This article was downloaded by: [Gazi University] On: 27 December 2014, At: 03:01 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Characteristics of nitrate removal in a bio-ceramsite reactor by aerobic denitrification a
a
a
b
c
Dan Chen , Kai Yang , Hongyu Wang , Bin Lv & Fang Ma a
School of Civil Engineering, Wuhan University, Wuhan 430072, People's Republic of China
b
School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, People's Republic of China c
State Key Laboratory of Urban Water Resources and Environment, Harbin Institute of Technology, Harbin 150090, People's Republic of China Accepted author version posted online: 02 Dec 2014.Published online: 23 Dec 2014.
Click for updates To cite this article: Dan Chen, Kai Yang, Hongyu Wang, Bin Lv & Fang Ma (2014): Characteristics of nitrate removal in a bioceramsite reactor by aerobic denitrification, Environmental Technology, DOI: 10.1080/09593330.2014.993729 To link to this article: http://dx.doi.org/10.1080/09593330.2014.993729
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 http://dx.doi.org/10.1080/09593330.2014.993729
Characteristics of nitrate removal in a bio-ceramsite reactor by aerobic denitrification Dan Chena , Kai Yanga , Hongyu Wanga∗ , Bin Lvb and Fang Mac of Civil Engineering, Wuhan University, Wuhan 430072, People’s Republic of China; b School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, People’s Republic of China; c State Key Laboratory of Urban Water Resources and Environment, Harbin Institute of Technology, Harbin 150090, People’s Republic of China
a School
Downloaded by [Gazi University] at 03:01 27 December 2014
(Received 12 September 2014; accepted 26 November 2014 ) A newly aerobic denitrifying bacterial strain, Pseudomonas sp. X31, which was isolated from activated sludge, was added to a newly developed aerobic denitrification bio-ceramsite reactor as an inoculum to treat nitrate-polluted water and the denitrification activities of this system under different air–water ratio, hydraulic loading, and C/N (carbon/nitrogen ratio) conditions were investigated. It demonstrated excellent capability for denitrification in the bio-ceramsite reactor at air– water ratios that varied from 6.5:1 to 8:1. The optimal hydraulic loading for the bio-ceramsite reactor was 0.75 m/h with the optimum denitrification efficiency of 95.18%. The optimal C/N was 4.5:1 with a maximum nitrate removal efficiency of 98.48%. COD could be completely removed under the most appropriate condition (air–water ratio 6.5:1–8:1, hydraulic loading 0.75 m/h, and C/N 4.5:1). The quantity of the biomass in the reactor decreased along with flow, which was in accordance with the variety of the available substrate concentrations in the water. However, the biofilm activity was not proportional to the biomass in the bio-ceramsite reactor, but increased with the quantity of the biomass up to a peak value and then decreased. Keywords: aerobic denitrifying bacterial strain; Pseudomonas sp; bio-ceramsite reactor; denitrification efficiency; biofilm activity
1. Introduction Currently, the most conventional, efficient, and costeffective approach to eliminate nitrogen from municipal and industrial wastewaters is a biological process which normally requires a two-step process, nitrification followed by denitrification.[1,2] However, this common method tends to be time-consuming because of the separation of aerobic and anoxic phases and low rate of nitrification.[3] Simultaneous nitrification and denitrification can solve the problem of separate tanks required in conventional treatment plants. It means largely reduction of cost for both system space and construction. According to this idea, aerobic denitrification is highly valued because denitrification occurs directly in aerated reactors, where nitrification takes place.[4] Denitrification is the biological process in which denitrifying bacteria use nitrate as electron acceptor and organic or inorganic substances as electron donor in order to convert nitrate into nitrogen gas. Denitrification gene expression occurs under anoxic or aerobic conditions and requires the presence of an N-oxide.[5] Large numbers of denitrifying bacteria have been isolated from activated sludge and soil including[6–13] Achromobacter, Aerobacter, Alcaligenes, Bacillus, Brevibacterium, Flavobacterium, Lactobacillus,
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Micrococcus, Proteus, Pseudomonas, and Spirillum. Aerobic denitrifying bacteria can be exemplified with efficient treatment of synthetic wastewater in an open aeration reactor with low process costs.[14–18] There were many recent reports of aerobic denitrifying species such as Alcaligenes faecalis,[18] Citrobacter diversus,[9] Pseudomonas aeruginosa,[19] Pseudomonas stutzeri,[12] and Thiosphaera pantotropha,[12] which were isolated from canals, ponds, soils, and activated sludge. In this work, pure cultures of Pseudomonas sp. X31 were inoculated into a newly developed bio-ceramsite reactor in order to form a biofilm on the surface of the ceramsite to treat nitrate-contaminated wastewater. The objective of this work was to investigate the characteristics of nitrate removal in the bio-ceramsite reactor under different air–water ratio, hydraulic loading, and C/N conditions. 2.
Materials and methods
2.1. Aerobic denitrifying bacterial strain and media The activated sludge samples for the isolation of aerobic denitrifying bacterial strain X31 (FJ480211) were collected from the steadily operating aerobic denitrification bioreactor in the laboratory (State Key Lab of
2
D. Chen et al.
Urban Water Resources and Environment, Harbin Institute of Technology, Harbin, China). Synthetic wastewater as the basic medium (COD 345.28–405.11 mg/L, NO− 3 −N 50.01–73.79 mg/L, NO− 2 −N 0.20–3.17 mg/L, TN 62.05– 90.88 mg/L, and pH 6.8–7.5) was prepared by dissolving NaNO3 , NaNO2 , NaAc, Na2 HPO4 , and KH2 PO4 , and 2 mL of trace mineral solution in distilled water (1 L). The trace mineral solution contains the following components (mg/L): MgSO4 ·7H2 O 90, MnSO4 70, ZnSO4 ·5H2 O 80, FeSO4 ·7H2 O 100, CuSO4 ·5H2 O 30, CoCl2 ·7H2 O 150, H3 BO3 20, and NaMoO4 ·2H2 O 60.
Experimental apparatus of the aerobic denitrification bio-ceramsite reactor A schematic of the reactor used in this work is shown in Figure 1. The main reactor compartment consisted of a plexiglass cylinder (diameter 100 mm, height 2000 mm). The heights of the supporting layer of gravel and the packing (ceramsite) were 100 and 1400 mm, respectively. At every 250 mm height of the packing, there was a sampling port along the periphery of the reactor. Compressed air was pumped into the bottom of the reactor using air diffuser and the aeration quantity was measured by gas measuring flowmeter. The synthetic wastewater was also pumped into the bottom of the reactor. After making good contact with the biofilm on the surface of the ceramsite, it would flow out of the top of the reactor. During the inoculation phase, the precultured Pseudomonas sp. X31 isolates were inoculated into the reactor amended with KNO3 (2 g/L), NaAc (3 g/L) and trace
element solution as described [13] in order to quickly form the biofilm. Aeration was applied consistently from the bottom of the reactor for seven days. After inoculation, the reactor was operated at a temperature of 25°C, C/N of 4.5:1, hydraulic loading of 0.75 m/h, air–water ratio of 6.5:1, and nitrate concentration of 30 mg/L for 33 days. The aerobic denitrification efficiency was perceived to be 80%, which demonstrated that the domestication stage was completed. It formed a dense dark brown biofilm on the surface of the ceramsite in the reactor at the end of the domestication stage. After the domestication stage, the experiment went into the steady operation phase. In order to compare nitrate and COD reduction rates at different air–water ratios, the applied air–water ratios were adjusted to 3:1, 5:1, 6.5:1, 8:1, and 10:1, with hydraulic loading, temperature, nitrate concentration, TN, and COD at steady values of 0.75 m/h, 25°C, 64.64–73.79, 80.99–90.88, and 345.28–382.43 mg/L, respectively. In order to compare nitrate and COD reduction rates at different hydraulic loadings, the applied hydraulic loadings were adjusted to 0.60, 0.75, and 0.90 m/h, with air–water ratio, temperature, C/N, nitrate concentration, and TN at steady values of 6.5:1, 25°C, 4.5:1, 50.01–59.04 mg/L, and 62.05–69.30 mg/L, respectively. In order to compare nitrate and COD reduction rates at different C/N ratios, the applied C/N ratios were adjusted to 1.8:1, 2.5:1, 3.5:1, 4.2:1, 4.5:1, and 5:1, with hydraulic loading, air–water ratio, temperature, NO− 3 −N, TN, and COD at steady values of 0.75 m/h, 6.5:1, 25°C, 63.01–73.63, 69.89–83.13, and 349.57– 401.02 mg/L, respectively.
