Effects of substrate shock on extracellular polymeric substance (EPS) excretion and characteristics of attached biofilm anammox granules

Abstract

Environmental stresses are assumed to significantly impact the content and concentration of produced extracellular polymeric substances (EPS) and therefore influence the performance of an ananmmox attached film expanded bed (AAFEB) reactor. In this study, a transient high substrate concentration of 2500 mg N L⁻¹ (calculated as the sum of NN₄⁺–N and NO₂⁻–N) for 24 hours stimulated abundant EPS excretion as well as deterioration of anammox granules. The results indicated that a high EPS concentration of 89.6 ± 48.3 mg g⁻¹ VSS resulted in 35.0 ± 0.8% decrease in the granule settling properties and 30.5 ± 0.9% reduction in the total VSS amount. The production of EPS was reasonably attributed to the impact of utilization-associated and stress-associated effects by the substrate. The results of a series of batch experiments indicated that a rapid increase of loosely-bound EPS (LB-EPS) from 41.2 to 114.6 mg g⁻¹ VSS occurred when the substrate concentration steadily increased from 400 to 1000 mg N L⁻¹, in contrast, the tightly-bound EPS (TB-EPS) remained stable at 32.5 ± 2.8 mg g⁻¹ VSS. Thus, the LB-EPS was considered the key factor for the deterioration of granule stability and the substrate concentration should be controlled below 400 mg N L⁻¹ to avoid triggering the accumulation of LB-EPS. Furthermore, the formation and disintegration mechanisms of attached film anammox granules were also elucidated in this study.

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1. Introduction

Over the past decade, the anammox (anaerobic ammonium oxidation) process has been widely accepted as an economical and sustainable bioprocess to treat nitrogen rich wastewater with low ratios of carbon : nitrogen (C : N), such as sludge digestion water and industrial wastewater.^1,2 Compared with the conventional nutrient removal process,³ the anammox process has clear advantages over the nitrification–denitrification process in terms of reduced electric consumption and lower organic carbon demand.^4,5 However, the physiological properties of anammox, including a very long doubling time (10–14 days), low-cell yield (0.11 g VSS per g NH₄⁺–N) and high sensitivity to environmental stress, have prevented the anammox process from widespread application.^5–7 As such, in order to maintain the stable operation of the anammox-based reactor, it is essential to enhance its ability to retain biomass and improve its resistance to environmental stresses. The anammox attached film expanded bed (AAFEB) reactor, which was developed as a new reactor configuration with a high nitrogen removal ability and operational stability has potential to popularize anammox process. The anammox attached film granules demonstrate high settling velocities and good resistance to environmental stresses.⁸ However, further research on the operation features of the AAFEB reactor is necessary.

It has been widely reported that both the content and concentration of extracellular polymeric substances (EPS) have significant impact on the structure, particle size, shear strength, filterability, and settling behavior of granular sludge in biological wastewater treatment processes and thus influence treatment performance.^9,10 From this perspective, determining the optimal EPS content has the potential to improve the granulation and stability of aggregates and to avoid cell dewatering and damage from toxic substances through a vast net-like structure with plenty of water.¹¹ However, excessive EPS may decrease the mass transfer efficiency in biofilm and granules and accordingly result in the deterioration of reactor performance.¹² Furthermore, since the EPS is always negatively charged, it can bind with positively charged organic pollutants and metals ions through electrostatic interaction,¹³ and thus inhibit anammox activity since the anammox bacteria is very sensitive to organic pollutants and heavy metals.^14–16 According to the above description, the content and concentration of EPS in the anammox biofilm and granules may also exert influence on reactor performance, and an excessively high or low concentration or an unsuitable composition may significantly compromise treatment performance, and even result in the loss of the stable operation of the anammox process.

