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Biological treatment of high NH4+-N wastewater using an ammonia-tolerant photosynthetic bacteria strain (ISASWR2014)
Biological treatment of high NH 4 + -N wastewater using an ammonia-tolerant photosynthetic bacteria strain (ISASWR2014) Qin Zhou, Guangming Zhang, Xiang Zheng, Guohua Liu PII: DOI: Reference:
S1004-9541(15)00294-3 doi: 10.1016/j.cjche.2015.08.018 CJCHE 373
To appear in: Received date: Revised date: Accepted date:
15 December 2014 16 June 2015 17 June 2015
Please cite this article as: Qin Zhou, Guangming Zhang, Xiang Zheng, Guohua Liu, Biological treatment of high NH4 + -N wastewater using an ammonia-tolerant photosynthetic bacteria strain (ISASWR2014), (2015), doi: 10.1016/j.cjche.2015.08.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Biological treatment of high NH4+-N wastewater using an ammonia-tolerant
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photosynthetic bacteria strain (ISASWR2014)
School of Environment & Natural Resource, Renmin University of China, Beijing 100872, China
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1
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Qin Zhou1, Guangming Zhang1*, Xiang Zheng1, Guohua Liu1
Abstract
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Wastewater with high NH4+-N is difficult to treat by traditional methods. So in this paper, a wild
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strain of photosynthetic bacteria was used for high NH4+-N wastewater treatment in together with
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biomass recovery. Isolation, identification and characterization of the microorganism were carried out. The strain was inoculated to the biological wastewater treatment unit. The impacts of important
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factors were examined, including temperature, dissolved oxygen and light intensity. Results showed that photosynthetic bacteria could effectively treat high NH4+-N wastewater. For wastewater with
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NH4+-N of 2300 mg·L-1, COD/N=1.0, 98.3% of COD was removed, and cell concentration increased by 43 times. The optimal conditions for the strain’s cell growth and wastewater treatment were 30 oC, dissolved oxygen of 0.5-1.5 mg·L-1 and light intensity of 4000 lux. Photosynthetic bacteria could bear a lower C/N ratio than bacteria in a traditional wastewater treatment process, but the NH4+-N removal was only 20%-40% because small molecule carbon source was used prior to NH4+-N. Also,
*
Corresponding Author: E-mail: [email protected]
Acknowledgments Supported by the National Natural Science Foundation of China (51278489). 1
ACCEPTED MANUSCRIPT the use of photosynthetic bacteria in chicken manure wastewater containing NH4+-N about 7000 mg·L-1 proved that photosynthetic bacteria could remove NH4+-N in a real case, finally, 83.2% of
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NH4+-N was removed and 66.3% of COD was removed.
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Graphic abstract
The isolated photosynthetic bacteria (PSB) were used to treat chicken manure wastewater contained recalcitrant organic compounds. The total COD value was about 3000 mg·L-1, and NH4+-N concentration was 7000 mg·L-1, with a C/N of 0.42. The use of PSB proved to be very efficient in treating such a high NH4+-N and low C/N wastewater. The results indicated that the PSB has special adaptations for survival and reducing the NH4+-N even with an NH4+-N concentration of 7000 mg·L-1. The COD removal reached 66.3% and the NH4+-N removal reached 83.2%.
[Keyword: High NH4+-N wastewater; C/N; Photosynthetic bacteria; Chicken manure wastewater] 2
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INTRODUCTION
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With the rapid development of fertilizer and petrochemical industry, a large number of high
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NH4+-N wastewater discharged. NH4+-N is usually oxidized to nitrate and nitrite, the nitrate and nitrite usually may cause serious eutrophication, and they are hazardous to humans. So NH4+-N wastewater treatment is essential for preventing eutrophication and human harm. High NH4+-N
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wastewaters, such as piggery wastewater and landfill leachate contain not only high concentrations
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of nitrogen compounds (up to 1000-3000 mg·L-1) but also high concentrations of organic matter. In high NH4+-N wastewaters, the C/N ration is often quite low and the ammonia concentration is
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50-100 times higher than that in municipal wastewater [1-3]. All these contributed to the difficulty of
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conventional biological treatment of high NH4+-N wastewaters.