Figure 1. Schematic of an aerobic denitrification bio-ceramsite reactor (1. intake pump; 2. liquid measuring flowmeter; 3. main reactor; 4. ceramsite; 5. air diffuser; 6. air pump; 7. gas measuring flowmeter; 8. sampling port; 9. temperature sensor; 10. electric heating tape; and 11. temperature controller).
2.3. Analytical methods − Total nitrogen (TN), nitrate (NO− 3 −N), nitrite (NO2 −N), + ammonium (NH4 −N), and chemical oxygen demand (COD) were measured according to the Standard Methods.[20] Total organic carbon (TOC) was determined by the TOC analyzer (TOC-V CPH, Shimadzu, Japan). Dissolved oxygen (DO) in the reactor was measured by the DO meter (AZ8403, AZ Instrument Corp, Taiwan). The pH was measured by the pH meter (PHS-3C, Kexiao Instrument, China). The sampling points were chosen above the supporting layers of 250, 750, and 1250 mm. The biomass was measured by the dry weight method,[21] and the biofilm activity was measured by the specific oxygen uptake rate (SOUR).[22] The bacteria were identified by 16S rRNA sequencing analysis by Sangon (Sangon Biotech Shanghai Co., Ltd., China) after isolation and purification. During the isolation and purification phase of X31, 30 mL turbid liquid of the activated sludge samples was added into 100 mL medium, and then the mixed liquid was cultured for 48 h at a temperature of 30°C in a thermostatic oscillator. This step was repeated a few times until nitrate could be reduced at a steady rate. After that, the culture solution was vaccinated onto the solid
Downloaded by [Gazi University] at 03:01 27 December 2014
2.2.
3
Environmental Technology tablet, and then cultured for seven days in a biochemical incubator of 30°C. Finally, the separation–purification phase was completed through plate streaking on the solid medium, forming smooth colonies. To ensure the purity of the isolated strains, plate streaking was repeated several times. The effects of the operating conditions including the air–water ratio, hydraulic loading, and C/N ratio on aerobic denitrification were analysed by variance analysis and significance analysis.
Downloaded by [Gazi University] at 03:01 27 December 2014
3. Results and discussion 3.1. The effect of air–water ratio on aerobic denitrification It is apparent from Table 1 that the concentrations of − NO− 3 −N, NO2 −N, TN, COD, and DO in influent and effluent of the aerobic denitrification bio-ceramsite reactor were changed with various air–water ratios. The applied air–water ratios were 3:1, 5:1, 6.5:1, 8:1, and 10:1, with hydraulic loading, temperature, nitrate concentration, TN, and COD at steady values of 0.75 m/h, 25°C, 64.64–73.79, 80.99–90.88, and 345.28–382.43 mg/L, respectively. It can be observed from Table 1 that the NO− 3 −N, TN, and COD removal efficiencies increased with increase in air–water ratio from 3:1 to 6.5:1. The highest efficiencies (NO− 3 −N 91.54%, TN 91.07%, and COD 96.57%) were observed at air–water ratio of 6.5:1. When air–water ratio increased to 8:1, the removal efficiencies of NO− 3 −N, TN, and COD remained at relatively high levels of 89.37%, 88.53%, and 94.90%, respectively. However, the removal efficiencies NO− 3 −N, TN, and COD dropped to 72.29%, 78.26%, and 87.12% when the air–water ratio was further increased to 10:1. During the whole air–water ratio experimental stage, the NO− 2 −N concentration varied from 0.39 to 2.85 mg/L. The effluent DO concentration increased
with increasing air–water ratio, which was in accordance with Li’s [23] report. The results demonstrated that the increase in the air– water ratio caused increase in NO− 3 −N, TN, and COD removal efficiencies within the range of 3:1–6.5:1; the reason was that the increase in air–water ratio increased DO concentration,[23] thus enhancing the turbulence degree of the mixed liquor in the reactor in order to improve the mass transfer on the surface of the biofilm. On the other hand, the bubble effect of the air was reinforced as the air–water ratio increased,[23,24] which contributed to accelerated scouring action of the biofilm on the big surface of the ceramsite; as a result the updating speed of the biofilm was faster and the activity of the biofilm was higher. However, the air–water ratio should not be too high because a very high air–water ratio (10:1) not only increased the operation cost, but also led to larger aeration quantity, causing extremely intensified scour of the biofilm so that the microorganism in the reactor could not grow well. In addition, a very high air–water ratio would result in a very high DO environment, which was harmful to the denitrification process.[25] In this study, the bio-ceramsite reactor performed well when the air–water ratio varied from 6.5:1 to 8:1.
3.2.
The effect of hydraulic loading on aerobic denitrification − Figure 2 shows the concentrations of NO− 3 −N, NO2 −N, TN, and COD in the influent and effluent of the aerobic denitrification bio-ceramsite reactor with various hydraulic loadings. The applied hydraulic loadings were 0.60, 0.75, and 0.90 m/h, with the air–water ratio, temperature, C/N, nitrate concentration, and TN at steady values of 6.5:1, 25°C, 4.5:1, 50.01–59.04 mg/L, and 62.05–69.30 mg/L, respectively.
− Table 1. Concentrations of NO− 3 −N,NO2 −N, TN, COD, and DO in the influent and effluent under different air–water ratio conditions {analysis results of significance test of the air–water ratio on aerobic denitrification SSA [Sum of Squares for factor A (air–water ratio)] = 4028.78, SSE (Sum of Squares for Error) = 218.25, SST (Sum of Squares for Total) = 4247.02, and F(level of significance) = 138.45 > F crit }.
Air–water ratio Test items NO− 3 −N NO− 2 −N TN COD DO
Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L)
3:1
5:1
6.5:1
8:1
73.79 27.77 62.37 1.94 1.44 90.88 31.06 65.82 382.43 62.42 83.68 2.71–3.82 2.54–4.01
64.64 15.98 75.28 2.85 0.70 80.99 18.01 77.76 345.29 49.57 85.64 3.27–4.71 3.18–4.57
66.76 5.65 91.54 0.87 0.39 81.16 7.25 91.07 362.43 12.43 96.57 3.76–5.51 3.85–5.25
68.56 7.29 89.37 2.20 0.84 84.92 9.74 88.53 383.86 19.57 94.90 4.02–6.14 4.11–6.20
10:1 67.91 18.82 72.29 1.64 1.30 83.45 18.14 78.26 373.86 48.14 87.12 4.36–6.79 4.25–6.86
Downloaded by [Gazi University] at 03:01 27 December 2014
4
D. Chen et al.
− Figure 2. Concentrations of NO− 3 −N, NO2 −N, TN, and COD in the influent and effluent under different hydraulic loading conditions {analysis results of significance test of hydraulic loading on aerobic denitrification SSB [Sum of Squares for factor B (hydraulic loading)] = 4741.39, SSE (Sum of Squares for Error) = 218.01, SST (Sum of Squares for Total) = 4959.40, F(level of significance) = 195.73 > F crit }.