Protein and polysaccharides are the major components of EPS, and typically account for 1–60% and 40–95% of content, respectively.¹⁷ It is known that EPS can be subdivided into soluble EPS forms and bound EPS forms: bound EPS is closely bound to cells, and are connected with the bio-aggregate characteristics such as shear strength, settling velocity, surface charge, dewaterability and hydrophilicity/hydrophobicity characteristics.^9,18 Bound EPS can be further subdivided into tightly-bound EPS (TB-EPS) and loosely-bound EPS (LB-EPS) according to their binding force to cells.⁹

The production behavior of EPS in anammox biofilm and granules is highly related to the reactor configurations, the type of biomass, and the operational conditions.^9,19 A number of investigations have been reported to determine the distribution of EPS in anammox granules and their important roles during anammox granulation process.^18,20,21 In addition, the protection effect of EPS to environmental stresses has been also widely reported.^22,23 Few studies have been focused on the variation in EPS content and composition under toxic substance stress and the adverse impact on reactor performance. To gain a better understanding of the key characteristics of the EPS in the attached biofilm anammox granules, with the final purpose of optimizing the conditions of the anammox process, this study evaluated the variation of the content and concentration of EPS in a long-term experiment under stable operation conditions and transient substrate shock condition. Furthermore, the shear strength of the granule and the EPS production characteristics under various substrate stresses were studied in a series of batch experiments.

2. Materials and methods

2.1. Reactor operation

The reactor configuration and biomass characteristics were the same as the previous study.⁸ There were five operation phases in the continuous experiment, during the stable operation period (phase I and II), the substrate TN concentration increased from 600–1250 mg N L⁻¹ with a constant HRT of 3 hours to increase the nitrogen loading rate (NLR) from 4.8–10.0 g N L⁻¹ d⁻¹. Then a transient high substrate concentration of 2500 mg N L⁻¹ was employed for 24 hours to evaluate the shock effects on the reactor performance as well as on EPS production. After that, the substrate concentration was decreased to 1250 mg N L⁻¹ (in phase III), 625 mg N L⁻¹ (in phase IV) and 313 mg N L⁻¹ (in phase V) step by step to recover the treatment performance. The variation of EPS content and concentration and its distribution along with reactor height were evaluated during the experiment.

2.2. Batch tests

The EPS production and related characteristics in the attached biofilm anammox granules were evaluated under different TN concentrations by batch tests. The TN concentration gradient was set as 100, 200, 400, 800 and 1000 mg N L⁻¹ using a mixture of (NH₄)₂SO₄, and NaNO₂ solution, the ratio of NO₂⁻–N/NH₄⁺–N was set as the reported anammox stoichiometric ratio of 1.32.⁶ The tests were performed in serum flasks and incubated in a thermostatic water bath shaker (110 rpm) in the dark at 35 °C. Each serum flake was inoculated with 5 g wet anammox granules, which were taken from the bottom of the AAFEB reactor. In order to ensure the sludge loading rate (SLR) was the same as that in the AAFEB reactor, the TN amount injected into each flask was kept at 30 mg. The produced N₂ gas was accumulated in the headspace and measured throughout the tests for subsequent processing. Specific anammox activity (SAA) was calculated based on the gas production rate.^8,24 Since the SAA is related to the substrate concentration, when the TN concentration is lower than the toxic threshold, the SAA raises as the substrate concentration increases, however, when the substrate concentration is higher than the toxic threshold concentration, the SAA is inhibited. The inhibition rate can be expressed as:

Where I is the inhibition rate (%), SAA is the specific anammox activity under a certain substrate concentration (g N per g VSS per d), and MSAA is the maximum SAA obtained at the optimum concentration in the gradient range that set above.

The anammox granules were taken out from the serum flasks to analyze the content and concentration of EPS as well as the granular shear strength when the accumulated gas production reached 21 mL, which is the theoretical value calculated by the anammox reaction formula.⁶ All the tests were performed in parallel samples to ensure reliability.

