Effect of C/N Ratio on Nitrogen Removal of A/O-MBBR Process for Treating Mariculture Wastewater

CHEN Xi, XIANG Zhuangzhuang, HUANG Xiao, RONG Huimin,BAI Jie,4), and ZHAO Yangguo,4),*

1) College of Marine Life Science, Ocean University of China, Qingdao 266003, China

2) College of Environmental Science and Engineering, Ocean University of China, Qingdao 266100, China

3) Nanjing University of Information Science and Technology, Collaborative Innovation Center of Atmospheric Environment and Equipment Technology, Jiangsu Key Laboratory of Atmospheric Environment Monitoring and Pollution Control, Nanjing 210044, China

4) Key Laboratory of Marine Environment and Ecology (Ocean University of China), Ministry of Education,Qingdao 266100, China

Abstract In order to explore the effect of carbon and nitrogen (C/N) ratio on the performance of anoxic/aerobic-moving bed biofilm reactor (A/O-MBBR) process for treating mariculture wastewater, a laboratory-scale A/O-MBBR was conducted. The results showed that the reduction of C/N ratio was conducive to improving the removal efficiency of chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N), while inhibiting that of nitrite nitrogen (NO2--N) and nitrate nitrogen (NO3--N). The extracellular polymeric substances (EPS) in anoxic zone were significantly higher in concentration than that in aerobic zone although they both declined with decrease of C/N ratio. The result provides solid support for better controlling the pollution of mariculture wastewater.

Key words mariculture wastewater; C/N ratio; A/O-MBBR process; EPS

1 Introduction

The rapid development of mariculture led to the continuous increase of wastewater. The discharge of mariculture wastewater without any treatment will cause serious damage to the surrounding estuary ecosystem(Zhenget al., 2016). Meanwhile, it has been well known that the high salinity (＞10 g NaCl L-1) of mariculture wastewater produce high osmotic pressure on bacterial cells, which could cause cytoplasmic decomposition and cell activity reduction, and low organic matters and nitrogen removal efficiencies of organic matters and nitrogen (Nget al., 2005; Wanget al., 2017). Zhanget al.(2016) reported that the removal rates of ammonia nitrogen (NH4+-N) and total phosphorus (TP) were only 56%and 70% at high salinity (30 g L-1NaCl) condition by using a sequencing batch bioreactor (SBR) reactor to treat saline wastewater. Brazil (2006) used a rotating biological contactor to treat mariculture wastewater, and its treatment efficiency of NH4+-N was only 31.5%. Domesticated biofilm reactor has been proved to be suitable for the treatment of mariculture wastewater (Songet al.,2018; Zhanget al., 2020), the microorganisms in the reactor have adapted to the high salinity conditions and presented high removal efficiency for various pollutants.In addition, as a common wastewater treatment process,moving bed biofilm reactor (MBBR) possessed the advantages of less excess sludge, relative small volume,high solid-liquid separation efficiency, thus it was then extensive studied (Mielcareket al., 2016; Khanet al.,2018).

The ratio of chemical oxygen demand (COD) to total nitrogen concentration (C/N) was often referred to as one of the most critical factors affecting the removal of nutrients in the process of nitrification and denitrification. A high C/N ratio was beneficial to the growth of heterotrophic bacteria. However, the faster growth rate of heterotrophic bacteria would cause inhibition for autotrophic bacteria (Bassinet al., 2015). Maet al. (2013) illustrated that the reduction of the influent C/N ratio improved the nitrification efficiency of membrane bioreactor (MBR).Zhouet al. (2020) evaluated the performance of an upflow anaerobic filter-biological aerated filter (UAF-BAF)process with C/N radio ranging from 10 to 3. Their results showed that the removal efficiency of COD and NH4+-N was basically stable, but the removal efficiency of total nitrogen (TN) decreased from 68.7% to 50.6%.