Figure 2 shows that the average denitrification efficiency reached the peak value of 95.18% at a hydraulic loading of 0.75 m/h; meanwhile the average TN and COD removal efficiencies also reached peak values of 92.68% and 90.45% under this condition. However, the removal efficiencies of NO− 3 −N, TN, and COD decreased to 71.00%, 71.44%, and 79.23% as hydraulic loading increased to 0.90 m/h. Also, the removal efficiencies decreased to 60.91%, 66.40%, and 76.86% with hydraulic loading of 0.60 m/h. Moreover, the effluent NO− 2 −N was always maintained at a low level below 4.09 mg/L during this phase. It is very important to determine the appropriate hydraulic loading for the reactor because the performance of denitrification was obviously associated with hydraulic loading.[26] If hydraulic loading was too low, the removal efficiencies decreased because of insufficient carbon source for the rapidly proliferative microorganism to remove the nitrate, and the construction cost increased. Nevertheless, a very high hydraulic loading also caused decrease in removal efficiencies, due to the fact that the synthetic wastewater stayed for a shorter time in the reactor, so that the substrate did not have enough time to make contact with the aerobic denitrifying bacteria; on the other hand, the biofilm on the surface of the ceramsite might be easily washed away under very high hydraulic loading condition so that the aerobic denitrifying bacteria could not proliferate well. In the present study, the optimal hydraulic loading for the bio-ceramsite reactor
was 0.75 m/h with the optimum denitrification efficiency of 95.18%.
3.3. The effect of C/N on aerobic denitrification It is apparent from Table 2 that the concentrations of − NO− 3 −N, NO2 −N, TN, COD, and DO in the influent and effluent of the aerobic denitrification bio-ceramsite reactor were changed with various C/N ratios. The applied C/N ratios were 1.8:1, 2.5:1, 3.5:1, 4.2:1, 4.5:1, and 5:1, with hydraulic loading, air–water ratio, temperature, NO− 3 −N, TN, and COD at steady values of 0.75 m/h, 6.5:1, 25°C, 63.01–73.63, 69.89–83.13, and 349.57– 401.02 mg/L, respectively. It can be observed from Table 2 that the C/N ratio has − significant effects on NO− 3 −N, NO2 −N, TN, and COD − removal. NO3 −N and TN removal efficiencies rapidly increased as the C/N ratio increased from 1.8:1 to 4.5:1. The highest NO− 3 −N and TN removal efficiencies (98.48% and 96.85%) were observed at a C/N ratio of 4.5:1. The removal efficiency of COD slowly increased from 85.62% to 94.04% when the C/N ratio varied from 1.8:1 to 4.5:1. If the C/N ratio was further increased to 5:1, the removal efficiencies of NO− 3 −N and TN reached roughly the same level as C/N = 4.5:1. However, the removal efficiency of COD dropped to 88.35%. During the experimental phase, the effluent NO− 2 −N concentration always remained at low level below 3.17 mg/L.
5
Environmental Technology
− Table 2. Concentrations of NO− 3 −N,NO2 −N, TN, COD, and DO in the influent and effluent under different C/N conditions (analysis results of significance test of C/N on aerobic denitrification SSC (Sum of Squares for factor C(C/N)) = 30508.34, SSE (Sum of Squares for Error) = 307.71, SST (Sum of Squares for Total) = 30816.05, and F(level of significance) = 713.84 > F crit ).
C/N Test items
1.8:1
NO− 3 −N
Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L)
NO− 2 −N TN COD
Downloaded by [Gazi University] at 03:01 27 December 2014
DO
63.01 47.81 24.12 0.53 0.20 69.89 57.62 17.56 378.14 54.36 85.62 3.26–4.67 3.08–4.56
2.5:1 69.54 38.66 44.41 2.39 2.10 79.12 48.91 38.19 373.86 46.36 87.60 2.93–4.86 3.73–4.99
It is important to optimize the C/N ratio for each denitrifying bacterium because carbon is essential for cell growth and nitrate reduction processes; the optimal quantity of carbon is a key parameter in the denitrification process.[27] When the carbon source was higher, nitrate and nitrite were easily converted into nitrogen gas due to sufficient energy.[11,28] However, an extremely high concentration of carbon source inhibited the aerobic denitrification process [9] and caused a high level of organic carbon source in the effluent. If the C/N ratio was maintained at an appropriate level in the aerobic denitrifica− tion system, the nitrogen oxides – NO− 3 −N, NO2 −N, + NH4 −N, etc. – could be completely converted to nitrogen gas, while guaranteeing the minimality of the effluent organic carbon source. According to some similar work, the NO− 3 −N removal efficiencies by aerobic denitrifying bacteria isolated from sequencing batch reactors were 30.5–60.5%.[17] In the present study, the optimal C/N ratio was 4.5:1 with a maximum nitrate removal efficiency of 98.48%. These results should be useful information for performing an aerobic denitrification process in wastewater treatment plants. 3.4.
Biofilm purification mechanism and biofilm activity analysis in the reactor
Biofilm had been successfully used in water treatment for over a century.[29] Wastewater biofilms are very complex Table 3.
66.60 10.72 83.91 3.07 3.17 76.63 19.00 75.21 363.86 39.99 89.01 3.89–4.90 3.90–5.10
4.2:1 73.63 6.31 91.43 1.94 2.87 83.13 11.01 86.76 349.57 26.71 92.36 3.19–4.82 3.06–4.75
4.5:1
5:1
70.36 1.07 98.48 0.84 0.99 78.31 2.47 96.85 352.43 21.00 94.04 3.89–5.09 3.88–5.13
71.18 1.24 98.26 0.77 0.70 79.15 2.61 96.08 401.02 46.71 88.35 3.52–5.09 3.23–4.98
systems consisting of microbial cells and colonies embedded in a polymer matrix, whose structure and composition was a function of biofilm age and environmental conditions.[30] The differences between the biofilm process and the traditional activated sludge process were not only the existing ways of the main microorganism, but also the behaviour characteristics of the biofilm dynamics during the diffusion process.[30] In the biofilm system, pollutants, DO, and necessary nutrients first diffused onto the surface of the biofilm through the liquid phase, and then transferred to the inner biofilm. Pollutants which diffused to the surface or interior of the biofilm were decomposed and converted to metabolite by microorganisms, while implementing complete sewage purification. Specifically, the compounds (COD, NO− 3 −N, etc.) in the synthetic wastewater were transferred to the aerobic layer of the biofilm through solute diffusion. After a long time reaction, the dead aerobic bacteria were utilized as nutrients for the anaerobic bacteria in the system. When the nutrients were further utilized and ran out, anaerobic bacteria which attached to the packing surface died, leading to more shedding of the biofilm. New biofilm was easily formed because more bacteria quickly attached to the exposed surface of the packing. As a result, the biofilm system maintained steady removal efficiency. Biomass quantity was one of the most important parameters in characterizing biofilms in the biological water treatment process. On the other hand, the key parameter
Biomass and biofilm activity in the biofilm reactor.