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Generally, the most common and cost-efficient approach for ammonia wastewater treatment is biological treatment [4, 5]. However, traditional nitrification process is not effective in such high
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ammonia environment with such a low C/N. One reason is that autotrophic bacteria are vulnerable to high concentrations of wastewater with C/N ratio of 0-2 [6]. Another reason is that excessive high ammonia exerts a toxic and inhibitory effect on microbial activity [7, 8]. Thus, traditional nitrification can be used only after pretreating the wastewater to increase C/N ratio [9] or decrease ammonia concentration of the wastewater [10, 11], which greatly increases the operation cost. Hence, the utilization of an ammonia-tolerant organism in biological treatment units seems to be a reasonable approach for high NH4+-N wastewater treatment. Application of photosynthetic bacteria (PSB) might be employed in degrading pollutants in high NH4+-N wastewater treatment. PSB are widely distributed in the ocean, lakes, soil and activated sludge also in 3
ACCEPTED MANUSCRIPT high-temperature, low-temperature, high-ammonia or low-ammonia environment. They grow in both aerobic and anaerobic light conditions. They are metabolically the most versatile among all
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prokaryotes [12]. PSB have been used to treat a variety of wastewaters such as cadmium wastewater,
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olive mill wastewater and sulfide containing wastewater [15-19]. And using PSB for wastewater treatment has been proven to be a cost effective method. This is because PSB do not only remove pollutants in wastewater, but also accumulate useful materials such as single-cell protein,
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biopolymers, antimicrobial agents, carotene, pantothenic acid and therapeutic compounds [13, 14].
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The biomass can be recycled as useful raw materials in food, medical and agriculture industries. In this work, a new process was investigated, a strain of PSB isolated from aqua-farm,
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demonstrated an amazing tolerance to high NH4+-N. The aim of the paper was to study the ability of
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PSB to treat high NH4+-N wastewater in together with biomass recovery.
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MATERIALS AND METHODS
Isolation and identification of ammonia-tolerant microorganism
Aqua-farm water
provided the medium for PSB isolation and selection. The bacteria strain was isolated with the medium [12] consisting of DL-malate: 4 g·L-1, MgSO4: 0.12 g·L-1, (NH4)2SO4: 1 g·L-1, CaCl2: 0.075 g·L-1, KH2PO3: 0.3 g·L-1, Na2EDTA: 0.020 g·L-1, VB1: 0.001 g·L-1. Nicotinic acid: 0.001 g·L-1, Biotin: 0.015 g·L-1. Trace element solution, 1 ml. The trace element solution contained ZnSO4•7H2O: 20 mg·L-1, MnCl2•4H2O: 6 mg·L-1, H3BO3: 60 mg·L-1, CoCl2•6H2O: 40 mg·L-1, CuCl2•2H2O: 2 mg·L-1. pH was 7.0. And the culture was anaerobically at 28-35 ºC and light intensity of 2000 lux conditions for 96 hours. 4
ACCEPTED MANUSCRIPT The mixed culture were examined microscopically, and they were purified and identified according to the biochemical reactions such as catalase test, tolerance to 3% NaCl, gram test, nitrate
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reduction test, indole test, H2S reaction and spectrum scanning. All experiments were carried out
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according to Bergey’s manual [20].
Artificial high NH4+-N wastewater was used for PSB
Wastewater and reactor operation
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treatment. And its characteristics were as follows: COD, NH4+-N and total phosphorus (TP) were
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2300, 2300, and 45 mg·L-1. The initial pH was around 7.0. The carbon to nitrogen ratio (C/N) was 1, which was much lower than that in the traditional wastewater treatment.
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The bioreactors were 500 mL glass flasks. These flasks were sterilized NH4+-N at 121 oC for 30
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min before use. Each time, 400 mL artificial high NH4+-N wastewater was added to the bioreactor.
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PSB were inoculated and the initial cell concentration in wastewater was 10 mg (dry weight)/L. The amount of PSB was 5% (by volume) in conducted experiments. The artificial high NH4+-N
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wastewater and PSB were rotated at 120 r·min-1 with temperature 10, 20, 30, 40 and 50 oC, respectively, in the five experimental groups. The dissolved oxygen (DO) was less than 0.5, 0.5-1.5 and 5.5-6.5 mg·L-1, respectively; the value was chosen according to previous studies. The illumination intensity were <500, 500, 1000, 2000, 4000 and 8000 lux, respectively. The dissolved oxygen in the control group (< 0.5 mg·L-1) condition was realized by micro aeration (the purity of nitrogen was 98.0%). After saturated with nitrogen, the bioreactors were sealed with sealing membrane to keep micro-oxygen or anaerobic condition. The dissolved oxygen (DO) of 0.5-1.5 mg·L-1 was realized without aeration, and the bioreactor was directly covered with oxygen enriching membranes. The dissolved oxygen of 5.5-6.5 mg·L-1 was realized with micro 5
ACCEPTED MANUSCRIPT aeration (the purity of oxygen was 98.5%).