2.3. EPS extraction and analysis

Attached biofilm anammox granules were taken from three sampling points along with different reactor height (top, middle and bottom) to analyze the respective EPS production during the whole experiment. Because the cation exchange resin (CER) method (Dowes Marathon C, 20–50 mesh, sodium form) has been found effective in avoiding cell lysis,²⁵ the method was modified to extract the EPS. In order to study the functions of the LB-EPS and TB-EPS, they were extracted separately. The attached film anammox granules were washed three times with a buffer solution (Na₃PO₄ of 2 mmol L⁻¹, NaH₂PO₄ of 4 mmol L⁻¹, NaCl of 9 mmol L⁻¹ and KCl of 1 mmol L⁻¹). Afterwards the granules were resuspended in 30 mL of the buffer solution and centrifuged at 8000G for 15 min. Then the supernatant was filtered through a 0.45 μm syringe filter: the filtrate was regarded as the LB-EPS. For the extraction of TB-EPS, the granules after the first centrifugation were resuspended in the phosphate buffer solution to its original volume of 30 mL, followed by the addition of CER, with a dosage of 60 g per g VSS. The mixed liquor was stirred by a magnetic stirrer at 900 rpm for 1 h, and then centrifuged at 12 000G for 15 min. The supernatant which was collected and filtered was regarded as the TB-EPS of the anammox granules.

The content of protein and polysaccharides was analyzed for both the LB-EPS and TB-EPS. Protein was measured using the Lowry method with bovine serum albumin as the standard,²⁶ and the polysaccharide content was measured using the phenol–sulfuric acid method with glucose as the standard.²⁷

2.4. Granular analysis

In the assessment of anammox granules, the size distribution, settling velocity and shear strength were evaluated. The size distribution of the granules was determined by image analysis of a photograph using the image analysis software ImageJ. (1.48v, USA) and was divided into five groups according to the particle size (mm): <1.0, 1.0–2.0, 2.0–3.0, 3.0–4.0 and >4.0. The granule settling velocity of each group was measured during the middle 1 m through a 2 m water column.²⁸ For each group, the granule settling velocity was measured in 10 replicates to ensure the reliability. The shear strength was presented in integrity coefficient according to the previous study,²⁰ 2 g of wet granules were added to 100 mL of the buffer solution. The mixed liquor was sheared in a thermostatic water bath shaker for 20 min at 200 rpm and 35 °C. After settlement for 1 min in a 1000 mL cylinder, the suspended solid (SS) of the supernatant and sediment were determined. The integrity coefficient can be used to quantify the shear strength, as follows:

Where, SS₀ is the total amount of the attached biofilm anammox granule, g, and SS_t is amount of the sludge in the supernatant after 1 min sedimentation, g.

The SEM observation of the anammox granule was also performed. The granules were fixed in a 2.5% glutaraldehyde solution for 4 hours and then dehydrated in a graded series of ethanol (50, 60, 70, 80, 90, 100%, 10 min per step). When dried, the samples were glued to an adhesive coated tab (EMS, Washington, USA), sputter coated with a palladium/gold alloy, and analyzed with a field emission scanning electron microscope (JEOL, JSM-7800F, Japan) at 15 kV.

2.5. Water quality analysis

Samples of the influent and effluent were collected every 2 days and filtered through a 0.45 μm syringe filter. The nitrogen concentration of NH₄⁺–N, NO₂⁻–N and NO₃⁻–N was analyzed by capillary electrophoresis (Agilent 7100), and the TN concentration was calculated as the sum of the three kinds of nitrogen compounds. All other analyses were carried out according to the standard method.²⁹

3. Results and discussion

3.1. Long-term performance of the AAFEB reactor

Previous study have indicated that varying the NLR and influent substrate concentration resulted in different nitrogen removal performance, SAA and substrate tolerance ability.⁸ The EPS production and biomass-effluent separation ability were also affected by variations in substrate. During the stable operation phases I and II, the substrate TN concentration increased from 600 mg N L⁻¹ to 1250 mg N L⁻¹ with a constant HRT of 3 hours (Fig. S1A†). Due to the effects of effluent recirculation, the diluted substrate concentration (SN_inf, calculated as the sum of the diluted NH₄⁺–N and NO₂⁻–N concentration) increased from 160 ± 9 to 297 ± 29 mg L⁻¹ (Fig. S1A†). Correspondingly, the NLR increased from 4.8 to 10.0 g N L⁻¹ d⁻¹ (Table 1) with a stable TN removal efficiency of 85–92% (Fig. 1B). Along with the change in NLR and substrate concentration, the average EPS concentration (average value of the three sampling points, calculated as the sum of proteins and polysaccharides) and effluent suspended solids slightly increased (Fig. 1A and B).