Sampling location (mm) 250 750 1250
3.5:1
Substrate concentration (mgNO− 3 −N/L) 43.55 21.12 9.75
Biomass (103 gMLSS/m3 ceramsite) 5.13 2.26 1.18
Biofilm activity (SOUR) (mgO2 /gMLSS/h) 25.26 48.94 34.73
Downloaded by [Gazi University] at 03:01 27 December 2014
6
D. Chen et al.
of a water and wastewater treatment process was the estimation of active biomass or biofilm activity, which had a direct influence on the substrate degradation rate.[30] Biofilm activity was not proportional to the quantity of fixed biomass, but increased with the thickness of biofilm up to a determined level of ‘active thickness’ and then decreased.[31] In this research, experiments were carried out to investigate the relationship between biomass and biofilm activity in the representative regions of the bio-ceramsite reactor. Table 3 shows the variation trends of biomass and biofilm activity in the reactor. It was observed that biomass decreased along with flow, which was in accordance with the variety of the available substrate concentrations in the water. The reason for this might be that a large number of suspended solids were accumulated through ceramsite at the bottom of the reactor, where the concentrations of the organic substrate and nitrate at the bottom of the ceramsite were higher than in other zones, so that a lot of aerobic denitrifying bacteria competed for oxygen and a carbon source and then these bacteria bred more rapidly. However, the biofilm activity (which was represented by SOUR) first increased to a peak value (48.94 mgO2 /gMLSS/h) and then decreased to 34.73 mgO2 /gMLSS/h with biomass decreasing from 5.13 to 1.18 103 gMLSS/m3 ceramsite. This result was similar to that of Bratbak’s [31] research, which demonstrated that biofilm activity was not proportional to the biomass in the bio-ceramsite reactor, but increased with the quantity of the biomass up to a peak value and then decreased along with increasing biomass quantity. 4. Conclusions A newly isolated bacterium Pseudomonas sp. X31 performed excellent aerobic denitrification in a bio-ceramsite reactor. NO− 3 −N, TN, and COD removal efficiencies increased with increase in the air–water ratio from 3:1 to 6.5:1 and the highest removal efficiencies (NO− 3 −N 91.54, TN 91.07%, and COD 96.57%) were observed at an air–water ratio of 6.5:1. The average NO− 3 −N, TN, and COD removal efficiencies reached the peak value of 95.18%, 92.68%, and 90.45% at hydraulic loading of 0.75 m/h. The C/N ratio has a significant effect on NO− 3 −N, TN, and COD removal and the optimal C/N ratio was 4.5:1 for the reactor. The biomass quantity decreased along with flow, but the biofilm activity first increased to a peak value (48.94 mgO2 /gMLSS/h) and then decreased to 34.73 mgO2 /gMLSS/h. Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was financially supported by the National Natural Science Foundation of China (NSFC) [ grant number 51008239],
[grant number 51378400]; the Natural Science Foundation of Hubei Province, China [grant number 2013CFB289], [grant number 2013CFB308]; and the opened fund of State Key Lab of Urban Water Resources and Environment (HIT) [grant number QAK201014].
References [1] Chen J, Strous M. Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution. Biochim Biophys Acta. 2013;1827:136–144. [2] Yao S, Ni J, Ma T, Li C. Heterotrophic nitrification and aerobic denitrification at low temperature by a newly isolated bacterium, Acinetobacter sp. HA2. Bioresour Technol. 2013;139:80–86. [3] Zhu G, Peng Y, Li B, Guo J, Yang Q, Wang S. Biological removal of nitrogen from wastewater. Rev Environ Contam Toxicol. 2008;192:159–195. [4] Bernat K, Wojnowska-Baryła I. Carbon source in aerobic denitrification. Biochem Eng J. 2007;36:116–122. [5] Zumft WG. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev. 1997;61:533–616. [6] Chèneby D, Philippot L, Hartmann A, Hénault C, Germon JC. 16S rDNA analysis for characterization of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol. 2000;34:121–128. [7] Frette L, Gejlsbjerg B, Westermann P. Aerobic denitrifiers isolated from an alternating activated sludge system. FEMS Microbiol Ecol. 1997;24:363–370. [8] Huang X, Li W, Zhang D, Qin W. Ammonium removal by a novel oligotrophic Acinetobacter sp. Y16 capable of heterotrophic nitrification-aerobic denitrification at low temperature. Bioresour Technol. 2013;146:44–50. [9] Huang HK, Tseng SK. Nitrate reduction by Citrobacter diversus under aerobic environment. Appl Microbiol Biotechnol. 2001;55:90–94. [10] Joo H-S, Hirai M, Shoda M. Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification. Water Res. 2006;40:3029–3036. [11] Kim M, Jeong S-Y, Yoon SJ, Cho SJ, Kim YH, Kim MJ, Ryu EY, Lee S-J. Aerobic denitrification of Pseudomonas putida AD-21 at different C/N ratios. J Biosci Bioeng. 2008;106:498–502. [12] Su J-J, Liu B-Y, Liu C-Y. Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. J Appl Microbiol. 2001;90:457–462. [13] Zhang J, Wu P, Hao B, Yu Z. Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001. Bioresour Technol. 2011;102:9866– 9869. [14] Kshirsagar M, Gupta AB, Gupta SK. Aerobic denitrification studies on activated sludge mixed with Thiosphaera pantotropha. Environ Technol. 2010;16:35–43. [15] Qin L, Liu Y. Aerobic granulation for organic carbon and nitrogen removal in alternating aerobic–anaerobic sequencing batch reactor. Chemosphere. 2006;63: 926–933. [16] Third KA, Gibbs B, Newland M, Cord-Ruwisch R. Long-term aeration management for improved N-removal via SND in a sequencing batch reactor. Water Res. 2005;39:3523–3530.
Downloaded by [Gazi University] at 03:01 27 December 2014
Environmental Technology [17] Wang P, Li X, Xiang M, Zhai Q. Characterization of efficient aerobic denitrifiers isolated from two different sequencing batch reactors by 16S-rRNA analysis. J Biosci Bioeng. 2007;103:563–567. [18] Joo H-S, Hirai M, Shoda M. Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4. J Biosci Bioeng. 2005;100:184–191. [19] Chen F, Xia Q, Ju L-K. Competition between oxygen and nitrate respirations in continuous culture of Pseudomonas aeruginosa performing aerobic denitrification. Biotechnol Bioeng. 2006;93:1069–1078. [20] Apha AW. Standard methods for the examination of water and wastewater. Washington, DC: American Public Health Association; 2005. [21] Bratbak G. Bacterial biovolume and biomass estimations. Appl Environ Microbiol. 1985;49:1488–1493. [22] Scaglia B, Erriquens FG, Gigliotti G, Taccari M, Ciani M, Genevini PL, Adani F. Precision determination for the specific oxygen uptake rate (SOUR) method used for biological stability evaluation of compost and biostabilized products. Bioresour Technol. 2007;98:706–713. [23] Li E, Zeng X, Fan Y. Air–water ratio as a characteristic criterion for fine bubble diffused aeration systems. Chem Eng J. 2008;137:214–224.
7
[24] Capela S, Roustan M, Héduit AC. Transfer number in fine bubble diffused aeration systems. Water Sci Technol. 2001;43:145–152. [25] Del Pozo R, Diez V. Integrated anaerobic–aerobic fixedfilm reactor for slaughterhouse wastewater treatment. Water Res. 2005;39:1114–1122. [26] Wang Q, Feng C, Zhao Y, Hao C. Denitrification of nitrate contaminated groundwater with a fiber-based biofilm reactor. Bioresour Technol. 2009;100:2223–2227. [27] Patureau D, Bernet N, Delgenès JP, Moletta R. Effect of dissolved oxygen and carbon-nitrogen loads on denitrification by an aerobic consortium. Appl Microbiol Biotechnol. 2000;54:535–542. [28] Chiu Y-C, Chung M-S. Determination of optimal COD/nitrate ratio for biological denitrification. Int Biodeterior Biodegrad. 2003;51:43–49. [29] Atkinson B. Biochemical reactors. London: Pion Press; 1975. [30] Lazarova V, Manem J. Biofilm characterization and activity analysis in water and wastewater treatment. Water Res. 1995;29:2227–2245. [31] LaMotta EJ. Kinetics of growth and substrate uptake in a biological film system. Appl Environ Microbiol. 1976;31:286–293.
Environmental Technology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tent20
Characteristics of nitrate removal in a bio-ceramsite reactor by aerobic denitrification a
a
a
b
c
Dan Chen , Kai Yang , Hongyu Wang , Bin Lv & Fang Ma a
School of Civil Engineering, Wuhan University, Wuhan 430072, People's Republic of China
b
School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, People's Republic of China c
State Key Laboratory of Urban Water Resources and Environment, Harbin Institute of Technology, Harbin 150090, People's Republic of China Accepted author version posted online: 02 Dec 2014.Published online: 23 Dec 2014.
