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Simultaneous removal of organic matter and nitrogen by a heterotrophic nitrifying–aerobic denitrifying bacterial strain in a membrane bioreactor
Bioresource Technology 143 (2013) 83–87
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Simultaneous removal of organic matter and nitrogen by a heterotrophic nitrifying–aerobic denitrifying bacterial strain in a membrane bioreactor Yan-Chun Yao a,b, Qing-Ling Zhang a, Ying Liu a, Zhi-Pei Liu a,⇑ a b
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
h i g h l i g h t s Bacillus methylotrophicus L7 was inoculated into a membrane bioreactor solely. This single bioreactor was applied for treating artificial sewage aerobically. A simultaneous removal of COD and TN was achieved efficiently. This was different from traditional O/A system and SBR.
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Article history: Received 12 April 2013 Received in revised form 27 May 2013 Accepted 29 May 2013 Available online 4 June 2013 Keywords: Bacillus methylotrophicus L7 MBR Heterotrophic nitrification–aerobic denitrification Simultaneous removal of organic matter and nitrogen
a b s t r a c t A heterotrophic nitrifying–aerobic denitrifying bacterial strain, Bacillus methylotrophicus L7, was inoculated solely into a submerged membrane bioreactor (MBR) for continuous treatment of artificial sewage. The running conditions were also optimized for improvement of the treatment efficiency. The results indicated that inoculation of this single strain in a single reactor under constant aerobic conditions resulted in simultaneous removal of organic matter and nitrogen, in striking contrast to traditional aerobic nitrification–anaerobic denitrification treatment system and the sequencing batch reactor (SBR) systems. The optimal running conditions for the MBR were dissolved oxygen (DO) 4.5 mg/L, pH 7.5, loading ammonia <100 mg/L, and C/N ratio 3.5. Under these conditions, the removal percentages of chemical oxygen demand (COD), NHþ 4 –N, and TN as high as 96%, 77.5% and 53%, respectively, were achieved without nitrite accumulation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Chemical oxygen demand (COD), reflecting organic matter content, and nitrogen content are two key parameters monitored during wastewater treatment. The most efficient method for removal of nitrogen from wastewater is based on the processes of nitrification (by autotrophic nitrifiers) and denitrification (by anoxic denitrifiers). Nitrifiers convert ammonia to nitrite and then to nitrate. Denitrifiers reduce nitrate to nitrite and then to N2 (Joo et al., 2005). Organic matter interferes with the activity of nitrifiers (Kulikowska et al., 2010) but is necessary for denitrifiers. Oxygen is required for the growth of nitrifiers but is toxic to denitrifiers (Lloyd et al., 1987). Because of these opposing tolerances to organic matter and oxygen, it is impossible to achieve simultaneous removal of these two components in a single bioreactor under ⇑ Corresponding author. Address: Institute of Microbiology, Chinese Academy of Sciences, West Beichen Road, Chaoyang District, Beijing 100101, PR China. Tel.: +86 10 62653757; fax: +86 10 62538564. E-mail address: [email protected] (Z.-P. Liu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.05.120
constant aerobic conditions. Removal strategies therefore require separate treatment systems and precise control of treatment conditions. Nitrification by autotrophic bacteria that have slow growth rates requires a long retention time of flowing wastewater in the reactor (Jetten et al., 1997; Muller et al., 2003). These factors all tend to increase the cost of wastewater treatment. An increasing number of studies have focused on new biological nitrogen removal technologies. One exciting development is the discovery of ‘‘heterotrophic nitrifying–aerobic denitrifying’’ bacteria. Bacteria of this type have been isolated from many different environments; examples include Arthrobacter sp. (Verstrae and Alexande, 1972), Alcaligenes faecalis No. 4 (Joo et al., 2005), Bacillus sp. strains (Yang et al., 2011), Pseudomonas sp. (Wan et al., 2011), and Bacillus methylotrophicus L7 (Zhang et al., 2012). In comparison with traditional methods, nitrogen removal by heterotrophic nitrification and aerobic denitrification has several advantages: (i) The utilization of organic substrates and tolerance to oxygen by these bacteria are compatible, making it possible to achieve the simultaneous removal of organic matter and nitrogen via simultaneous nitrification and denitrification (SND) in a single reactor (Third
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et al., 2005). (ii) The process of denitrification counters the acidification caused by nitrification and tends to maintain a stable pH in the reactor. (iii) The diversity of possible substrates and products of heterotrophic nitrification facilitates mixed culture with a variety of bacterial strains and expands the scope of possible applications (Marazioti et al., 2003). Recent studies of heterotrophic nitrifying–aerobic denitrifying bacterial have focused on substrate removal, accumulation of intermediates, and production of gaseous nitrogen compounds (Wan et al., 2011; Zhang et al., 2012). The characteristics and optimal reaction conditions of these bacteria were investigated in sequencing batch flask culture (Kulkarni, 2013). However, no study to date has addressed the removal of organic matter and nitrogen during the continuous sewage treatment via SND by these bacteria. Previously, the heterotrophic nitrification–aerobic denitrification abilities of B. methylotrophicus L7 were described (Zhang et al., 2012). In the present study, these abilities of strain L7 were further assessed and its potential of simultaneous removal of organic matter and nitrogen was also investigated, by inoculating the strain into a membrane bioreactor designed for continuous wastewater treatment. The results suggest a basis for an alternative nutrient removal method for domestic wastewater. 2. Methods 2.1. Bacterial strain and culture condition The bacterial strain B. methylotrophicus L7 was cultured in LB broth at 30 °C for 24 h. Cells were harvested by centrifugation at 9000g for 30 min, washed twice with sterile saline solution, and the cell mass was resuspended in sterile saline solution to 3000 mg/L (wet weight) for use as the inoculum in a membrane bioreactor. 2.2. Membrane bioreactor and startup and operating conditions The laboratory-scale submerged membrane bioreactor (MBR) with a total effective volume of 29 L (Fig. 1) was fitted with a polypropylene hollow fiber membrane module (average pore diameter 0.2 lm; Hangzhou Kaihong Membrane Technology Co. Ltd., Hang Zhou, China) was used. Polypropylene spherical suspended fillers were used as a biological carrier, with a filling rate of 30% (v/v). A continuous flow of wastewater was provided by a delivery pump installed between the storage tank and the reaction tank. A fully aerobic operation throughout the experimental period was ensured by the introduction of compressed air via aerator pipes at
