not negatively affect ammonia removal

efficiency of the system, which was confirmed by the unchanged ammonia removal efficiency and settling

ability of the nitrifying granules after the disintegration. It’s reported that different populations had large

variations in their micro-colony strength and in their resistance to micro-colony break-up due to

physical-chemical effects from various chemicals [33], [34]. Nitrifiers are known to grow in dense

micro-colonies and micro-colonies generally seem to form the strongest fraction of the flocs. Gilda reported

that the overall nitrogen removal in their system was not affected by the physical separation of ammonium

and nitrite oxidation, in which most NOB was located in the granules and AOB in flocs [35]. Considering

that nitrifying granules in this study are dominant of AOB and NOB (data not shown), it is reasonable to

speculate that the disintegration of the nitrifying granules could be mainly the separation of the AOB and

NOB inside, which have high flexibility in the forms of cooperation. Overall nitrogen removal efficiency

was thus not much negatively affected.

Loss of granule stability and deterioration of the whole system was one of the major barriers to practical

applications for long-term operation. However, in this study, the disintegration of the nitrifying granules did

not lead to the deterioration of the system accordingly. On the contrary, the system maintained a good

performance through self-adjustments of size of the nitrifying granules, showing stronger self-healing ability

than that of the heterotrophic granules. This discrepancy of the performances of the granules may be

attributed to the microbial difference. Not as the loosely aggregating form of the nitrifying bacteria (AOB

and NOB) in nitrifying granules mentioned above, most granules with complicated bacteria have closely

related microbial communities. The physical break-up of the micro-colonies could lead to big shock to

microbial communities who depend closely on each other for balanced life and hence the deterioration of the

whole system. Therefore, nitrifying granules may have a higher self-healing ability than that of the

heterotrophic granules, which make it a good option for stable and effective nitrification under high

ammonia loading conditions.

However, it should be noted that the disintegration of the nitrifying granules in the development is not

the doomed process. Without the outgrowth of size, the nitrifying granules in our parallel experiment

experienced a stepwise growth in terms of size of the granules [36]. In addition, the DGGE results clearly

showed that the main microbial communities in the two experiments were almost same under the same

operational conditions, though there was a big difference on the evolution process of the physical

characteristics of the nitrifying granules. These results clearly demonstrated that nitrifying granules have a

more flexible developing mode than heterotrophic granules due to their stronger self-healing ability, which

make the system more stable and promising in practice.

5. Conclusions

The nitrifying granules able to treat ammonia as high as 1000 mg N/L were successfully cultivated in the

reactor of SBR. During the operation period, the nitrifying granules disintegrated first and then recovered by

themselves. However, the main characteristics of the nitrifying granules, such as treatment capacity, settling

ability, biomass concentration did not deteriorate. These results demonstrated that nitrifying granules have

good performance and stability under high loading rates. At the same time, they have a more flexible

developing mode than mix-cultured granules due to their stronger self-healing ability. These good attributes

of the nitrifying granules make their nitrification more stable and promising in practice.

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