Click for updates To cite this article: Dan Chen, Kai Yang, Hongyu Wang, Bin Lv & Fang Ma (2014): Characteristics of nitrate removal in a bioceramsite reactor by aerobic denitrification, Environmental Technology, DOI: 10.1080/09593330.2014.993729 To link to this article: http://dx.doi.org/10.1080/09593330.2014.993729
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Environmental Technology, 2014 http://dx.doi.org/10.1080/09593330.2014.993729
Characteristics of nitrate removal in a bio-ceramsite reactor by aerobic denitrification Dan Chena , Kai Yanga , Hongyu Wanga∗ , Bin Lvb and Fang Mac of Civil Engineering, Wuhan University, Wuhan 430072, People’s Republic of China; b School of Environmental Engineering, Wuhan Textile University, Wuhan 430073, People’s Republic of China; c State Key Laboratory of Urban Water Resources and Environment, Harbin Institute of Technology, Harbin 150090, People’s Republic of China
a School
Downloaded by [Gazi University] at 03:01 27 December 2014
(Received 12 September 2014; accepted 26 November 2014 ) A newly aerobic denitrifying bacterial strain, Pseudomonas sp. X31, which was isolated from activated sludge, was added to a newly developed aerobic denitrification bio-ceramsite reactor as an inoculum to treat nitrate-polluted water and the denitrification activities of this system under different air–water ratio, hydraulic loading, and C/N (carbon/nitrogen ratio) conditions were investigated. It demonstrated excellent capability for denitrification in the bio-ceramsite reactor at air– water ratios that varied from 6.5:1 to 8:1. The optimal hydraulic loading for the bio-ceramsite reactor was 0.75 m/h with the optimum denitrification efficiency of 95.18%. The optimal C/N was 4.5:1 with a maximum nitrate removal efficiency of 98.48%. COD could be completely removed under the most appropriate condition (air–water ratio 6.5:1–8:1, hydraulic loading 0.75 m/h, and C/N 4.5:1). The quantity of the biomass in the reactor decreased along with flow, which was in accordance with the variety of the available substrate concentrations in the water. However, the biofilm activity was not proportional to the biomass in the bio-ceramsite reactor, but increased with the quantity of the biomass up to a peak value and then decreased. Keywords: aerobic denitrifying bacterial strain; Pseudomonas sp; bio-ceramsite reactor; denitrification efficiency; biofilm activity
1. Introduction Currently, the most conventional, efficient, and costeffective approach to eliminate nitrogen from municipal and industrial wastewaters is a biological process which normally requires a two-step process, nitrification followed by denitrification.[1,2] However, this common method tends to be time-consuming because of the separation of aerobic and anoxic phases and low rate of nitrification.[3] Simultaneous nitrification and denitrification can solve the problem of separate tanks required in conventional treatment plants. It means largely reduction of cost for both system space and construction. According to this idea, aerobic denitrification is highly valued because denitrification occurs directly in aerated reactors, where nitrification takes place.[4] Denitrification is the biological process in which denitrifying bacteria use nitrate as electron acceptor and organic or inorganic substances as electron donor in order to convert nitrate into nitrogen gas. Denitrification gene expression occurs under anoxic or aerobic conditions and requires the presence of an N-oxide.[5] Large numbers of denitrifying bacteria have been isolated from activated sludge and soil including[6–13] Achromobacter, Aerobacter, Alcaligenes, Bacillus, Brevibacterium, Flavobacterium, Lactobacillus,
*Corresponding author. Email: [email protected] © 2014 Taylor & Francis
Micrococcus, Proteus, Pseudomonas, and Spirillum. Aerobic denitrifying bacteria can be exemplified with efficient treatment of synthetic wastewater in an open aeration reactor with low process costs.[14–18] There were many recent reports of aerobic denitrifying species such as Alcaligenes faecalis,[18] Citrobacter diversus,[9] Pseudomonas aeruginosa,[19] Pseudomonas stutzeri,[12] and Thiosphaera pantotropha,[12] which were isolated from canals, ponds, soils, and activated sludge. In this work, pure cultures of Pseudomonas sp. X31 were inoculated into a newly developed bio-ceramsite reactor in order to form a biofilm on the surface of the ceramsite to treat nitrate-contaminated wastewater. The objective of this work was to investigate the characteristics of nitrate removal in the bio-ceramsite reactor under different air–water ratio, hydraulic loading, and C/N conditions. 2.
Materials and methods
2.1. Aerobic denitrifying bacterial strain and media The activated sludge samples for the isolation of aerobic denitrifying bacterial strain X31 (FJ480211) were collected from the steadily operating aerobic denitrification bioreactor in the laboratory (State Key Lab of
2
D. Chen et al.
Urban Water Resources and Environment, Harbin Institute of Technology, Harbin, China). Synthetic wastewater as the basic medium (COD 345.28–405.11 mg/L, NO− 3 −N 50.01–73.79 mg/L, NO− 2 −N 0.20–3.17 mg/L, TN 62.05– 90.88 mg/L, and pH 6.8–7.5) was prepared by dissolving NaNO3 , NaNO2 , NaAc, Na2 HPO4 , and KH2 PO4 , and 2 mL of trace mineral solution in distilled water (1 L). The trace mineral solution contains the following components (mg/L): MgSO4 ·7H2 O 90, MnSO4 70, ZnSO4 ·5H2 O 80, FeSO4 ·7H2 O 100, CuSO4 ·5H2 O 30, CoCl2 ·7H2 O 150, H3 BO3 20, and NaMoO4 ·2H2 O 60.
Experimental apparatus of the aerobic denitrification bio-ceramsite reactor A schematic of the reactor used in this work is shown in Figure 1. The main reactor compartment consisted of a plexiglass cylinder (diameter 100 mm, height 2000 mm). The heights of the supporting layer of gravel and the packing (ceramsite) were 100 and 1400 mm, respectively. At every 250 mm height of the packing, there was a sampling port along the periphery of the reactor. Compressed air was pumped into the bottom of the reactor using air diffuser and the aeration quantity was measured by gas measuring flowmeter. The synthetic wastewater was also pumped into the bottom of the reactor. After making good contact with the biofilm on the surface of the ceramsite, it would flow out of the top of the reactor. During the inoculation phase, the precultured Pseudomonas sp. X31 isolates were inoculated into the reactor amended with KNO3 (2 g/L), NaAc (3 g/L) and trace
element solution as described [13] in order to quickly form the biofilm. Aeration was applied consistently from the bottom of the reactor for seven days. After inoculation, the reactor was operated at a temperature of 25°C, C/N of 4.5:1, hydraulic loading of 0.75 m/h, air–water ratio of 6.5:1, and nitrate concentration of 30 mg/L for 33 days. The aerobic denitrification efficiency was perceived to be 80%, which demonstrated that the domestication stage was completed. It formed a dense dark brown biofilm on the surface of the ceramsite in the reactor at the end of the domestication stage. After the domestication stage, the experiment went into the steady operation phase. In order to compare nitrate and COD reduction rates at different air–water ratios, the applied air–water ratios were adjusted to 3:1, 5:1, 6.5:1, 8:1, and 10:1, with hydraulic loading, temperature, nitrate concentration, TN, and COD at steady values of 0.75 m/h, 25°C, 64.64–73.79, 80.99–90.88, and 345.28–382.43 mg/L, respectively. In order to compare nitrate and COD reduction rates at different hydraulic loadings, the applied hydraulic loadings were adjusted to 0.60, 0.75, and 0.90 m/h, with air–water ratio, temperature, C/N, nitrate concentration, and TN at steady values of 6.5:1, 25°C, 4.5:1, 50.01–59.04 mg/L, and 62.05–69.30 mg/L, respectively. In order to compare nitrate and COD reduction rates at different C/N ratios, the applied C/N ratios were adjusted to 1.8:1, 2.5:1, 3.5:1, 4.2:1, 4.5:1, and 5:1, with hydraulic loading, air–water ratio, temperature, NO− 3 −N, TN, and COD at steady values of 0.75 m/h, 6.5:1, 25°C, 63.01–73.63, 69.89–83.13, and 349.57– 401.02 mg/L, respectively.