Table 1 The quality of artificial wastewater. Items
COD (mg/L)
NHþ 4 –N (mg/L)
TP (mg/L)
pH
Value
1000
120
7.8–9.4
7.5
the bottom of the reactor. The air bubbles provided oxygen for biological needs and a hydrodynamic scouring effect to reduce membrane fouling (Chang et al., 2011). For startup, the reactor was inoculated with 6 L of strain L7 cell suspension prepared as above and run at room temperature. The influent was controlled to hydraulic retention time (HRT) 24 h, dissolved oxygen (DO) 3.5–4.5 mg/L, and pH 7.5, ammonia content 30–40, and 150–300 mg/L. The startup period was approximately 30 days. For continuous treatment of artificial wastewater after startup, the running conditions were: temperature 25–30 °C, HRT 24 h; DO 4.5 mg/L; influent pH 7.5, influent NHþ 4 –N 120 mg/L, C/N ratio 3.5, or otherwise stated. 2.3. Artifical wastewater Artificial wastewater, used as an imitation of sanitary, was composed of glucose, peptone, KH2PO4, NH4Cl and tapwater (Table 1), plus appropriate trace amounts of calcium chloride, copper sulfate, and magnesium sulfate. 2.4. Analytical methods Bacterial growth was determined by monitoring the optical density at 600 nm (OD600) with a spectrophotometer (model UV7200, UNICO, Shanghai, China). NO 2 –N level was determined by N-(1-naphthyl)-1, 2-diaminoethane dihydrochloride spectrophotometry (Mahmood et al., 2009). Ammonia level was determined by Nessler assay (Zhang, 2009). Total nitrogen (TN) content was determined by peroxydisulfate oxidation with a UV spectrophotometric method (Ebina et al., 1983). COD was determined using a COD instrument COD (model CTL-12, Chengde Huatong Environmental Protection Equipment Co. Ltd., Chengde, China). DO was determined with a DO meter (model JPSJ-605Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China). pH was measured with a pH meter (model PB-10, Sartorius, Germany). 3. Results and discussion 3.1. Reactor startup During the 30-day startup period, the removal percentages of COD, ammonia, and total nitrogen (TN) increased gradually to 80.0%, 66.7% and 48.6%, respectively, on day 30 (data not shown). These findings indicate that a successful startup was achieved, according to the criteria of Trigo et al. (2006) and Xue et al. (2008). 3.2. Factors that affected the removal efficiency To improve the treating performance of the MBR, several factors as described below were evaluated for their effects on the removal efficiency of COD and nitrogen.
Fig. 1. Schematic diagram of the membrane bioreactor (MBR) system. (1) Storage tank; (2) delivery pump; (3) MBR; (4) membrane model; (5) air pump; (6) suction pump.
3.2.1. Effect of DO DO was decreased gradually from 6.0 to 1.5 mg/L during a period of 72 days (Fig. 2). A stable, high average COD removal percentage (85%) was maintained throughout this period (Fig. 2A), indicating a good biological degradation capability with consequent high effluent quality. In contrast, the removal percentages
85
6
50 40 30 20
+
3 2 1
10 0 42
50
58
66
74
82
90
-
4
20 3
15
DO
5
25
2
10
-
-
NO2 -N, NO3 -N in effluent (mg/L)
6
30
1
5 0
0 34
42
50
58
66 74 82 Run time (d)
90
7.5 7 6.5 6 110
114
118
122
126
130
134
Run time (d)
7
B
35
8
98
Run time (d)
40
8.5
98
Fig. 2. Effect of DO on wastewater treatment efficiency. (A) COD and nitrogen removal ratios. (B) Nitrite and nitrate concentration in effluent. }, COD removal percentage. 4, NHþ 4 –N removal percentage. h, TN removal percentage. , DO concentration. j, NO 3 –N concentration. N, NO2 –N concentration.
of ammonia and TN were 20–25% and 12–20%, respectively (Fig. 2A); much lower than during the startup period. This finding may be due to the much higher influent ammonia content during the experimental period (120 vs. 25–30 mg/L). Nitrogen removal tended to vary with decrease DO. The NHþ 4 –N removal percentage did not vary significantly within the DO range of 6.0–2.8 mg/L. When DO fell below 2.0 mg/L, the NHþ 4 –N removal percentage decreased sharply (Fig. 2A). These findings indicate that the ammonia-oxidizing ability of strain L7 was greatly inhibited by low DO (<2.0 mg/L). Thus, oxygen is required for heterotrophic ammonia oxidation, as for autotrophic ammonia oxidation. The removal percentages of TN did not undergo changes as great as those of ammonia within the range of DO tested (Fig. 2A), suggesting that the aerobic denitrification performance of strain L7 was influenced to a lesser degree by DO. This result was consistent with those reported previously for certain aerobic denitrifying strains (Lloyd et al., 1987; Patureau et al., 2000), which maintained their denitrification ability when DO was close to or greater than oxygen saturation in the air. DO has been described as an important factor for aerobic denitrification in a few strains (Huang and Tseng, 2001). The NO 2 –N concentration in effluent remained low throughout the experimental period, whereas the NO 3 –N concentration increased when DO was >6.5 mg/L or <4 mg/L (Fig. 2B). This finding was consistent with the denitrification properties of strain L7 as observed in our previous study (Zhang et al., 2012). NO 2 –N is a product of ammonia oxidation and a connecting point between nitrification and denitrificaion, and can thus be converted not only to nitrogen gas but also to nitrate via nitrification. Such coordination varies depending on the bacterial species and the culture conditions (Sakai et al., 1997). In this case, the accumulation of nitrate at DO >6.5 mg/L or <4.0 mg/L may have contributed to the slightly higher conversion of nitrite to nitrate under these conditions, the TN removal was relatively stable.