Table 1 Operation conditions and reactor performance during the experiment

	Phase I	Phase II	Substrate shock	Phase III	Phase IV	Phase V
Periods (d)	0–25	26–59	60	61–88	89–159	160–213
TN (mg N L^−1)	600	1250	2500	1250	625	313
SNinf (mg N L^−1)	160 ± 9	297 ± 29	1048	542 ± 129	288 ± 50	98 ± 27
HRT (h)	3	3	3	3	3	1.5
NLR (g N L^−1 d^−1)	4.8	10.0	20.0	10.0	5.0	5.0
NRR (g N L^−1 d^−1)	4.20 ± 0.11	8.93 ± 0.16	14.77	6.99 ± 1.58	3.11 ± 0.51	3.90 ± 0.47
Free ammonia (mg L^−1)	6.68–23.22	18.34–48.13	131.98	33.00–74.00	15.27–61.48	6.12–27.21
Free nitrous acid (μg L^−1)	1.94–7.55	2.73–8.99	13.06	4.84–16.28	1.04–8.74	0.26–6.38
Average VSS (g L^−1)	56.3 ± 1.2	67.2 ± 1.8	67.2 ± 1.8	56.3 ± 2.1	48.1 ± 1.2	48 ± 1.1
Sludge blanket height (cm)	65	68	68	64	66	69
Total VSS (g)	229.8 ± 4.2	287.0 ± 7.6	287.0 ± 7.6	226.3 ± 7.4	199.4 ± 3.6	208.0 ± 3.3

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Fig. 1 Variation of the average EPS concentration (A) and effluent suspended solids concentration (B) during the experiment.

A substrate shock of 2500 mg N L⁻¹ over a period of 24 hours was performed on day 60, with operation conditions immediately recovered to the levels applied in phase II. As shown in Fig. S1† and 1, the transient high substrate concentration profoundly inhibited the anammox reaction. In phase III, the TN removal efficiency gradually decreased to 40%, resulting in an increase in SN_inf from 320 to 721 mg N L⁻¹ and the continuous inhibition of the anammox reaction. The average EPS and effluent suspended solids (as SS) rapidly increased to peak values of 89.6 ± 48.3 mg g⁻¹ VSS and 0.083 g L⁻¹, respectively.

In order to recover anammox activity and investigate the effects of substrate concentration on EPS production, the substrate concentration was decreased to 625 mg N L⁻¹ from the 89^th day with a constant HRT of 3 hours. Accordingly, the SN_inf and NLR decreased to 294 mg N L⁻¹ and 5.0 g N L⁻¹ d⁻¹, respectively. As shown in Fig. S1,† even though the TN removal efficiency recovered to around 80% as soon as the operation conditions were changed, anammox activity remained inhibited due to the high substrate concentration in phase IV. This resulted in a gradual decrease in TN removal efficiency to 40%. Even though the anammox reaction remained inhibited, the average EPS concentration and effluent SS decreased gradually until they reached the level observed in stable operation phase II (Fig. 1). Following the further decrease in substrate concentration to 313 mg N L⁻¹ in phase V, the SN_inf immediately decreased to 200 mg N L⁻¹, and the TN removal efficiency gradually increased to around 85% and remained stable (Fig. S1†). The results obtained from the long-term experiment indicate that besides significantly inhibiting the anammox reaction, the transient high substrate concentration of 2500 mg N L⁻¹ (SN_inf of 1048 mg N L⁻¹) led to a considerable increase in the EPS concentration and effluent SS. It should also be noted that although the anammox reaction was in the inhibition condition during phase III and IV, EPS excretion proceeded more efficiently when the SN_inf was at a high level of 542 ± 129 mg N L⁻¹ (phase III). This had dramatically reduced during phase IV when the SN_inf was at a lower level of 288 ± 50 mg N L⁻¹.