Figure 1. Schematic of an aerobic denitrification bio-ceramsite reactor (1. intake pump; 2. liquid measuring flowmeter; 3. main reactor; 4. ceramsite; 5. air diffuser; 6. air pump; 7. gas measuring flowmeter; 8. sampling port; 9. temperature sensor; 10. electric heating tape; and 11. temperature controller).
2.3. Analytical methods − Total nitrogen (TN), nitrate (NO− 3 −N), nitrite (NO2 −N), + ammonium (NH4 −N), and chemical oxygen demand (COD) were measured according to the Standard Methods.[20] Total organic carbon (TOC) was determined by the TOC analyzer (TOC-V CPH, Shimadzu, Japan). Dissolved oxygen (DO) in the reactor was measured by the DO meter (AZ8403, AZ Instrument Corp, Taiwan). The pH was measured by the pH meter (PHS-3C, Kexiao Instrument, China). The sampling points were chosen above the supporting layers of 250, 750, and 1250 mm. The biomass was measured by the dry weight method,[21] and the biofilm activity was measured by the specific oxygen uptake rate (SOUR).[22] The bacteria were identified by 16S rRNA sequencing analysis by Sangon (Sangon Biotech Shanghai Co., Ltd., China) after isolation and purification. During the isolation and purification phase of X31, 30 mL turbid liquid of the activated sludge samples was added into 100 mL medium, and then the mixed liquid was cultured for 48 h at a temperature of 30°C in a thermostatic oscillator. This step was repeated a few times until nitrate could be reduced at a steady rate. After that, the culture solution was vaccinated onto the solid
Downloaded by [Gazi University] at 03:01 27 December 2014
2.2.
3
Environmental Technology tablet, and then cultured for seven days in a biochemical incubator of 30°C. Finally, the separation–purification phase was completed through plate streaking on the solid medium, forming smooth colonies. To ensure the purity of the isolated strains, plate streaking was repeated several times. The effects of the operating conditions including the air–water ratio, hydraulic loading, and C/N ratio on aerobic denitrification were analysed by variance analysis and significance analysis.
Downloaded by [Gazi University] at 03:01 27 December 2014
3. Results and discussion 3.1. The effect of air–water ratio on aerobic denitrification It is apparent from Table 1 that the concentrations of − NO− 3 −N, NO2 −N, TN, COD, and DO in influent and effluent of the aerobic denitrification bio-ceramsite reactor were changed with various air–water ratios. The applied air–water ratios were 3:1, 5:1, 6.5:1, 8:1, and 10:1, with hydraulic loading, temperature, nitrate concentration, TN, and COD at steady values of 0.75 m/h, 25°C, 64.64–73.79, 80.99–90.88, and 345.28–382.43 mg/L, respectively. It can be observed from Table 1 that the NO− 3 −N, TN, and COD removal efficiencies increased with increase in air–water ratio from 3:1 to 6.5:1. The highest efficiencies (NO− 3 −N 91.54%, TN 91.07%, and COD 96.57%) were observed at air–water ratio of 6.5:1. When air–water ratio increased to 8:1, the removal efficiencies of NO− 3 −N, TN, and COD remained at relatively high levels of 89.37%, 88.53%, and 94.90%, respectively. However, the removal efficiencies NO− 3 −N, TN, and COD dropped to 72.29%, 78.26%, and 87.12% when the air–water ratio was further increased to 10:1. During the whole air–water ratio experimental stage, the NO− 2 −N concentration varied from 0.39 to 2.85 mg/L. The effluent DO concentration increased
with increasing air–water ratio, which was in accordance with Li’s [23] report. The results demonstrated that the increase in the air– water ratio caused increase in NO− 3 −N, TN, and COD removal efficiencies within the range of 3:1–6.5:1; the reason was that the increase in air–water ratio increased DO concentration,[23] thus enhancing the turbulence degree of the mixed liquor in the reactor in order to improve the mass transfer on the surface of the biofilm. On the other hand, the bubble effect of the air was reinforced as the air–water ratio increased,[23,24] which contributed to accelerated scouring action of the biofilm on the big surface of the ceramsite; as a result the updating speed of the biofilm was faster and the activity of the biofilm was higher. However, the air–water ratio should not be too high because a very high air–water ratio (10:1) not only increased the operation cost, but also led to larger aeration quantity, causing extremely intensified scour of the biofilm so that the microorganism in the reactor could not grow well. In addition, a very high air–water ratio would result in a very high DO environment, which was harmful to the denitrification process.[25] In this study, the bio-ceramsite reactor performed well when the air–water ratio varied from 6.5:1 to 8:1.
3.2.
The effect of hydraulic loading on aerobic denitrification − Figure 2 shows the concentrations of NO− 3 −N, NO2 −N, TN, and COD in the influent and effluent of the aerobic denitrification bio-ceramsite reactor with various hydraulic loadings. The applied hydraulic loadings were 0.60, 0.75, and 0.90 m/h, with the air–water ratio, temperature, C/N, nitrate concentration, and TN at steady values of 6.5:1, 25°C, 4.5:1, 50.01–59.04 mg/L, and 62.05–69.30 mg/L, respectively.
− Table 1. Concentrations of NO− 3 −N,NO2 −N, TN, COD, and DO in the influent and effluent under different air–water ratio conditions {analysis results of significance test of the air–water ratio on aerobic denitrification SSA [Sum of Squares for factor A (air–water ratio)] = 4028.78, SSE (Sum of Squares for Error) = 218.25, SST (Sum of Squares for Total) = 4247.02, and F(level of significance) = 138.45 > F crit }.
Air–water ratio Test items NO− 3 −N NO− 2 −N TN COD DO
Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L)
3:1
5:1
6.5:1
8:1
73.79 27.77 62.37 1.94 1.44 90.88 31.06 65.82 382.43 62.42 83.68 2.71–3.82 2.54–4.01
64.64 15.98 75.28 2.85 0.70 80.99 18.01 77.76 345.29 49.57 85.64 3.27–4.71 3.18–4.57
66.76 5.65 91.54 0.87 0.39 81.16 7.25 91.07 362.43 12.43 96.57 3.76–5.51 3.85–5.25
68.56 7.29 89.37 2.20 0.84 84.92 9.74 88.53 383.86 19.57 94.90 4.02–6.14 4.11–6.20
10:1 67.91 18.82 72.29 1.64 1.30 83.45 18.14 78.26 373.86 48.14 87.12 4.36–6.79 4.25–6.86
Downloaded by [Gazi University] at 03:01 27 December 2014
4
D. Chen et al.
− Figure 2. Concentrations of NO− 3 −N, NO2 −N, TN, and COD in the influent and effluent under different hydraulic loading conditions {analysis results of significance test of hydraulic loading on aerobic denitrification SSB [Sum of Squares for factor B (hydraulic loading)] = 4741.39, SSE (Sum of Squares for Error) = 218.01, SST (Sum of Squares for Total) = 4959.40, F(level of significance) = 195.73 > F crit }.