40
10
B
35
9.5
30
9
25
8.5
20
8
15
7.5
-
34
9.5 9
106
0
10
A
pH
4
DO
5
70 60
100 90 80 70 60 50 40 30 20 10 0
pH
7
A
90 80
COD, NH4 -N, TN removal (%)
100
NO2 -N, NO3 -N in effluent (mg/L)
+
COD, NH4 -N, TN removal (%)
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10 5
7
0
6.5
-5
6 106
110
114
118
122
126
130
134
Run time (d) Fig. 3. Effect of pH on wastewater treatment efficiency. (A) COD and nitrogen removal ratios. (B) Nitrite and nitrate concentrations in effluent. }, COD removal percentage. 4, NHþ 4 –N removal percentage. h, TN removal percentage. d, pH. j, NO 3 –N concentration. N, NO2 –N concentration.
Taken together, these results suggest that an optimal DO level may enhance the activity of heterotrophic nitrification and aerobic dentrification, resulting in thorough biodegradation of organic matter. Taking into account nitrification and denitrification, COD and TN removal, and running cost, the optimal DO for strain L7 was estimated as 4.5 mg/L. 3.2.2. Effect of influent pH Efficient COD and nitrogen removal were observed in our system at an influent pH of 7.0–9.0, with removal percentages of 89.0% for COD and 23.4% for NHþ 4 –N (Fig. 3A). A slightly alkaline environment was conducive to heterotrophic nitrification because more free ammonia (NH3) was present in the wastewater; presumably, the substrate utilized by ammonia monooxygenase (AMO) was NH3 rather than NHþ 4 (Mevel and Prieur, 2000). The concentration of NO 2 –N in effluent was negligible, while the concentration of NO 3 –N ranged 13–35 mg/L (Fig. 3B). The NO 3 –N concentration was low within the effluent pH ranged from 7.0–9.0 and minimal at pH 7.5, therefore pH 7.5 was selected as optimal. 3.2.3. Effect of C/N ratio in influent The influent concentration of NHþ 4 –N was 60 mg/L in our experiment. C/N ratios of 0.7, 1.2, 1.6, 3.2 and 5.0 were established by adding appropriate amounts of glucose. The COD removal percentages were high (90–96.2%) at each of these C/N ratios (Fig. 4A). The removal percentages of ammonia and TN were correlated with C/N ratio (Fig. 4A) and were strikingly higher for C/N ratio >3.2. The removal percentages of NHþ 4 –N and TN were <55% and <22% at C/N ratio <1.6 and increased to 75% and 53% at C/N ratio 3.2 and 5.0, respectively. Thus, there was a significantly improvement in ammonia and TN removal at the higher C/N ratios. A previous study found that sufficient carbon in wastewater was essential
Y.-C. Yao et al. / Bioresource Technology 143 (2013) 83–87
120
Table 2 Effects of loading ammonia content on the removal of ammonia.
A
100
Influent average NHþ 4 –N (mg/L)
Effluent average NHþ 4 –(mg/L)
NHþ 4 –N removal (%)
Removal efficiency (g/m3/ d)
59.2 107.6 184 300
15.8 48.2 117 213.4
73.3 55.2 36.4 28.9
43.4 59.4 67.0 86.7
80 60 40 20 0 150 155 Run time (d)
160
165
70
NH4 -N (mg/L)
B
16
+
14 12 10 8 6 4
40 30 20
0 200
2
204
208
204
208
0 140
145
150 155 Run time (d)
160
165
Fig. 4. Effect of C/N on wastewater treatment efficiency. (A) COD and nitrogen removal ratios. (B) Nitrite and nitrate concentrations in effluent. }, COD removal percentage. 4, NHþ 4 –N removal percentage. h, TN removal percentage. j, NO3 –N concentration. N, NO –N concentration. 2
for the efficient removal of ammonia by Alcaligenes faecalis strain No. 4 (Joo et al., 2006), perhaps because an insufficient carbon inhibits the growth and nitrification ability of heterotrophic nitrifying strains, resulting in reduced ammonia removal. In terms of ammonia removal, heterotrophic nitrification is quite different from the autotrophic nitrification. In another study, autotrophic nitrification was greatly reduced or eliminated at C/N ratios >0.25 (Okabe et al., 1996). The efficient nitrogen removal by heterotrophic nitrifying strains at high C/N ratios results in simultaneous removal of COD and nitrogen. This ability is highly advantageous in wastewater treatment because it eliminates the need to reduce organic matter content prior to nitrification (as in traditional nitrogen removal methods). Nitrite levels in effluent were low at all C/N ratios tested (Fig. 4B). Nitrate levels varied depending on the C/N ratio and were minimal (<13 mg/L) at the ratio 3.2 (Fig. 4B). These findings suggest that a sufficient amount of carbon is also essential for the denitrification. The limited formation of NO 2 –N (<1 mg/L) by strain L7 at various C/N ratios satisfies the criteria for a well-functioning SND system with minimal nitrite accumulation (Third et al., 2005). Based on considerations of COD removal, ammonia removal, TN removal, and running cost, C/N ratio of 3.5 was selected as optimal. 3.2.4. Effect of influent ammonia concentration In this laboratory-scale system, a higher influent ammonia concentration promoted NHþ 4 –N removal, concentrations of 59.2, 107.6, 184, 300 mg/L were associated with removal rate of 43.4, 59.4, 67.0, 86.7 g/m3/d, respectively (Table 2). Strain L7 is tolerant of high ammonia loads (Zhang et al., 2012). In contrast, the ammonia removal percentages were 73.3% and 28.9% at influent ammonia concentrations of 59.2 and 300 mg/L, respectively, resulting in a clear increase of effluent ammonia concentration in the latter case. Based on consideration of the effluent ammonia concentration in effluent and the removal percentage of NHþ 4 –N
800 700 COD (mg/L)
-
50
10
-
NO2 -N, NO3 -N in effluent (mg/L)
18
20 18 16 14 12 10 8 6 4 2 0 -2
A
60
212 216 Run time (d)
220
224
228
220
224
228
-
145
-
140
NO2 -N, NO3 -N (mg/L)
+
COD, NH4 -N, TN removal (%)
86
B
600 500 400 300 200 100 0 200
212
216
Run time (d) Fig. 5. Wastewater treatment efficiency under optimal conditions as described in þ the text. (A) The NHþ 4 –N concentration in influent and NH4 –N, nitrate, and nitrite concentrations in effluent. (B) COD in influent and effluent. }, COD in influent. , þ COD in effluent. s, NHþ 4 –N in influent. d, NH4 –N in effluent. j, NO3 –N concentration. N, NO 2 –N concentration.