3.2. Changes in biomass and EPS distribution

3.2.1. Biomass washout and sludge distribution. Accompanying the increase in the effluent suspended solids, biomass washout and variations in sludge distribution were inevitable. The distribution of sludge (including SS and VSS) in different operation stages (stable, inhibited and recovered) along with reactor height is shown in Fig. 2. The VSS and SS concentrations at the bottom of the reactor were much higher than those in the middle and top parts (Fig. 2A and B), indicating that both the biomass and inorganic matters had accumulated at the bottom part. On the 55^th day in the stable operation phase II, the VSS concentrations at different heights were in the range of 60–79 g L⁻¹. However, after the transient substrate shock, the sharp increase in SS in the effluent was noticed (Fig. 1B), resulting in a considerable decrease in the VSS concentrations to 45–54 g L⁻¹ in the reactor (on the 127^th day). It can be inferred that the transient substrate shock led to considerable variations in the sludge distribution. When anammox treatment performance had totally recovered, the VSS concentrations had recovered to the range of 47–58 g L⁻¹ (at 205^th day), but remained significantly lower than the level observed in stable operation phase II. In none of the phases, however, was any significant change noted in the SS concentration: it was maintained in the range of 196–238 g L⁻¹ (Fig. 2B). In contrast, the ratio of VSS/SS decreased sharply from a range of 28.7–33.2% (on the 55^th day) to a range of 22.3–23.9% (on the 127^th day) then slightly recovered to a range of 24.1–25.1% (on the 205^th day) (Fig. 2C). The results indicate that the anammox biofilm peeled off from the carrier, causing 30.5 ± 0.9% of the total VSS to be washed out from the reactor (Table 1). The inorganic matter was retained in the reactor as carriers.

Fig. 2 Sludge distribution along with reactor height of different operation stages. (A) VSS distribution; (B) SS distribution; and (C) VSS/SS distribution.

3.2.2. Variation of EPS production and distribution. In general, EPS was produced with the growth and metabolism of bacteria. Previous studies indicated that the production of EPS is either growth-synonymous or growth-associated and is affected by genotype as well as the physical, chemical and biological conditions.¹⁷ The contents and concentration of EPS of different aggregate types and reactor configurations are summarized in Table 2. For anammox aggregates (e.g., granules and biofilms), the EPS content of both proteins and polysaccharides is relative high compared to anaerobic methanogenic granules and aerobic granules. This difference indicates that anammox bacteria is more effective in the excretion of EPS. Furthermore, EPS is usually biodegradable and can be used by heterotrophic bacteria as a source of carbon and energy. Autotrophic bacteria like anammox, however, is unable to utilize organic matters and therefore accumulates more EPS.^17,19 In this study, as shown in Fig. 3, EPS accumulated at the bottom of the reactor during the stable operation period (phase I and II). The concentration variation of EPS was consisted with that of the substrate distribution trend (as shown in Fig. S2†), which also decreased along with increasing reactor height. When the NLR increased from 4.8 to 10.0 g N L⁻¹ d⁻¹, the average EPS concentration increased slightly from 22.3 ± 3.2 (at 20^th day) to 35.2 ± 7.3 mg g⁻¹ VSS (at 48^th and 55^th day), indicating that EPS excretion was effected by substrate feeding. In other words, EPS production was substrate utilization-associated under stable operation stages.