Figure 2 shows that the average denitrification efficiency reached the peak value of 95.18% at a hydraulic loading of 0.75 m/h; meanwhile the average TN and COD removal efficiencies also reached peak values of 92.68% and 90.45% under this condition. However, the removal efficiencies of NO− 3 −N, TN, and COD decreased to 71.00%, 71.44%, and 79.23% as hydraulic loading increased to 0.90 m/h. Also, the removal efficiencies decreased to 60.91%, 66.40%, and 76.86% with hydraulic loading of 0.60 m/h. Moreover, the effluent NO− 2 −N was always maintained at a low level below 4.09 mg/L during this phase. It is very important to determine the appropriate hydraulic loading for the reactor because the performance of denitrification was obviously associated with hydraulic loading.[26] If hydraulic loading was too low, the removal efficiencies decreased because of insufficient carbon source for the rapidly proliferative microorganism to remove the nitrate, and the construction cost increased. Nevertheless, a very high hydraulic loading also caused decrease in removal efficiencies, due to the fact that the synthetic wastewater stayed for a shorter time in the reactor, so that the substrate did not have enough time to make contact with the aerobic denitrifying bacteria; on the other hand, the biofilm on the surface of the ceramsite might be easily washed away under very high hydraulic loading condition so that the aerobic denitrifying bacteria could not proliferate well. In the present study, the optimal hydraulic loading for the bio-ceramsite reactor
was 0.75 m/h with the optimum denitrification efficiency of 95.18%.
3.3. The effect of C/N on aerobic denitrification It is apparent from Table 2 that the concentrations of − NO− 3 −N, NO2 −N, TN, COD, and DO in the influent and effluent of the aerobic denitrification bio-ceramsite reactor were changed with various C/N ratios. The applied C/N ratios were 1.8:1, 2.5:1, 3.5:1, 4.2:1, 4.5:1, and 5:1, with hydraulic loading, air–water ratio, temperature, NO− 3 −N, TN, and COD at steady values of 0.75 m/h, 6.5:1, 25°C, 63.01–73.63, 69.89–83.13, and 349.57– 401.02 mg/L, respectively. It can be observed from Table 2 that the C/N ratio has − significant effects on NO− 3 −N, NO2 −N, TN, and COD − removal. NO3 −N and TN removal efficiencies rapidly increased as the C/N ratio increased from 1.8:1 to 4.5:1. The highest NO− 3 −N and TN removal efficiencies (98.48% and 96.85%) were observed at a C/N ratio of 4.5:1. The removal efficiency of COD slowly increased from 85.62% to 94.04% when the C/N ratio varied from 1.8:1 to 4.5:1. If the C/N ratio was further increased to 5:1, the removal efficiencies of NO− 3 −N and TN reached roughly the same level as C/N = 4.5:1. However, the removal efficiency of COD dropped to 88.35%. During the experimental phase, the effluent NO− 2 −N concentration always remained at low level below 3.17 mg/L.
5
Environmental Technology
− Table 2. Concentrations of NO− 3 −N,NO2 −N, TN, COD, and DO in the influent and effluent under different C/N conditions (analysis results of significance test of C/N on aerobic denitrification SSC (Sum of Squares for factor C(C/N)) = 30508.34, SSE (Sum of Squares for Error) = 307.71, SST (Sum of Squares for Total) = 30816.05, and F(level of significance) = 713.84 > F crit ).
C/N Test items
1.8:1
NO− 3 −N
Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L) Efficiency (%) Influent (mg/L) Effluent (mg/L)
NO− 2 −N TN COD
Downloaded by [Gazi University] at 03:01 27 December 2014
DO
63.01 47.81 24.12 0.53 0.20 69.89 57.62 17.56 378.14 54.36 85.62 3.26–4.67 3.08–4.56
2.5:1 69.54 38.66 44.41 2.39 2.10 79.12 48.91 38.19 373.86 46.36 87.60 2.93–4.86 3.73–4.99
It is important to optimize the C/N ratio for each denitrifying bacterium because carbon is essential for cell growth and nitrate reduction processes; the optimal quantity of carbon is a key parameter in the denitrification process.[27] When the carbon source was higher, nitrate and nitrite were easily converted into nitrogen gas due to sufficient energy.[11,28] However, an extremely high concentration of carbon source inhibited the aerobic denitrification process [9] and caused a high level of organic carbon source in the effluent. If the C/N ratio was maintained at an appropriate level in the aerobic denitrifica− tion system, the nitrogen oxides – NO− 3 −N, NO2 −N, + NH4 −N, etc. – could be completely converted to nitrogen gas, while guaranteeing the minimality of the effluent organic carbon source. According to some similar work, the NO− 3 −N removal efficiencies by aerobic denitrifying bacteria isolated from sequencing batch reactors were 30.5–60.5%.[17] In the present study, the optimal C/N ratio was 4.5:1 with a maximum nitrate removal efficiency of 98.48%. These results should be useful information for performing an aerobic denitrification process in wastewater treatment plants. 3.4.
Biofilm purification mechanism and biofilm activity analysis in the reactor
Biofilm had been successfully used in water treatment for over a century.[29] Wastewater biofilms are very complex Table 3.
66.60 10.72 83.91 3.07 3.17 76.63 19.00 75.21 363.86 39.99 89.01 3.89–4.90 3.90–5.10
4.2:1 73.63 6.31 91.43 1.94 2.87 83.13 11.01 86.76 349.57 26.71 92.36 3.19–4.82 3.06–4.75
4.5:1
5:1
70.36 1.07 98.48 0.84 0.99 78.31 2.47 96.85 352.43 21.00 94.04 3.89–5.09 3.88–5.13
71.18 1.24 98.26 0.77 0.70 79.15 2.61 96.08 401.02 46.71 88.35 3.52–5.09 3.23–4.98
systems consisting of microbial cells and colonies embedded in a polymer matrix, whose structure and composition was a function of biofilm age and environmental conditions.[30] The differences between the biofilm process and the traditional activated sludge process were not only the existing ways of the main microorganism, but also the behaviour characteristics of the biofilm dynamics during the diffusion process.[30] In the biofilm system, pollutants, DO, and necessary nutrients first diffused onto the surface of the biofilm through the liquid phase, and then transferred to the inner biofilm. Pollutants which diffused to the surface or interior of the biofilm were decomposed and converted to metabolite by microorganisms, while implementing complete sewage purification. Specifically, the compounds (COD, NO− 3 −N, etc.) in the synthetic wastewater were transferred to the aerobic layer of the biofilm through solute diffusion. After a long time reaction, the dead aerobic bacteria were utilized as nutrients for the anaerobic bacteria in the system. When the nutrients were further utilized and ran out, anaerobic bacteria which attached to the packing surface died, leading to more shedding of the biofilm. New biofilm was easily formed because more bacteria quickly attached to the exposed surface of the packing. As a result, the biofilm system maintained steady removal efficiency. Biomass quantity was one of the most important parameters in characterizing biofilms in the biological water treatment process. On the other hand, the key parameter
Biomass and biofilm activity in the biofilm reactor.