as determining factors for system treatment efficiency, the optimal ammonia load for the bioreactor is <100 mg/L. 3.3. Wastewater treatment efficiency under the optimal conditions The running parameters used in this experiment were: loading NHþ 4 –N concentration 60 mg/L, C/N ratio 3.5, pH 7.5, DO 4.5 mg/L. The results obtained under these optimal conditions are shown in Fig. 5. An average COD removal percentage of 92.3% was maintained during the entire experimental period (Fig. 5B). The maximal NHþ 4 –N removal percentage was 77.5%, and the average TN removal percentage was 52% (Fig. 5A). Nitrite concentration was maintained at a low level (<0.2 mg/L; Fig. 5A). These findings all indicate that the system was stable, feasible and had excellent treatment efficiency. In this study, simultaneous removal of COD and nitrogen was achieved using a single strain under relatively constant conditions. This phenomenon presents a striking contrast to traditional methods of aerobic–anaerobic nitrogen removal treatment. In the traditional methods, (i) ammonia is transformed to nitrite and then to nitrate (but TN is not removed) in the aerobic phase, and this
Y.-C. Yao et al. / Bioresource Technology 143 (2013) 83–87
process in inhibited by organic matter; (ii) nitrogen is removed only in the anaerobic phase, through consumption of a large amount of organic compounds (Joo et al., 2006; Kulikowska et al., 2010). This method also differs from the sequencing batch reactor (SBR) system in that it does not require alternation of running conditions and is thus more easily to be controlled and stable. In view of the above considerations, it appears that strain L7 might provide a less expensive alternative for treatment of urban sewage. Typical parameters of sewage (COD 300–600 mg/L, ammonia concentration 40–80 mg/L, pH 7.5) are consistent with efficient nitrogen removal performance by strain L7. 4. Conclusion Artificial sewage was treated by a submerged membrane bioreactor (MBR) with a heterotrophic nitrifying–aerobic denitrifying strain, Bacillus methylotrophicus L7, as inoculum. This single strain achieved simultaneous removal of organic matter and nitrogen from the sewage. The optimal running conditions for the MBR were DO 4.5 mg/L, pH 7.5, loading ammonia <100 mg/L, and C/N ratio 3.5. Under these conditions, the removal percentages of COD, NHþ 4 –N and TN as high as 96%, 77.5%, and 53%, respectively, were achieved without nitrite accumulation. Strain L7 provides an effective and economical alternative method for treatment of urban sewage. Acknowledgements This study was supported by Grants from the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KJCX2YW-L08). We thank Dr. S. Anderson for English language editing of the manuscript. References Chang, C.-Y., Tanong, K., Xu, J., Shon, H., 2011. Microbial community analysis of an aerobic nitrifying–denitrifying MBR treating ABS resin wastewater. Bioresour. Technol. 102, 5337–5344. Ebina, J., Tsutsui, T., Shirai, T., 1983. Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Water Res. 17, 1721–1726. Huang, H.K., Tseng, S.K., 2001. Nitrate reduction by Citrobacter diversus under aerobic environment. Appl. Microbiol. Biotechnol. 55, 90–94. Jetten, M.S.M., Logemann, S., Muyzer, G., Robertson, L.A., de Vries, S., van Loosdrecht, M.C.M., Kuenen, J.G., 1997. Novel principles in the microbial conversion of nitrogen compounds. Ant. Leeuwenhoek 71, 75–93.
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Joo, H.S., Hirai, M., Shoda, M., 2005. Characteristics of ammonium removal by heterotrophic nitrification–aerobic denitrification by Alcaligenes faecalis No. 4. J. Biosci. Bioeng. 100, 184–191. Joo, H.-S., Hirai, M., Shoda, M., 2006. Piggery wastewater treating using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification. Water Res. 40, 3029–3036. Kulikowska, D., Jozwiak, T., Kowal, P., Ciesielski, S., 2010. Municipal landfill leachate nitrification in RBC biofilm-process efficiency and molecular analysis of microbial structure. Bioresour. Technol. 101, 3400–3405. Kulkarni, P., 2013. Nitrophenol removal by simultaneous nitrification denitrification (SND) using T. pantotropha in sequencing batch reactors (SBR). Bioresour. Technol. 127, 273–280. Lloyd, D., Boddy, L., Davies, K.J.P., 1987. Persistence of bacterial denitrifications capacity under aerobic conditions-the rule rather than the exception. FEMS Microbiol. Ecol. 45, 185–190. Mahmood, Q., Zheng, P., Hayat, Y., Jin, R.C., Azim, M.R., Jilani, G., Islam, E., Ahmed, M., 2009. Effect of nitrite to sulfide ratios on the performance of anoxic sulfide oxidizing reactor. Arabian J. Sci. Eng. 34 (1A), 45–54. Marazioti, C., Kornaros, M., Lyberatos, G., 2003. Kinetic modeling of a mixed culture of Pseudomonas denitrificans and Bacillus subtilis under aerobic and anoxic operating conditions. Water Res. 37, 1239–1251. Mevel, G., Prieur, D., 2000. Heterotrophic nitrification by a thermophilic Bacillus species as influenced by different culture conditions. Can. J. Microbiol. 46, 465– 473. Muller, A., Wentzel, M.C., Loewenthal, R.E., Ekama, G.A., 2003. Heterotroph anoxic yield in anoxic aerobic activated sludge system treating municipal wastewater. Water Res. 37, 2435–2441. Okabe, S., Oozawa, Y., Hirata, K., Watanabe, Y., 1996. Relationship between population dynamics of nitrifiers in biofilms and reactors performance at various C:N ratios. Water Res. 30, 1563–1572. Patureau, D., Bernet, N., Delgenès, J.P., Moletta, R., 2000. Effect of dissolved oxygen and carbon–nitrogen loads on denitrification by an aerobic consortium. Appl. Microbiol. Biotechnol. 54, 535–542. Sakai, K., Nakamura, K., Wakayama, M., Moriguchi, M., 1997. Change in nitrite conversion direction from oxidation to reduction in heterotrophic bacteria depending on the aeration conditions. J. Ferment Bioeng. 