C/N ratio affects functional microorganism populations and thus changes the structure of microbial communities.In the biological reactor, microorganisms existed in the form of microbial aggregates, such as biofilms, sludge flocs and particles (Niet al., 2009; Lenget al., 2015),while extracellular polymeric substances (EPS) was a three-dimensional matrix that surrounds and protects the microbial aggregates (Flemming and Wingender, 2001).EPS originated from microbial metabolism, cell lysis and sorption of constituents from wastewater, largely determined the characteristics and functions of biofilm and affected the performance of biological wastewater treatment (Shiet al., 2017). The EPS production of microorganisms was closely related to the environmental factors,such as pH, C/N ratio, temperature and dissolved oxygen(DO),etc. Among the factors, C/N ratio was one of the critical factors affecting EPS production and composition,and it was also an important factor affecting pollutant removal efficiency of water treatment process (Wanget al.,2015). Therefore, exploring the relationship between C/N ratio, EPS and pollutant removal efficiency will help to regulate wastewater biofilm and improve wastewater treatment effect. Wanget al. (2014) reported that proteins(PN) in loosely bound EPS (LB-EPS) increased with the decrease of C/N ratio, while polysaccharides (PS) in LB-EPS decreased. In addition, the production of EPS also has been considered as a crucial method to protect the microorganisms against the variation of influent salinity in biological wastewater treatment systems (Zhanget al., 2011). Janget al. (2013) reported that the concentration of PS and PN in EPS increased significantly with the improvement of salinity. Although the effect of C/N ratio on removal efficiency and EPS has been reported in activated sludge and biofilms treating saline wastewater, the removal efficiency of anoxic/aerobic-moving bed biofilm reactor (A/O-MBBR) treating mariculture wastewater and the effect of EPS of different functional units have not been well investigated.

In this study, the C/N ratio in the influent was changed by adjusting the COD, and the removal efficiency of various pollutants and the change of extracellular polymer content in different functional units during the reduction of the C/N ratio were investigated, so as to provided technical support for the application of A/O-MBBR process in mariculture wastewater treatment.

2 Materials and Methods

2.1 Experimental Equipment

The schematic diagram of A/O-MBBR device is shown in Fig.1, the device was made of plexiglass splices with an effective volume of 10 L. The reaction device was divided into anoxic compartment, aerobic compartment and sedimentation zone by cross wall, wherein an agitator was arranged in the anoxic zone. K3 cylindrical hollow filler made of polypropylene (PP) material was used for carrier,the diameter was 2.5 cm, the height was 1.2 cm, the specific gravity is greater than 0.96 g cm-3, and the filling rate of the carrier in aerobic zone and anoxic zone was 60%.The mariculture wastewater was pumped into the A/OMBBR device from the bottom of the anoxic zone, and stirred by a stirrer in the anoxic zone, then flowed upward to the overflow weir and then into the aerobic zone. The effluent of aerobic zone flowed into the sedimentation zone through the overflow weir, and then discharged. In addition, the reactor was also provided with a peristaltic pump to reflux nitration liquid to an anoxic zone, and the reflux ratio is set to 100%.

Fig.1 The schematic diagram of the A/O-MBBR-DMBR process test system.

2.2 Seed Sludge and Synthetic Wastewater

The sludge was collected from the secondary sedimentation tank in Qingdao Tuandao Sewage Treatment Plant.The influent was artificially prepared as shown in Table 1.The average concentrations of COD, NH4+-N, NO2--N and NO3--N in the influent were 163.8, 4.7, 0.6 and 4.9 mg L-1respectively.

Table 1 Component of simulated mariculture wastewater (mg L-1)

2.3 Reactor Operation

During the process of start-up, the ambient temperature was maintained at 25℃, and the salinity was increased from 5‰ to 30‰ at a rate of 1‰ per day. When the domestication of the reactor was basically completed (the C/N was 16, HRT was 8 h, salinity was 30‰), the influent COD concentration was reduced to control the C/N ratio at 18, 16, 14, 12, 10, 8, 6 and 4, respectively, and then the bioreactor was performed with the constant C/N ratio for 15 days in each stage.