Table 2 EPS composition in different systems

Aggregate type	Reactor configuration	Protein	Polysaccharide	PN/PS	Extraction methods	Reference
a Dry wet.b The sum of TB-EPS and LB-EPS.
Methanogenic granules	UASB	87 mg per g DW^a	68 mg per g DW^a	1.28	Sonication/centrifugation	37
Methanogenic granules	UASB	41 mg per g TS	14 mg per g TS	2.93	CER	38
Methanogenic granules	SRUSB	100–130 mg per g VSS	100–130 mg per g VSS	1.0–1.16	Formaldehyde NaOH	39
Aerobic granules	SBR	253.8 mg per g VSS	20.8 mg per g VSS	12.20	EDTA	40
Aerobic granules	SBR	101.1 mg per g VSS	15.8 mg per g VSS	6.40	Heating	41
Aerobic granules	SBR	30.12–47.86 mg per g VSS^b	26.52–25.70 mg per g VSS^b	1.14–1.86	CER	42
Anammox granules	UCR	63.8–82.9 mg per g VSS	151–310 mg per g VSS	0.34–0.52	CER	43
Anammox granules	Three-bio-electrode reactor	63.8–307 mg per g VSS	151–287 mg per g VSS	0.42–1.07	CER	43
Anammox granules	UASB	55.6 ± 3.2 mg per g VSS	70.8 ± 6.5 mg per g VSS	0.8	CER	20
Anammox granules	UASB	164.4 ± 9.3 mg per g VSS	71.8 ± 2.3 mg per g VSS	2.29	EDTA	33
Biofilm	MBR	79.51 ± 3.61 mg per g VSS	30.12 ± 1.52 mg per g VSS	2.64 ± 0.12	Heating	44
Attached biofilm anammox granule	AAFEB	21.1 ± 8.7 mg per g VSS (stable operation stage)	10.2 ± 0.3 mg per g VSS (stable operation stage)	2.6 ± 1.4 (stable operation stage)	CER	This study
		46.2 ± 24.9 mg per g VSS (non-stable operation stage)	10.8 ± 2.6 mg per g VSS (non-stable operation stage)	5.4 ± 2.1 (non-stable operation stage)

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Fig. 3 Variation of EPS composition along with reactor height during different operation stages (stable operation period, 1^st inhibition period, 2^nd inhibition period, recovered period).

Because appropriate EPS production benefits the biomass granulation and flocculation process, it enhances the effluent–solids separation and biomass retention in biological wastewater treatment processes.¹⁷ However, excessive EPS has been shown to weaken biomass retention ability.³⁰ In this study, transient substrate shock led to extreme variations in EPS production and distribution, which further compromised the reactor performance. As shown in Fig. 3, in the first inhibition period, protein concentration sharply increased to 77.2 ± 33.1 mg g⁻¹ VSS with a stable polysaccharides concentration of 10.8 ± 2.6 mg g⁻¹ VSS. The highest concentration of EPS had accumulated in the top part of the reactor, whereas it was at the bottom of the reactor during the stable operation period. In the second inhibition period, even though the anammox reaction remained inhibited, after the sudden decrease of substrate concentration in phase IV, the protein concentration in EPS gradually decreased. This continued until the protein concentration was at the level of the stable operation stage. It should be noted that concentration of polysaccharides remained stable. In addition, the distribution characteristic of EPS along with reactor height recovered to conditions similar to those noted in previous stable operation phases. Yang et al. (2009)³¹ proposed that excessive EPS had adverse effects on the sludge settleability and cell attaching ability due to the bound water brought into the aggregates by the excessive EPS, which results in the production of highly porous flocs with a low density. In this study, the high substrate concentration led to the excessive excretion of EPS and also a decrease in the density of anammox granules, followed by a biomass wash out from the reactor that aggravated the reactor performance. Corresponding to the variation in the EPS content and concentration, the average proteins/polysaccharides (PN/PC) ratio also sharply increased from 2.6 ± 1.4 (stable period) to 5.4 ± 2.1 after the transient substrate shock and then gradually recovered (Fig. 3). As an important parameter to assess the granule characteristics, the PN/PC ratio played a significant role in granule settleability, shear strength and substrate transfer. This is further discussed in the following sections. The variation in EPS content and concentration during the inhibitory phases indicated that EPS production was also substrate stress-associated.

3.2.3. Variation of particle size and settleability. The transient substrate shock resulted in unstable treatment performance and marked variations in the characteristics of the granules. In the images of the anammox granules in different phases shown in Fig. S3,† variations in the size and color of the granules are clear. As shown in Fig. 4A, a considerable variation in particle size distribution was observed in the different operation stages (before inhibition, and in the inhibited and recovered phases). The average particle size on the 55^th, 127^th and 205^th day were 2.41, 2.31 and 2.32 mm, respectively. Moreover, the percentage of granules with a particle size smaller than 1.0 mm significantly increased from 11.2 to 24.1% during the inhibitory phases, and then decreased to 13.1% when the anammox reaction had recovered. Considering that the average particle size of the inorganic carrier did not change significantly during the experiment, the increase in the ratio of small granules (particle size < 1 mm) can likely be attributed to the biomass peeling off from the carrier.