84 (1), 47–52. Third, K.A., Gibbs, B., Newland, M., Cord-Ruwisch, R., 2005. Long-term aeration management for improved N-removal via SND in a sequencing batch reactor. Water Res. 39, 3523–3530. Trigo, C., Campos, J.L., Garrido, J.M., Mendez, R., 2006. Start-up of the anammox process in a membrane bioreactor. J. Biotechnol. 126, 475–487. Verstrae, W., Alexande, M., 1972. Heterotrophic nitrification by Arthrobacter sp. J. Bacteriol. 110, 955–959. Wan, C., Yang, X., Lee, D.-J., Du, M., Wan, F., Chen, C., 2011. Aerobic denitrification by novel isolated strain using as nitrogen source. Bioresour. Technol. 102, 7244– 7248. Xue, Y., Yang, F.-L., Liu, S.-T., Fu, Z.-M., 2008. The influence of controlling factors on the start-up and operation for partial nitrification in membrane bioreactor. Bioresour. Technol. 100, 1055–1060. Yang, X.-P., Wang, S.-M., Zhang, D.-W., Zhou, L.-X., 2011. Isolation and nitrogen removal characteristics of an aerobic heterotrophic nitrifying–denitrifying bacterium, Bacillus subtilis A1. Bioresour. Technol. 102, 854–862. Zhang, Q., 2009. Research on key issues in determination of ammonia nitrogen in water and wastewater by Nessler’s reagent spectrophotometry. Environ. Eng. 27, 85. Zhang, Q.-L., Liu, Y., Ai, G.-M., Miao, L.-L., Zheng, H.-Y., Liu, Z.-P., 2012. The characteristics of a novel heterotrophic nitrification–aerobic denitrification bacterium, Bacillus methylotrophicus strain L7. Bioresour. Technol. 108, 35–44.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Simultaneous removal of organic matter and nitrogen by a heterotrophic nitrifying–aerobic denitrifying bacterial strain in a membrane bioreactor Yan-Chun Yao a,b, Qing-Ling Zhang a, Ying Liu a, Zhi-Pei Liu a,⇑ a b
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China
h i g h l i g h t s Bacillus methylotrophicus L7 was inoculated into a membrane bioreactor solely. This single bioreactor was applied for treating artificial sewage aerobically. A simultaneous removal of COD and TN was achieved efficiently. This was different from traditional O/A system and SBR.
a r t i c l e
i n f o
Article history: Received 12 April 2013 Received in revised form 27 May 2013 Accepted 29 May 2013 Available online 4 June 2013 Keywords: Bacillus methylotrophicus L7 MBR Heterotrophic nitrification–aerobic denitrification Simultaneous removal of organic matter and nitrogen
a b s t r a c t A heterotrophic nitrifying–aerobic denitrifying bacterial strain, Bacillus methylotrophicus L7, was inoculated solely into a submerged membrane bioreactor (MBR) for continuous treatment of artificial sewage. The running conditions were also optimized for improvement of the treatment efficiency. The results indicated that inoculation of this single strain in a single reactor under constant aerobic conditions resulted in simultaneous removal of organic matter and nitrogen, in striking contrast to traditional aerobic nitrification–anaerobic denitrification treatment system and the sequencing batch reactor (SBR) systems. The optimal running conditions for the MBR were dissolved oxygen (DO) 4.5 mg/L, pH 7.5, loading ammonia <100 mg/L, and C/N ratio 3.5. Under these conditions, the removal percentages of chemical oxygen demand (COD), NHþ 4 –N, and TN as high as 96%, 77.5% and 53%, respectively, were achieved without nitrite accumulation. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Chemical oxygen demand (COD), reflecting organic matter content, and nitrogen content are two key parameters monitored during wastewater treatment. The most efficient method for removal of nitrogen from wastewater is based on the processes of nitrification (by autotrophic nitrifiers) and denitrification (by anoxic denitrifiers). Nitrifiers convert ammonia to nitrite and then to nitrate. Denitrifiers reduce nitrate to nitrite and then to N2 (Joo et al., 2005). Organic matter interferes with the activity of nitrifiers (Kulikowska et al., 2010) but is necessary for denitrifiers. Oxygen is required for the growth of nitrifiers but is toxic to denitrifiers (Lloyd et al., 1987). Because of these opposing tolerances to organic matter and oxygen, it is impossible to achieve simultaneous removal of these two components in a single bioreactor under ⇑ Corresponding author. Address: Institute of Microbiology, Chinese Academy of Sciences, West Beichen Road, Chaoyang District, Beijing 100101, PR China. Tel.: +86 10 62653757; fax: +86 10 62538564. E-mail address: [email protected] (Z.-P. Liu). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.05.120
constant aerobic conditions. Removal strategies therefore require separate treatment systems and precise control of treatment conditions. Nitrification by autotrophic bacteria that have slow growth rates requires a long retention time of flowing wastewater in the reactor (Jetten et al., 1997; Muller et al., 2003). These factors all tend to increase the cost of wastewater treatment. An increasing number of studies have focused on new biological nitrogen removal technologies. One exciting development is the discovery of ‘‘heterotrophic nitrifying–aerobic denitrifying’’ bacteria. Bacteria of this type have been isolated from many different environments; examples include Arthrobacter sp. (Verstrae and Alexande, 1972), Alcaligenes faecalis No. 4 (Joo et al., 2005), Bacillus sp. strains (Yang et al., 2011), Pseudomonas sp. (Wan et al., 2011), and Bacillus methylotrophicus L7 (Zhang et al., 2012). In comparison with traditional methods, nitrogen removal by heterotrophic nitrification and aerobic denitrification has several advantages: (i) The utilization of organic substrates and tolerance to oxygen by these bacteria are compatible, making it possible to achieve the simultaneous removal of organic matter and nitrogen via simultaneous nitrification and denitrification (SND) in a single reactor (Third