Chicken manure wastewater from a plant was treated biologically using the
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Case study
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All experiments were repeated twice.
isolated PSB. The characteristics of the chicken manure wastewater were as below: total COD, total nitrogen (TN), NH4+-N and TP were about 3000, 8000, 7000 and 15 mg·L-1, respectively. The pH
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Analysis
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was about 9.6.
Samples were collected from bioreactors and were centrifuged at 9000 rpm for 10
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min. The supernatant was used to test the COD with APHA standard methods [22]; the collected PSB
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were used to measure the biomass (dry weight). The pH was measured using pH tester and the DO
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was measured using dissolved oxygen meter. The biomass was tested by APHA standard methods. NH4+-N levels were analyzed by the Nesslerization method at an absorbance of 420 nm using a
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TU-1900 spectrophotometer.
RESULTS AND DISCUSSIONS
Isolation and identification of the ammonia-tolerant microorganism
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ammonia-tolerant microorganism was isolated from an eutrophic aqua-farm. The identification of bacteria was based on cultural and biochemical characteristics, the results were shown in Tab. 1. The isolate strain was examined microscopically as gram-negative bacteria. After 48 h culture under anaerobic light conditions, cell suspensions were red and the absorption spectra of living cells 6
ACCEPTED MANUSCRIPT suspension showed maxima absorbance peaks at 270, 280, 375, 800 and 860 nm. The main peak at 860 nm was closely related to bacteriochlorin a, and the main peaks at 280, 375 and 800 nm was
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closely related to carotenoids. The contained pigments of the isolate were characteristics of purple
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non-sulfur photosynthetic bacteria [12]. Also the strain was able to grow in the presence of 3% NaCl. The strain had a negative indole reaction, H2S positive and catalase-positive in the test. According to Bergey's Manual of Determinative Bacteriology, the strain was identified as Pseudomonas sp.
Features Bacteriochlorin A Carotenoid Catalase reaction
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Gram straining
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Growth in 3% NaCl
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Properties and biochemical tests
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Tab. 1. Identification and biochemical tests of the strain
Nitrate reduction Indole test
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H2S reaction
Results + + + + — + — +
Utilization for different carbon sources
—
Ethyl alcohol
— — — — — +
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Sodium thiosulfate Methylcellulose Sodium oleate
Protein Powder Glycerinum Glucose
+ + + + +
Mannose Sodium acetate Mannitol Sorbitol Fructose
Feasibility study of the strain in high NH4+-N wastewater treatment 7
The isolated PSB
ACCEPTED MANUSCRIPT were inoculated to the biological wastewater treatment unit. The feasibility was studied and the results were shown in Fig.1. PSB could effectively treat high NH4+-N wastewater. 98.3% of COD
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was removed at the 96th hour. Meanwhile, biomass grew well in high NH4+-N wastewater. The final
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cell concentration was 435 mg ·L-1 at the 96th hour, which increased by 43 times. There was a decrease in PSB cell concentration during the 72th to 96th hour. This could be explained by the quick breeding of PSB in the first 72 hours, and the left organic compounds were insufficient for all cells,
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and bacteria began to enter into a stagnant stage for lack of food and energy. Thus PSB started to
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dissolve, and the corresponding cell concentration decreased. The COD reduction of the high NH4+-N wastewater followed a pseudo first order reaction with a first order rate constant (k) of
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0.042/d and R2 of 0.996.
Fig. 1. COD removal and PSB biomass production in high NH4+-N wastewater, DO=0.5-1.5 mg·L-1, 30 oC, 2000 lux. 8
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Biological treatment of high NH4+-N wastewater
Temperature is one of the most
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important factors that affect the growth of bacteria. Previous studies have shown that the optimum
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temperature for PSB nutrient medium was between 30-35 oC [21]. According to the biochemistry theory, DO and light intensity are key factors for PSB growth. In order to study the effects of these factors, the temperature varied between 10 to 50 oC, DO varied between 0.0 to 6.5 mg·L-1, and light
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intensity varied between 0 to 8000 lux were studied. The results were shown in Fig. 2.