2.4 Analytical Methods

COD, NH4+-N, NO2--N and NO3--N were measured according to the Standard Methods (Sunet al., 2017).EPS from the biofilm was extracted using a heat extraction method at 80℃ for 30 min. The PN and PS were measured using the Lowry procedure (Zhanget al., 2019)and phenol-sulfuric acid method (Dinget al., 2018) with bovine serum albumin and glucose as the standard, respectively. All the tests were performed in triplicates and the average of data was reported.

3 Results and Discussion

3.1 Effect of Decrease of C/N on Organic Matter Removal

The removal efficiency of organic pollutants in A/OMBBR process reactor under different C/N ratios is shown in Fig.2. Under C/N ratio of 18, the average effluent COD concentrations were 17.86 mg L-1with average efficiency levels of 90.26%. The removal of COD was enhanced by the bioreactor with the decrease of effluent carbon source, in which COD removal efficiency reached to the highest value of 99.10% and the average effluent was less than 1 mg L-1when C/N ratio was 8. Under all C/N ratio conditions, the reactor kept a high removal efficiency of COD, with an average removal rate of 94.16%.The reduction of C/N within proper range was helpful to improve the ability of degrading organic matter by A/OMBBR process.

Fig.2 Effect of reducing C/N ratio on COD removal rate.

Although the high salinity stress and the influent C/N ratio change, the A/O-MBBR reactor still maintained a high COD removal efficiency (94.16%) compared to MBBR process under municipal wastewater (Leyva-Díazet al., 2016). These observations could be ascribed to the fact that the microorganisms in reactor were cultivated gradually using sewage sludge seed and seawater. The diverse halotolerant microorganisms in reactor laid the foundation for stable and efficient organics removal performance as well as capability for nitrogen removal (Songet al., 2020). Research showed that the COD in A/OMBBR system was mainly removed by denitrification in the pre-anoxic zone when the organic load was about 3 g(m-2d-1). When the organic load reached 5.3 g (m-2d-1) or higher, the aerobic zone played a more important role in the whole COD removal process (Limaet al., 2016).Hence, in this study, high COD removal rate might also be due to the existence of pre-anoxic zone by which various organic removals were enhanced.

3.2 Effect of Reducing C/N Ratio on Nitrogen Removal Performance

3.2.1 Nitrification process

Fig.3 demonstrated the NH4+-N removal performance of A/O-MBBR reactor during the whole experiment. It was found that the NH4+-N removal efficiency was improved with the decreasing C/N ratio. Meanwhile, the effluent average NH4+-N removal efficient of reactor remained at high levels (93.68%) throughout the experiment. The optimal NH4+-N removal efficient occurred at C/N ratio of 6 and the removal efficiency was 99.47%.The above results reveal that the reduction of C/N ratio is an effective measure to improve the removal rate of NH4+-N within a certain range. It was noticeable that most of the NH4+-N was removed by biological degradation in aerobic zone. These data indicated that aerobic zone could provide a consistent high removal efficiency of NH4+-N.

Fig.3 Effect of reducing C/N ratio on NH4+-N removal rate.

In the phase of the decrease of C/N ratio, the system showed higher nitrification efficiency with an average of 93.68%. This is different from the findings of Liuet al.(2020), who found a decrease of removal efficiency of NH4+-N for the reducing C/N ratio above 9 in single-stage MBBR reactor. Limaet al. (2016) reported that nitrification of single-stage MBBR process was inhibited under the condition of relatively high C/N ratio of influent water.The biofilm in the anoxic zone in the present study reduced influent COD concentration of aerobic zone, which might have enhanced nitrification even at the higher C/N ratio.

Carbon source is an important factor for denitrification,and the lack of organic matter would lead to insufficient electron donor for denitrifying bacteria, while high concentration of organic matter would inhibit the activity of nitrifying bacteria (Heet al., 2009). In this study, the C/N ratio was changed by reducing the influent COD concentration, and the influent NH4+-N concentration remained unaltered. The decrease of influent COD concentration and the consumption of COD in the anoxic zone established a relatively low C/N ratio environment in the subsequent aerobic zone. Under such conditions, the competition for the space and substrate between fast-growing heterotrophic bacteria and slow-growing nitrifying bacteria in the biofilm became less intense (Nogueiraet al.,2002; Carreraet al., 2004). Afterwards, nitrifying bacteria grew better and the NH4+-N removal rate increased continuously. Therefore, the lower C/N ratio improved the NH4+-N removal rates in A/O-MBBR process. These results are in good agreement with previous investigations on A/O-MBBR system treating municipal wastewater(Limaet al., 2016).