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et al., 2005). (ii) The process of denitrification counters the acidification caused by nitrification and tends to maintain a stable pH in the reactor. (iii) The diversity of possible substrates and products of heterotrophic nitrification facilitates mixed culture with a variety of bacterial strains and expands the scope of possible applications (Marazioti et al., 2003). Recent studies of heterotrophic nitrifying–aerobic denitrifying bacterial have focused on substrate removal, accumulation of intermediates, and production of gaseous nitrogen compounds (Wan et al., 2011; Zhang et al., 2012). The characteristics and optimal reaction conditions of these bacteria were investigated in sequencing batch flask culture (Kulkarni, 2013). However, no study to date has addressed the removal of organic matter and nitrogen during the continuous sewage treatment via SND by these bacteria. Previously, the heterotrophic nitrification–aerobic denitrification abilities of B. methylotrophicus L7 were described (Zhang et al., 2012). In the present study, these abilities of strain L7 were further assessed and its potential of simultaneous removal of organic matter and nitrogen was also investigated, by inoculating the strain into a membrane bioreactor designed for continuous wastewater treatment. The results suggest a basis for an alternative nutrient removal method for domestic wastewater. 2. Methods 2.1. Bacterial strain and culture condition The bacterial strain B. methylotrophicus L7 was cultured in LB broth at 30 °C for 24 h. Cells were harvested by centrifugation at 9000g for 30 min, washed twice with sterile saline solution, and the cell mass was resuspended in sterile saline solution to 3000 mg/L (wet weight) for use as the inoculum in a membrane bioreactor. 2.2. Membrane bioreactor and startup and operating conditions The laboratory-scale submerged membrane bioreactor (MBR) with a total effective volume of 29 L (Fig. 1) was fitted with a polypropylene hollow fiber membrane module (average pore diameter 0.2 lm; Hangzhou Kaihong Membrane Technology Co. Ltd., Hang Zhou, China) was used. Polypropylene spherical suspended fillers were used as a biological carrier, with a filling rate of 30% (v/v). A continuous flow of wastewater was provided by a delivery pump installed between the storage tank and the reaction tank. A fully aerobic operation throughout the experimental period was ensured by the introduction of compressed air via aerator pipes at
Table 1 The quality of artificial wastewater. Items
COD (mg/L)
NHþ 4 –N (mg/L)
TP (mg/L)
pH
Value
1000
120
7.8–9.4
7.5
the bottom of the reactor. The air bubbles provided oxygen for biological needs and a hydrodynamic scouring effect to reduce membrane fouling (Chang et al., 2011). For startup, the reactor was inoculated with 6 L of strain L7 cell suspension prepared as above and run at room temperature. The influent was controlled to hydraulic retention time (HRT) 24 h, dissolved oxygen (DO) 3.5–4.5 mg/L, and pH 7.5, ammonia content 30–40, and 150–300 mg/L. The startup period was approximately 30 days. For continuous treatment of artificial wastewater after startup, the running conditions were: temperature 25–30 °C, HRT 24 h; DO 4.5 mg/L; influent pH 7.5, influent NHþ 4 –N 120 mg/L, C/N ratio 3.5, or otherwise stated. 2.3. Artifical wastewater Artificial wastewater, used as an imitation of sanitary, was composed of glucose, peptone, KH2PO4, NH4Cl and tapwater (Table 1), plus appropriate trace amounts of calcium chloride, copper sulfate, and magnesium sulfate. 2.4. Analytical methods Bacterial growth was determined by monitoring the optical density at 600 nm (OD600) with a spectrophotometer (model UV7200, UNICO, Shanghai, China). NO 2 –N level was determined by N-(1-naphthyl)-1, 2-diaminoethane dihydrochloride spectrophotometry (Mahmood et al., 2009). Ammonia level was determined by Nessler assay (Zhang, 2009). Total nitrogen (TN) content was determined by peroxydisulfate oxidation with a UV spectrophotometric method (Ebina et al., 1983). COD was determined using a COD instrument COD (model CTL-12, Chengde Huatong Environmental Protection Equipment Co. Ltd., Chengde, China). DO was determined with a DO meter (model JPSJ-605Shanghai Precision & Scientific Instrument Co., Ltd, Shanghai, China). pH was measured with a pH meter (model PB-10, Sartorius, Germany). 3. Results and discussion 3.1. Reactor startup During the 30-day startup period, the removal percentages of COD, ammonia, and total nitrogen (TN) increased gradually to 80.0%, 66.7% and 48.6%, respectively, on day 30 (data not shown). These findings indicate that a successful startup was achieved, according to the criteria of Trigo et al. (2006) and Xue et al. (2008). 3.2. Factors that affected the removal efficiency To improve the treating performance of the MBR, several factors as described below were evaluated for their effects on the removal efficiency of COD and nitrogen.
Fig. 1. Schematic diagram of the membrane bioreactor (MBR) system. (1) Storage tank; (2) delivery pump; (3) MBR; (4) membrane model; (5) air pump; (6) suction pump.
3.2.1. Effect of DO DO was decreased gradually from 6.0 to 1.5 mg/L during a period of 72 days (Fig. 2). A stable, high average COD removal percentage (85%) was maintained throughout this period (Fig. 2A), indicating a good biological degradation capability with consequent high effluent quality. In contrast, the removal percentages
85
6
50 40 30 20
+
3 2 1
10 0 42
50
58
66
74
82
90
-
4
20 3
15
DO
5
25
2
10
-
-
NO2 -N, NO3 -N in effluent (mg/L)
6
30
1
5 0
0 34
42
50
58
66 74 82 Run time (d)
90
7.5 7 6.5 6 110
114
118
122
126
130
134
Run time (d)
7
B
35
8
98
Run time (d)
40
8.5
98
Fig. 2. Effect of DO on wastewater treatment efficiency. (A) COD and nitrogen removal ratios. (B) Nitrite and nitrate concentration in effluent. }, COD removal percentage. 4, NHþ 4 –N removal percentage. h, TN removal percentage. , DO concentration. j, NO 3 –N concentration. N, NO2 –N concentration.