Fig. 4 The anammox particle size distribution (A) and granule setting velocity of each particle size group (B) under different operation conditions (before inhibition, inhibited and recovered).

The correlation between granule settleability and particle size, density, geometrical shape, surface charge and hydrophilicity/hydrophobicity has been noted in previous studies.^9,32 As shown in Fig. 4B, the settling velocity of the granules in the stable phase was proportional to particle size and in the range of 185–560 m h⁻¹. It should be noted that this is considerably faster than the velocities reported in previous studies.³³ It is apparent that the ideal settling velocity would significantly enhance biomass retention ability. Transient substrate shock led to a marked reduction in the settling velocity, reducing it by approximately one third to 121–359 m h⁻¹. Many studies have confirmed that the negative charge of EPS has an adverse effect on settleability.³⁴ Furthermore, Tang et al. (2011)³³ indicated that a higher PN/PC resulted in lower shear strength and higher fluid viscosity of anammox granules, which weakened the settleability and raised the risk of biomass washout. Under the multi-functions of the increase in small granules (particle size <1 mm) and the decrease in settleability, obvious biomass washout accounting for 30.5 ± 0.9% of the total amount of VSS washed out from the reactor, which further deteriorated the reactor performance.

3.3. Batch tests for EPS production under different substrate concentrations

Excessive EPS is usually excreted under extreme environmental stress to protect bacteria from damage.^9,35 While this self-defense behavior serves to maintain bacteria activity, it also results in unstable reactor operation performance. The stimulation effects of substrate on anammox activity, EPS production and granule shear strength are shown in Fig. 5. Accompanying the increase in TN concentration, the SAA increased to the maximum value of 0.63 g N per g VSS per d when the TN concentration was 200 mg N L⁻¹, and was 97% inhibited when the TN concentration increased to 1000 mg N L⁻¹, the half maximal inhibitory concentration of TN (IC₅₀) was calculated to be 620 mg N L⁻¹ (Fig. 5A). At the end of the batch experiment, a considerable quantity of EPS had been produced and had accumulated on the surface of the granules (see Fig. S4†). Further analysis showed that with the increase in TN concentration, the content and concentration of TB-EPS remained stable at 32.5 ± 2.8 mg g⁻¹ VSS (Fig. 5B). However, the LB-EPS slightly increased when the TN concentration increased to 400 mg N L⁻¹, and a further rapid increase to 114.6 mg g⁻¹ VSS occurred when the TN concentration steadily increased from 400 to 1000 mg N L⁻¹ (Fig. 5C). In addition, with increasing LB-EPS concentration, the polysaccharide concentration remained stable at 9.4 ± 1.7 mg g⁻¹ VSS while the protein concentration increased rapidly from 20.4 mg g⁻¹ VSS to 103.2 mg g⁻¹ VSS. Corresponding to the variation of TB-EPS and LB-EPS, the PN/PC ratio of TB-EPS remained stable at 2.14 ± 0.31, but that of LB-EPS increased rapidly from 2.17 to 11.58.

Fig. 5 SAA inhibition rate along with increase of TN concentration (A); variation of TB-EPS (B) and LB-EPS (C) under TN concentration stimulation; relationship between the content of LB-EPS and granular shear strength (D).

The shear strength tests indicate an inverse correlation between LB-EPS and granule shear strength (Fig. 5D). As a parameter to describe granule shear strength, the integrity coefficient decreased from 98.8 to 88.6% with an increase in the LB-EPS concentration from 26.2 to 103.3 mg g⁻¹ VSS. The results indicate that excessive LB-EPS plays a negative role in granule stability. Li et al. (2007)³⁰ indicated a positive correlation between LB-EPS and bioflocculation ability when the LB-EPS was low. In this study, a deterioration of attachment ability between cells and the aggregates was observed when the LB-EPS was high, but there was insufficient evidence to conclude the existence of any relationship between TB-EPS and granule stability. The LB-EPS concentration was considerably higher than TB-EPS when the anammox granules were affected by excessively high TN concentrations, which resulted in an obvious negative effect on granule stability. Zhang et al. (2016)⁸ indicated an safe substrate concentration of lower than 320 mg N L⁻¹ should be maintained in the reactor, in this study, a TN concentration of higher than 400 mg N L⁻¹ resulted in the deterioration of anammox granules and the further damage of reactor performance.