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As Fig. 2(a) showed, the highest COD removal and the best biomass growth were observed at 30 oC, with a COD reduction of about 98.3% and the cell concentration was 430 mg·L-1. Thus, the
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optimal temperature was 30 oC. Either higher or lower temperatures were bad for COD removal and
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cell accumulation.
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As Fig. 2(b) showed, COD removals under DO 0.5-1.5 or 5.5-6.5 mg·L-1 were good (>96%). The finding could be attributed to the fact that PSB thrives better in a higher DO environment.
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Meanwhile, the highest biomass production was obtained under DO of 0.5-1.5 mg·L-1, with a cell concentration of about 410 mg·L-1. Thus, the optimal DO was 0.5-1.5 mg·L-1. Lower dissolved oxygen was inadequate for photosynthetic bacteria growth, but higher dissolved oxygen did not help. Light intensity is an important factor that affects photophosphorylation. As Fig. 2(c) showed, the highest COD removal was 96.0% under 4000 lux. Meanwhile, the highest cell concentration was 505 mg·L-1 and it was improved by 74.1% than the group without a light intensity less than 500 lux. Note that the optimal light intensity was 4000 lux. Too much or too little light were both unfavorable for PSB growth. In summary, the optimal conditions for PSB growth and wastewater treatment were temperature 9
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of 30 oC, DO of 0.5-1.5 mg·L-1 and light intensity of 4000 lux.
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Fig. 2. Effects of temperature (A), DO (B) and light intensity (C) on the COD removal and cell
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concentration in high NH4+-N wastewater
The high NH4+-N wastewater has a
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The NH4+-N removal in PSB wastewater treatment
NH4+-N value of about 2300 mg·L-1, which was with a C/N ratio of 1. The characteristics of high NH4+-N and low C/N contributed to the difficulty in conventional biological treatment of this kind of wastewater. The COD removal, cell concentration and NH4+-N removal were the major considerations for PSB to treat this wastewater. Through the feasibility and optimization study, the COD removal was good (>96%), and the cell grew well in the wastewater. However, the NH4+-N removal was still about 20%-40%, which became the major problem for PSB to treat this wastewater. The reason could be that sodium acetate was the carbon source in the artificial wastewater, and sodium acetate was easily degraded by PSB. So when sodium acetate existed, this small molecule 11
ACCEPTED MANUSCRIPT carbon source was used prior to NH4+-N. Previous studies have shown that PSB were easy to degrade small molecular substances, but hard to degrade large molecular or complicated substances.
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So we suspected that if the carbon source was hard-biodegraded, there was a chance that NH4+-N
Case study
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might be degraded by PSB firstly.
Based on the results obtained with artificial high NH4+-N wastewater, the
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isolated PSB were used to treat chicken manure wastewater as a case study. The chicken manure
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wastewater contained recalcitrant organic compounds which was resistant to biodegradation. The total COD value was about 3000 mg·L-1, and NH4+-N concentration was 7000 mg·L-1. The results
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were shown in Fig. 3. The use of PSB proved to be very efficient in treating such a high NH4+-N and
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low C/N wastewater. The results indicated that the PSB has special adaptations for survival at high
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NH4+-N wastewater and it can degrade the COD and NH4+-N even at NH4+-N concentration of 7000 mg·L-1. The COD was degraded from 3000 to 1000 mg·L-1 (COD removal was 66.3%), NH4+-N was
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degraded from 7000 to 1000 mg·L-1 (NH4+-N removal was 83.2%). The COD reduction of the chicken manure wastewater for the first 96 h followed a pseudo first order reaction with a first order rate constant (k) of 0.015d-1 and R2 of 0.96.
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ACCEPTED MANUSCRIPT
This work analyzed the potential of PSB for high NH4+-N wastewater
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Conclusions
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Fig. 3. The changes of COD and NH4+-N concentration in PSB chicken manure wastewater treatment
treatment, and evaluated the important factors: temperature, DO and light intensity. Also, a case
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study using chicken manure wastewater containing NH4+-N about 7000 mg·L-1 was treated using the isolated PSB. The results were as follows: (1) PSB demonstrated an amazing tolerance to high NH4+-N wastewater and could treat high NH4+-N wastewater at low C/N effectively. (2) For wastewater with NH4+-N of 2300 mg·L-1, COD/N=1, 98.3% of COD was removed, and biomass increased by 43 times. (3) The optimal conditions for PSB growth and wastewater treatment were temperature of 30 oC, DO of 0.5-1.5 mg·L-1 and light intensity of 4000 lux. (4) The PSB results in a significant improvement in the removal of NH4+-N in the chicken manure 13
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wastewater containing NH4+-N about 7000 mg·L-1, the NH4+-N removal was 83.2% .