of ammonia and TN were 20–25% and 12–20%, respectively (Fig. 2A); much lower than during the startup period. This finding may be due to the much higher influent ammonia content during the experimental period (120 vs. 25–30 mg/L). Nitrogen removal tended to vary with decrease DO. The NHþ 4 –N removal percentage did not vary significantly within the DO range of 6.0–2.8 mg/L. When DO fell below 2.0 mg/L, the NHþ 4 –N removal percentage decreased sharply (Fig. 2A). These findings indicate that the ammonia-oxidizing ability of strain L7 was greatly inhibited by low DO (<2.0 mg/L). Thus, oxygen is required for heterotrophic ammonia oxidation, as for autotrophic ammonia oxidation. The removal percentages of TN did not undergo changes as great as those of ammonia within the range of DO tested (Fig. 2A), suggesting that the aerobic denitrification performance of strain L7 was influenced to a lesser degree by DO. This result was consistent with those reported previously for certain aerobic denitrifying strains (Lloyd et al., 1987; Patureau et al., 2000), which maintained their denitrification ability when DO was close to or greater than oxygen saturation in the air. DO has been described as an important factor for aerobic denitrification in a few strains (Huang and Tseng, 2001). The NO 2 –N concentration in effluent remained low throughout the experimental period, whereas the NO 3 –N concentration increased when DO was >6.5 mg/L or <4 mg/L (Fig. 2B). This finding was consistent with the denitrification properties of strain L7 as observed in our previous study (Zhang et al., 2012). NO 2 –N is a product of ammonia oxidation and a connecting point between nitrification and denitrificaion, and can thus be converted not only to nitrogen gas but also to nitrate via nitrification. Such coordination varies depending on the bacterial species and the culture conditions (Sakai et al., 1997). In this case, the accumulation of nitrate at DO >6.5 mg/L or <4.0 mg/L may have contributed to the slightly higher conversion of nitrite to nitrate under these conditions, the TN removal was relatively stable.
40
10
B
35
9.5
30
9
25
8.5
20
8
15
7.5
-
34
9.5 9
106
0
10
A
pH
4
DO
5
70 60
100 90 80 70 60 50 40 30 20 10 0
pH
7
A
90 80
COD, NH4 -N, TN removal (%)
100
NO2 -N, NO3 -N in effluent (mg/L)
+
COD, NH4 -N, TN removal (%)
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10 5
7
0
6.5
-5
6 106
110
114
118
122
126
130
134
Run time (d) Fig. 3. Effect of pH on wastewater treatment efficiency. (A) COD and nitrogen removal ratios. (B) Nitrite and nitrate concentrations in effluent. }, COD removal percentage. 4, NHþ 4 –N removal percentage. h, TN removal percentage. d, pH. j, NO 3 –N concentration. N, NO2 –N concentration.
Taken together, these results suggest that an optimal DO level may enhance the activity of heterotrophic nitrification and aerobic dentrification, resulting in thorough biodegradation of organic matter. Taking into account nitrification and denitrification, COD and TN removal, and running cost, the optimal DO for strain L7 was estimated as 4.5 mg/L. 3.2.2. Effect of influent pH Efficient COD and nitrogen removal were observed in our system at an influent pH of 7.0–9.0, with removal percentages of 89.0% for COD and 23.4% for NHþ 4 –N (Fig. 3A). A slightly alkaline environment was conducive to heterotrophic nitrification because more free ammonia (NH3) was present in the wastewater; presumably, the substrate utilized by ammonia monooxygenase (AMO) was NH3 rather than NHþ 4 (Mevel and Prieur, 2000). The concentration of NO 2 –N in effluent was negligible, while the concentration of NO 3 –N ranged 13–35 mg/L (Fig. 3B). The NO 3 –N concentration was low within the effluent pH ranged from 7.0–9.0 and minimal at pH 7.5, therefore pH 7.5 was selected as optimal. 3.2.3. Effect of C/N ratio in influent The influent concentration of NHþ 4 –N was 60 mg/L in our experiment. C/N ratios of 0.7, 1.2, 1.6, 3.2 and 5.0 were established by adding appropriate amounts of glucose. The COD removal percentages were high (90–96.2%) at each of these C/N ratios (Fig. 4A). The removal percentages of ammonia and TN were correlated with C/N ratio (Fig. 4A) and were strikingly higher for C/N ratio >3.2. The removal percentages of NHþ 4 –N and TN were <55% and <22% at C/N ratio <1.6 and increased to 75% and 53% at C/N ratio 3.2 and 5.0, respectively. Thus, there was a significantly improvement in ammonia and TN removal at the higher C/N ratios. A previous study found that sufficient carbon in wastewater was essential
Y.-C. Yao et al. / Bioresource Technology 143 (2013) 83–87
120
Table 2 Effects of loading ammonia content on the removal of ammonia.