3.2.2 Denitrification process

The influence of reducing C/N ratio on denitrification performance of A/O-MBBR reactor is shown in Fig.4 and Fig.5. During the C/N ratio of 18, there was 0.28 mg L-1of NO3--N left in the effluent, and removal efficiency was 94.25%. The reduction of C/N ratio had a significant impact on NO3--N elimination and the removal rate decreased to below 80% when the C/N ratio was lower than 14. The worst NO3--N removal occurred at C/N ratio of 8,where the average removal efficiency and average effluent concentration were 56.71% and 2.11 mg L-1. The effect of the reducing C/N ratio on NO3--N and NO2--N was synchronous, and the removal efficiency was positively correlated with C/N ratio. The average removal rate of NO2--N was reduced to -102.61% and the effluent concentration was 1.23 mg L-1, resulting in a significant nitrite accumulation at the C/N ratio of 4.

Fig.4 Effect of reducing C/N ratio on NO3--N removal rate.

Fig.5 Effect of reducing C/N ratio on NO2--N removal rate.

The C/N ratio decrease caused an obvious drop of denitrification efficiency, mainly due to carbon limitation for the heterotrophic denitrification. Owing to most of denitrifying bacteria were heterotrophic microorganisms,and a high C/N ratio can provide sufficient organic matters as electron donors for denitrification (Renet al.,2014). The organic matter was gradually insufficient as the decrease of C/N ratio and affected the activity of denitrifying bacteria, resulted in the reduction of removal efficiency, which was in accordance with previous research (Panet al., 2020).

As an intermediate product of denitrification, NO2--N was accumulated in reactors during denitrification process. Isakaet al. (2012) found that NO3--N reduction required lower activation compared to NO2--N reduction,so the reduction process of NO3--N occurred easier than NO2--N under the same conditions. Geet al. (2012) have proposed that the activity of NO2--N reductase was inhibited due to the electronic competition between NO2--N reductase and NO3--N. It can be concluded that when carbon source is insufficient, NO3--N will be preferentially removed and NO2--N will accumulate. As shown in the present study, with C/N of 4, the effluent NO2--N concentration was 1.23 mg L-1and accumulation of NO2--N occurred due to insufficient carbon source supply.However, with relative higher C/N ratio of 6, the average effluent concentration of NO2--N dropped to less than 0.2 mg L-1, and the accumulated NO2--N was completely removed.

3.3 Changes of EPS Under Different C/N Ratio

3.3.1 Changes of polysaccharides in different parts of bioreactor

PN and PS were the main components of EPS matrix(Sponza, 2003). For this study, the changes of PN and PS contents in different parts of bioreactor under different C/N conditions are analyzed. Fig.6 showed that PS content in anoxic compartment was richer than that in aerobic zone. The highest C/N ratio resulted in the highest content of PS (201.3 mg g-1SS). Afterwards, as the C/N was decreased, the content of PS in anoxic zone was gradually reduced up to 73.7 mg g-1SS. Compared with C/N ratio of 18, the PS content under C/N ratio of 6 declined by almost 63%. The trend of the content of PS in aerobic zone with varying C/N ratio was different from that in anoxic zone. When the C/N ratio was above 10, the reduction of C/N ratio had no obvious effect on PS in the aerobic zone,but the average content of PS was steeply decreased to 49.04 mg g-1SS when the C/N ratio was decreased to 8.After that, the content of PS began to gradually increase,but did not return to the previous level.

Fig.6 Effect of reducing C/N ratio on PS in (A) anoxic zone and (B) aerobic zone.