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References
[1] Ra, C. S., Lo, K.V., Shin, J. S., Oh, J. S., Hong, B. J., “Biological nutrient removal with an internal organic carbon source in piggery wastewater treatment”, Water Res., 34,
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965–973(2000).
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[2] Carrera, J., Baeza, J. A., Vicent, T., Lafuente, J., “Biological nitrogen removal of high-strength ammonium industrial wastewater with two-sludge system”, Water Res., 37, 4211–4221(2003). Uygur, A., Kargi, F., “Biological nutrient removal from pre-treated landfill leachate in a
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sequencing batch reactor” J. Environ. Manage, 71, 9–14(2004).
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[4] Wang, K., Wang, S. Y., Zhu, R. L., Miao, L., Peng. Y. Z., “Advanced nitrogen removal from landfill leachate without addition of external carbon using a novel system coupling ASBR and
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modified SBR”, Bioresour. Technol., 134, 212–218(2013). [5] Zhu, G., Peng, Y., Li, B., Guo, J., Yang, Q., Wang, S., “Biological removal of nitrogen from wastewater”, Rev. Environ. Contam. Toxicol., 192, 159–195(2008). [6] Tokawa, H., Hanaki, K., Matsuo, T., “Nitrous oxide production in high-loading biological nitrogen removal process under low COD/N ratio condition”, Water Res., 35, 657–664(2001). [7] Sung, S., Liu, T., “Ammonia inhibition on thermophilic anaerobic digestion”, Chemosphere, 53, 43–52(2003). [8] Rajinikanth, R., Daniel, I. M., Gursharan, S., “A critical review on inhibition of anaerobic digestion process by excess ammonia”, Bioresour. Technol., 143, 632–641(2013). 14
ACCEPTED MANUSCRIPT [9] Khin, T., Annachhatre, A. P., “Novel microbial nitrogen removal processes”. Biotechnol. Adv., 22, 519–532(2004).
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[10] Rostron, W. M., Stuckey, D. C., Young, A. A., “Nitrification of high strength ammonia
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wastewaters: comparative study of immobilization media”, Water Res., 35, 1169–1178(2001). [11] Jung, J. Y., Chung, Y. C., Shin, H. S., Son, D. H., “Enhanced ammonia nitrogen removal using
process”, Water Res., 38, 347–354(2004).
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consistent biological re-generation and ammonium exchange of zeolite in modified SBR
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[12] Madukasi E. I., Dai X., He C. H., Zhou J., “Potentials of phototrophic bacteria in treating pharmaceutical wastewater”, Int. J. Environ. Sci. Tech., 7 (1), 165–174(2010).
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[13] Kuo, F. S., Chien, Y. H., Chen, C. J., “Effects of light sources on growth and carotenoid content
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of PSB Rhodopseudomonas palustris”, Bioresour. Technol., 113, 315–318(2012).
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[14] Olga, K. R. O., O’Kane, S. R., Nitschke, W., Léger, C., Baymann, F., Soulimane, T., “Biochemical and biophysical characterization of succinate: Quinone reductase from Thermus
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thermophilus”,Biochimica et. Biophysica Acta (BBA) – Bioenergetics, 1807 (1), 68–79(2011). [15] Myung, K. K., Choi, K. M., Yin, C. R., Lee, K.Y., Im, W. T., Lim, J. H., Lee, S. T., “Odorous swine wastewater treatment by purple non-sulfur bacteria, Rhodopseupdomonas pulustris, Isolated from eutrophicated ponds”, Biotechnol. Lett., 26, 819–822(2004). [16] Nagadomi, H., Kitamura, T., Watanabe, M., Sasaki, K., “Simultaneous removal of chemical oxygen demand (COD), phosphate, nitrate and H2S in the synthetic sewage wastewater using porous ceramic immobilized PSB”, Biotechnol. Lett., 22, 1369–1374(2000). [17] Lu, H. F., Zhang, G. M., Dai, X., He, C. H., “PSB treatment of synthetic soybean wastewater: direct degradation of macromolecules”, Bioresour. Technol., 101 (19), 7672–7674(2010). 15
ACCEPTED MANUSCRIPT [18] Anam, K., Habibi, M.S., Harwati, T.U., Susilaningsih, D., “Photofermentative hydrogen production using Rhodobium marinum from bagasse and soy sauce wastewater”, Int. J.