A
100
Influent average NHþ 4 –N (mg/L)
Effluent average NHþ 4 –(mg/L)
NHþ 4 –N removal (%)
Removal efficiency (g/m3/ d)
59.2 107.6 184 300
15.8 48.2 117 213.4
73.3 55.2 36.4 28.9
43.4 59.4 67.0 86.7
80 60 40 20 0 150 155 Run time (d)
160
165
70
NH4 -N (mg/L)
B
16
+
14 12 10 8 6 4
40 30 20
0 200
2
204
208
204
208
0 140
145
150 155 Run time (d)
160
165
Fig. 4. Effect of C/N on wastewater treatment efficiency. (A) COD and nitrogen removal ratios. (B) Nitrite and nitrate concentrations in effluent. }, COD removal percentage. 4, NHþ 4 –N removal percentage. h, TN removal percentage. j, NO3 –N concentration. N, NO –N concentration. 2
for the efficient removal of ammonia by Alcaligenes faecalis strain No. 4 (Joo et al., 2006), perhaps because an insufficient carbon inhibits the growth and nitrification ability of heterotrophic nitrifying strains, resulting in reduced ammonia removal. In terms of ammonia removal, heterotrophic nitrification is quite different from the autotrophic nitrification. In another study, autotrophic nitrification was greatly reduced or eliminated at C/N ratios >0.25 (Okabe et al., 1996). The efficient nitrogen removal by heterotrophic nitrifying strains at high C/N ratios results in simultaneous removal of COD and nitrogen. This ability is highly advantageous in wastewater treatment because it eliminates the need to reduce organic matter content prior to nitrification (as in traditional nitrogen removal methods). Nitrite levels in effluent were low at all C/N ratios tested (Fig. 4B). Nitrate levels varied depending on the C/N ratio and were minimal (<13 mg/L) at the ratio 3.2 (Fig. 4B). These findings suggest that a sufficient amount of carbon is also essential for the denitrification. The limited formation of NO 2 –N (<1 mg/L) by strain L7 at various C/N ratios satisfies the criteria for a well-functioning SND system with minimal nitrite accumulation (Third et al., 2005). Based on considerations of COD removal, ammonia removal, TN removal, and running cost, C/N ratio of 3.5 was selected as optimal. 3.2.4. Effect of influent ammonia concentration In this laboratory-scale system, a higher influent ammonia concentration promoted NHþ 4 –N removal, concentrations of 59.2, 107.6, 184, 300 mg/L were associated with removal rate of 43.4, 59.4, 67.0, 86.7 g/m3/d, respectively (Table 2). Strain L7 is tolerant of high ammonia loads (Zhang et al., 2012). In contrast, the ammonia removal percentages were 73.3% and 28.9% at influent ammonia concentrations of 59.2 and 300 mg/L, respectively, resulting in a clear increase of effluent ammonia concentration in the latter case. Based on consideration of the effluent ammonia concentration in effluent and the removal percentage of NHþ 4 –N
800 700 COD (mg/L)
-
50
10
-
NO2 -N, NO3 -N in effluent (mg/L)
18
20 18 16 14 12 10 8 6 4 2 0 -2
A
60
212 216 Run time (d)
220
224
228
220
224
228
-
145
-
140
NO2 -N, NO3 -N (mg/L)
+
COD, NH4 -N, TN removal (%)
86
B
600 500 400 300 200 100 0 200
212
216
Run time (d) Fig. 5. Wastewater treatment efficiency under optimal conditions as described in þ the text. (A) The NHþ 4 –N concentration in influent and NH4 –N, nitrate, and nitrite concentrations in effluent. (B) COD in influent and effluent. }, COD in influent. , þ COD in effluent. s, NHþ 4 –N in influent. d, NH4 –N in effluent. j, NO3 –N concentration. N, NO 2 –N concentration.
as determining factors for system treatment efficiency, the optimal ammonia load for the bioreactor is <100 mg/L. 3.3. Wastewater treatment efficiency under the optimal conditions The running parameters used in this experiment were: loading NHþ 4 –N concentration 60 mg/L, C/N ratio 3.5, pH 7.5, DO 4.5 mg/L. The results obtained under these optimal conditions are shown in Fig. 5. An average COD removal percentage of 92.3% was maintained during the entire experimental period (Fig. 5B). The maximal NHþ 4 –N removal percentage was 77.5%, and the average TN removal percentage was 52% (Fig. 5A). Nitrite concentration was maintained at a low level (<0.2 mg/L; Fig. 5A). These findings all indicate that the system was stable, feasible and had excellent treatment efficiency. In this study, simultaneous removal of COD and nitrogen was achieved using a single strain under relatively constant conditions. This phenomenon presents a striking contrast to traditional methods of aerobic–anaerobic nitrogen removal treatment. In the traditional methods, (i) ammonia is transformed to nitrite and then to nitrate (but TN is not removed) in the aerobic phase, and this
Y.-C. Yao et al. / Bioresource Technology 143 (2013) 83–87
process in inhibited by organic matter; (ii) nitrogen is removed only in the anaerobic phase, through consumption of a large amount of organic compounds (Joo et al., 2006; Kulikowska et al., 2010). This method also differs from the sequencing batch reactor (SBR) system in that it does not require alternation of running conditions and is thus more easily to be controlled and stable. In view of the above considerations, it appears that strain L7 might provide a less expensive alternative for treatment of urban sewage. Typical parameters of sewage (COD 300–600 mg/L, ammonia concentration 40–80 mg/L, pH 7.5) are consistent with efficient nitrogen removal performance by strain L7. 4. Conclusion Artificial sewage was treated by a submerged membrane bioreactor (MBR) with a heterotrophic nitrifying–aerobic denitrifying strain, Bacillus methylotrophicus L7, as inoculum. This single strain achieved simultaneous removal of organic matter and nitrogen from the sewage. The optimal running conditions for the MBR were DO 4.5 mg/L, pH 7.5, loading ammonia <100 mg/L, and C/N ratio 3.5. Under these conditions, the removal percentages of COD, NHþ 4 –N and TN as high as 96%, 77.5%, and 53%, respectively, were achieved without nitrite accumulation. Strain L7 provides an effective and economical alternative method for treatment of urban sewage. Acknowledgements This study was supported by Grants from the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KJCX2YW-L08). We thank Dr. S. Anderson for English language editing of the manuscript. References Chang, C.-Y., Tanong, K., Xu, J., Shon, H., 2011. Microbial community analysis of an aerobic nitrifying–denitrifying MBR treating ABS resin wastewater. Bioresour. Technol. 102, 5337–5344. Ebina, J., Tsutsui, T., Shirai, T., 1983. Simultaneous determination of total nitrogen and total phosphorus in water using peroxodisulfate oxidation. Water Res. 17, 1721–1726. Huang, H.K., Tseng, S.K., 2001. Nitrate reduction by Citrobacter diversus under aerobic environment. Appl. Microbiol. Biotechnol. 55, 90–94. Jetten, M.S.M., Logemann, S., Muyzer, G., Robertson, L.A., de Vries, S., van Loosdrecht, M.C.M., Kuenen, J.G., 1997. Novel principles in the microbial conversion of nitrogen compounds. Ant. Leeuwenhoek 71, 75–93.
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