The PS content in anoxic zone was higher than that in aerobic zone, which might be related to the different EPS production between heterotrophic denitrifying bacteria and autotrophic nitrifying bacteria. Sepehri and Sarrafzadeh (2018) found that nitrifying bacteria produce less EPS than heterotrophic bacteria. Compared with aerobic zone, the fact of higher PS content in anoxic zone depended on the higher richness of heterotrophic denitrifying bacteria predominating in anoxic zone.

In biofilm system, PS was essential for the stability of granules because they formed a matrix that favored the deposition of proteins, lipids, α-polysaccharides and cells(Karunakaran and Biggs, 2011). Mengistuet al. (1994)found that microorganisms lacked necessary nutrients,such as nitrogen, would restrict cell growth and use excess carbon sources for PS biosynthesis. Carbon sources excessive, adenosine triphosphate (ATP) produced by microorganisms increased, which was helpful for PS biosynthesis (Miqueletoet al., 2010). According to the previous studies, it could be inferred that the reduction of available carbon source of microorganisms led to the gradual decrease of PS production. However, the content of PS in the aerobic zone remained basically unchanged,possibly because autotrophic nitrifying bacteria were dominant in aerobic zone, and more autotrophic metabolism can be used to obtain energy at low C/N ratio (Maet al.,2013).

3.3.2 Changes of proteins in different parts of bioreactors

Fig.7 showed that the reduction of C/N ratio caused the change of PN content in biofilm. The content of PN in aerobic zone exhibited a similar trend to that in anoxic zone. The content of PN depended on C/N ratio and was positively correlated with C/N ratio. When the C/N ratio decreased from 18 to 4, the PN content in aerobic zone and anoxic zone decreased from 322.2 mg g-1SS and 393.5 mg g-1SS to 155.5 mg g-1SS and 180.8 mg g-1SS respectively. No matter in anoxic or aerobic zone, the content of PN was obviously higher than that of PS, and the content of PN in anoxic zone was slightly higher than that in aerobic zone.

Fig.7 Effect of reducing C/N ratio on PN in (A) anoxic zone and (B) aerobic zone.

Miqueletoet al. (2010) reported that a high C/N ratio promoted more production of EPS. EPS production is always considered to be governed by the factors such as organic loading, carbon source, and limited nutrients(Fenget al., 2012). With the stepwise reduction of influent COD, there are less and less bio-available carbon sources, resulting in the decrease of EPS. This result is applicable for PN in the present study. The insufficient carbon source (low C/N ratio) would promote microorganisms degrade EPS into small molecular substances as carbon source and energy for their own growth (Shenget al., 2010), resulting in a decrease of PN production. On the other hand, the decline of C/N ratio would lead to the decrease of active biomass in the biofilm (Zhou and Xu,2020), and the PN content was thereby decreased.

Under the condition of limited influent organic matter,most of the organic carbon was used for biosynthesis instead of extracellular PS generation (Yeet al., 2011). The content of PN in biofilm was significantly higher than that of PS. This finding was in accordance with previous study (Mielcareket al., 2020). It might be due to the fact that a significant role of proteins in developing a highly proliferating biofilm. Higher PN content was also helpful for microorganism adhesion and promoting biofilm growth (Zhanget al., 2019).

4 Conclusions

This study investigated the effect of C/N ratio on pollutant removal performance of A/O-MBBR reactor in treating mariculture wastewater. When the C/N ratio decreased from 18 to 4, the reactor kept high COD and NH4+-N removal efficiency of 94.16% and 93.68%. However, in order to ensure the acceptable denitrification of A/O-MBBR process, it is necessary to keep the influent C/N above 14. In addition, the changes of C/N ratio affected EPS, the content of PN was positively correlated with C/N ratio, while the content of PS in aerobic zone was less affected by C/N ratio. In each zone PN constituted the dominant part of EPS, and the content of PN and PS in anoxic zone was higher than that in aerobic zone.

Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2018 YFC1407601), the Start-up Foundation for Introducing Talent of NUIST and Guangxi Innovation Driven Development Project (major science and technology project).