3.4. Mechanisms for granule formation and disintegration

The formation of the anammox granules suggests the self-immobilization of anammox bacteria on the surface of the carriers, resulting in a compact structure and a super-high settling velocity. However, transient substrate shock led to the disintegration of the attached anammox biofilm from the granules, which led to a further deterioration in reactor performance. The proposed mechanisms for granule formation and disintegration are shown in Fig. 6. The formation process of the anammox granules is in good agreement with the model provided by Liu et al.³⁶ for granulation mechanisms. Firstly, initial cell-to-cell (e.g. anammox and filamentous bacteria) and cell-to-carrier movement, which involves physical forces and cell mobility, attracts the cells to each other, then the cellular clusters attach to the surface of carriers though hydrogen bonds and filamentous bridging. Following the effects of the polymeric chain of EPS, biomass accumulation and hydrodynamic shear force, the granules become stable and compact. In this process, physical, chemical and biological factors are associated with the granule formation. The anammox granule has a multiple-layer structure with two distinct regions. The plane structure is shown in Fig. 6B, the outer region is the attached anammox biofilm bound by EPS. The inner region is a stable carrier formed in the reactor. The image from the SEM observations supports the view that LB-EPS (Fig. 6F), TB-EPS (Fig. 6G) and filamentous bacteria (Fig. 6H) coexist in the granule and play a role in the granulation process.

Fig. 6 Mechanism of the attached biofilm anammox granule formation and disintegration (A) and the section of the anammox granule (B). Washed out sludge from the reactor after the transient substrate shock (C). Excessive EPS encasing the surface of anammox granule (D), and the formed granule sludge cakes (E). LB-EPS (F), TB-EPS (G) and filamentous bacteria (H) coexisting in the aggregate and responsible for the granulation process.

It has been reported that the attached film is structured with communities of cells enclosed in an EPS matrix and adhered to the surface of the carriers.^9,30 Therefore, EPS is viewed as an important mediator in the adhesion process. However, the adverse effects of excessive EPS on granule stability have rarely been reported. In this study, along with the increase in EPS concentration after transient substrate shock, the amount of SS in the effluent increased sharply. According to the results, some of the excessive EPS peeled off from the carrier, removing an abundance of anammox cells, as can be seen in the washed out sludge shown in Fig. 6C. Moreover, because the granules were encased in the excessive EPS (Fig. 6D) and were bonded to each other (Fig. 6E), the mass transfer efficiency was extremely reduced since the substrate must pass through the EPS layer to reach the anammox cells. In addition, because the produced bubbles accumulated in the granules agglomeration rather than being immediately discharged from the reactor, the density of the aggregates was reduced and the up-floating of granules occurred.

4. Conclusions

While appropriate EPS production benefits the biomass granulation and flocculation process, the adverse effect of excessive EPS caused by substrate chock was investigated in this study. A transient substrate shock of 2500 mg N L⁻¹ for 24 hours significantly decreased the treatment performance of the AAFEB reactor and destroyed the structure of the formed anammox granules. Accompanying with the transient substrate shock, excessive EPS of 89.6 ± 48.3 mg g⁻¹ VSS accumulated in the reactor and led to 35 ± 0.8% reduction in the granule settling velocity and 30.5 ± 0.9% reduction in the amount of total VSS, which led to the further deterioration of the reactor performance. EPS production was shown to be both substrate utilization-associated and substrate stress-associated. Depend on the results of batch experiment, the influent TN concentrate shock of higher than 400 mg N L⁻¹ should be avoided in case of the accumulation of LB-EPS. The batch experiment also indicated that the excreted LB-EPS under various substrate concentrations was responsible for variations in the granule settleability and hydrodynamic shear strength.

Acknowledgements

The authors wish to thank the Japan Society for the Promotion of Science (JSPS) for financial supporting this study.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra20097d

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