Sampling location (mm) 250 750 1250
3.5:1
Substrate concentration (mgNO− 3 −N/L) 43.55 21.12 9.75
Biomass (103 gMLSS/m3 ceramsite) 5.13 2.26 1.18
Biofilm activity (SOUR) (mgO2 /gMLSS/h) 25.26 48.94 34.73
Downloaded by [Gazi University] at 03:01 27 December 2014
6
D. Chen et al.
of a water and wastewater treatment process was the estimation of active biomass or biofilm activity, which had a direct influence on the substrate degradation rate.[30] Biofilm activity was not proportional to the quantity of fixed biomass, but increased with the thickness of biofilm up to a determined level of ‘active thickness’ and then decreased.[31] In this research, experiments were carried out to investigate the relationship between biomass and biofilm activity in the representative regions of the bio-ceramsite reactor. Table 3 shows the variation trends of biomass and biofilm activity in the reactor. It was observed that biomass decreased along with flow, which was in accordance with the variety of the available substrate concentrations in the water. The reason for this might be that a large number of suspended solids were accumulated through ceramsite at the bottom of the reactor, where the concentrations of the organic substrate and nitrate at the bottom of the ceramsite were higher than in other zones, so that a lot of aerobic denitrifying bacteria competed for oxygen and a carbon source and then these bacteria bred more rapidly. However, the biofilm activity (which was represented by SOUR) first increased to a peak value (48.94 mgO2 /gMLSS/h) and then decreased to 34.73 mgO2 /gMLSS/h with biomass decreasing from 5.13 to 1.18 103 gMLSS/m3 ceramsite. This result was similar to that of Bratbak’s [31] research, which demonstrated that biofilm activity was not proportional to the biomass in the bio-ceramsite reactor, but increased with the quantity of the biomass up to a peak value and then decreased along with increasing biomass quantity. 4. Conclusions A newly isolated bacterium Pseudomonas sp. X31 performed excellent aerobic denitrification in a bio-ceramsite reactor. NO− 3 −N, TN, and COD removal efficiencies increased with increase in the air–water ratio from 3:1 to 6.5:1 and the highest removal efficiencies (NO− 3 −N 91.54, TN 91.07%, and COD 96.57%) were observed at an air–water ratio of 6.5:1. The average NO− 3 −N, TN, and COD removal efficiencies reached the peak value of 95.18%, 92.68%, and 90.45% at hydraulic loading of 0.75 m/h. The C/N ratio has a significant effect on NO− 3 −N, TN, and COD removal and the optimal C/N ratio was 4.5:1 for the reactor. The biomass quantity decreased along with flow, but the biofilm activity first increased to a peak value (48.94 mgO2 /gMLSS/h) and then decreased to 34.73 mgO2 /gMLSS/h. Disclosure statement No potential conflict of interest was reported by the authors.
Funding This work was financially supported by the National Natural Science Foundation of China (NSFC) [ grant number 51008239],
[grant number 51378400]; the Natural Science Foundation of Hubei Province, China [grant number 2013CFB289], [grant number 2013CFB308]; and the opened fund of State Key Lab of Urban Water Resources and Environment (HIT) [grant number QAK201014].
References [1] Chen J, Strous M. Denitrification and aerobic respiration, hybrid electron transport chains and co-evolution. Biochim Biophys Acta. 2013;1827:136–144. [2] Yao S, Ni J, Ma T, Li C. Heterotrophic nitrification and aerobic denitrification at low temperature by a newly isolated bacterium, Acinetobacter sp. HA2. Bioresour Technol. 2013;139:80–86. [3] Zhu G, Peng Y, Li B, Guo J, Yang Q, Wang S. Biological removal of nitrogen from wastewater. Rev Environ Contam Toxicol. 2008;192:159–195. [4] Bernat K, Wojnowska-Baryła I. Carbon source in aerobic denitrification. Biochem Eng J. 2007;36:116–122. [5] Zumft WG. Cell biology and molecular basis of denitrification. Microbiol Mol Biol Rev. 1997;61:533–616. [6] Chèneby D, Philippot L, Hartmann A, Hénault C, Germon JC. 16S rDNA analysis for characterization of denitrifying bacteria isolated from three agricultural soils. FEMS Microbiol Ecol. 2000;34:121–128. [7] Frette L, Gejlsbjerg B, Westermann P. Aerobic denitrifiers isolated from an alternating activated sludge system. FEMS Microbiol Ecol. 1997;24:363–370. [8] Huang X, Li W, Zhang D, Qin W. Ammonium removal by a novel oligotrophic Acinetobacter sp. Y16 capable of heterotrophic nitrification-aerobic denitrification at low temperature. Bioresour Technol. 2013;146:44–50. [9] Huang HK, Tseng SK. Nitrate reduction by Citrobacter diversus under aerobic environment. Appl Microbiol Biotechnol. 2001;55:90–94. [10] Joo H-S, Hirai M, Shoda M. Piggery wastewater treatment using Alcaligenes faecalis strain No. 4 with heterotrophic nitrification and aerobic denitrification. Water Res. 2006;40:3029–3036. [11] Kim M, Jeong S-Y, Yoon SJ, Cho SJ, Kim YH, Kim MJ, Ryu EY, Lee S-J. Aerobic denitrification of Pseudomonas putida AD-21 at different C/N ratios. J Biosci Bioeng. 2008;106:498–502. [12] Su J-J, Liu B-Y, Liu C-Y. Comparison of aerobic denitrification under high oxygen atmosphere by Thiosphaera pantotropha ATCC 35512 and Pseudomonas stutzeri SU2 newly isolated from the activated sludge of a piggery wastewater treatment system. J Appl Microbiol. 2001;90:457–462. [13] Zhang J, Wu P, Hao B, Yu Z. Heterotrophic nitrification and aerobic denitrification by the bacterium Pseudomonas stutzeri YZN-001. Bioresour Technol. 2011;102:9866– 9869. [14] Kshirsagar M, Gupta AB, Gupta SK. Aerobic denitrification studies on activated sludge mixed with Thiosphaera pantotropha. Environ Technol. 2010;16:35–43. [15] Qin L, Liu Y. Aerobic granulation for organic carbon and nitrogen removal in alternating aerobic–anaerobic sequencing batch reactor. Chemosphere. 2006;63: 926–933. [16] Third KA, Gibbs B, Newland M, Cord-Ruwisch R. Long-term aeration management for improved N-removal via SND in a sequencing batch reactor. Water Res. 2005;39:3523–3530.
Downloaded by [Gazi University] at 03:01 27 December 2014
Environmental Technology [17] Wang P, Li X, Xiang M, Zhai Q. Characterization of efficient aerobic denitrifiers isolated from two different sequencing batch reactors by 16S-rRNA analysis. J Biosci Bioeng. 2007;103:563–567. [18] Joo H-S, Hirai M, Shoda M. Characteristics of ammonium removal by heterotrophic nitrification-aerobic denitrification by Alcaligenes faecalis No. 4. J Biosci Bioeng. 2005;100:184–191. [19] Chen F, Xia Q, Ju L-K. Competition between oxygen and nitrate respirations in continuous culture of Pseudomonas aeruginosa performing aerobic denitrification. Biotechnol Bioeng. 2006;93:1069–1078. [20] Apha AW. Standard methods for the examination of water and wastewater. Washington, DC: American Public Health Association; 2005. [21] Bratbak G. Bacterial biovolume and biomass estimations. Appl Environ Microbiol. 1985;49:1488–1493. [22] Scaglia B, Erriquens FG, Gigliotti G, Taccari M, Ciani M, Genevini PL, Adani F. Precision determination for the specific oxygen uptake rate (SOUR) method used for biological stability evaluation of compost and biostabilized products. Bioresour Technol. 2007;98:706–713. [23] Li E, Zeng X, Fan Y. Air–water ratio as a characteristic criterion for fine bubble diffused aeration systems. Chem Eng J. 2008;137:214–224.
7
[24] Capela S, Roustan M, Héduit AC. Transfer number in fine bubble diffused aeration systems. Water Sci Technol. 2001;43:145–152. [25] Del Pozo R, Diez V. Integrated anaerobic–aerobic fixedfilm reactor for slaughterhouse wastewater treatment. Water Res. 2005;39:1114–1122. [26] Wang Q, Feng C, Zhao Y, Hao C. Denitrification of nitrate contaminated groundwater with a fiber-based biofilm reactor. Bioresour Technol. 2009;100:2223–2227. [27] Patureau D, Bernet N, Delgenès JP, Moletta R. Effect of dissolved oxygen and carbon-nitrogen loads on denitrification by an aerobic consortium. Appl Microbiol Biotechnol. 2000;54:535–542. [28] Chiu Y-C, Chung M-S. Determination of optimal COD/nitrate ratio for biological denitrification. Int Biodeterior Biodegrad. 2003;51:43–49. [29] Atkinson B. Biochemical reactors. London: Pion Press; 1975. [30] Lazarova V, Manem J. Biofilm characterization and activity analysis in water and wastewater treatment. Water Res. 1995;29:2227–2245. [31] LaMotta EJ. Kinetics of growth and substrate uptake in a biological film system. Appl Environ Microbiol. 1976;31:286–293.