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Hydrogen Energy, 37 (20), 15436–15442(2012).
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[19] Chitapornpan, S., Chiemchaisri, C., Chiemchaisri, W., Honda, R., Yamamoto, K., “Organic
carbon recovery and PSB population in an anaerobic membrane photo-bioreactor treating food processing wastewater”, Bioresour. Technol., 141, 65–74(2013).
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[20] Imhoff, J. F., Trüper, H. G., “Purple nonsulfur bacteria. In: Staley, J. T. (Ed.), Bergey’s manual of
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systematic bacteriology”, Baltimore, Williams and Wilkins. 3, 9th. Ed. 1904–1910(1989). [21] Zhao, W., He, C. H., Zhang, G. M., “Culture medium optimization for PSB”, Advanced Materials Research Vols., 113-114, 1443–1446(2010).
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[22] Eaton, A. D., Clesceri, L. S., Rice, E. W., Greenberg A. E., “Standard methods for the
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examination of water and wastewater", 21st ed. American Public Health Association, American
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Water Works Association & Water Environment Federation, Washington, DC, USA(2005).
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S1004-9541(15)00294-3 doi: 10.1016/j.cjche.2015.08.018 CJCHE 373
To appear in: Received date: Revised date: Accepted date:
15 December 2014 16 June 2015 17 June 2015
Please cite this article as: Qin Zhou, Guangming Zhang, Xiang Zheng, Guohua Liu, Biological treatment of high NH4 + -N wastewater using an ammonia-tolerant photosynthetic bacteria strain (ISASWR2014), (2015), doi: 10.1016/j.cjche.2015.08.018
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Biological treatment of high NH4+-N wastewater using an ammonia-tolerant
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photosynthetic bacteria strain (ISASWR2014)
School of Environment & Natural Resource, Renmin University of China, Beijing 100872, China
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1
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Qin Zhou1, Guangming Zhang1*, Xiang Zheng1, Guohua Liu1
Abstract
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Wastewater with high NH4+-N is difficult to treat by traditional methods. So in this paper, a wild
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strain of photosynthetic bacteria was used for high NH4+-N wastewater treatment in together with
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biomass recovery. Isolation, identification and characterization of the microorganism were carried out. The strain was inoculated to the biological wastewater treatment unit. The impacts of important
CE P
factors were examined, including temperature, dissolved oxygen and light intensity. Results showed that photosynthetic bacteria could effectively treat high NH4+-N wastewater. For wastewater with
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NH4+-N of 2300 mg·L-1, COD/N=1.0, 98.3% of COD was removed, and cell concentration increased by 43 times. The optimal conditions for the strain’s cell growth and wastewater treatment were 30 oC, dissolved oxygen of 0.5-1.5 mg·L-1 and light intensity of 4000 lux. Photosynthetic bacteria could bear a lower C/N ratio than bacteria in a traditional wastewater treatment process, but the NH4+-N removal was only 20%-40% because small molecule carbon source was used prior to NH4+-N. Also,
*
Corresponding Author: E-mail: [email protected]
Acknowledgments Supported by the National Natural Science Foundation of China (51278489). 1
ACCEPTED MANUSCRIPT the use of photosynthetic bacteria in chicken manure wastewater containing NH4+-N about 7000 mg·L-1 proved that photosynthetic bacteria could remove NH4+-N in a real case, finally, 83.2% of
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NH4+-N was removed and 66.3% of COD was removed.
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Graphic abstract
The isolated photosynthetic bacteria (PSB) were used to treat chicken manure wastewater contained recalcitrant organic compounds. The total COD value was about 3000 mg·L-1, and NH4+-N concentration was 7000 mg·L-1, with a C/N of 0.42. The use of PSB proved to be very efficient in treating such a high NH4+-N and low C/N wastewater. The results indicated that the PSB has special adaptations for survival and reducing the NH4+-N even with an NH4+-N concentration of 7000 mg·L-1. The COD removal reached 66.3% and the NH4+-N removal reached 83.2%.
[Keyword: High NH4+-N wastewater; C/N; Photosynthetic bacteria; Chicken manure wastewater] 2
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INTRODUCTION
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With the rapid development of fertilizer and petrochemical industry, a large number of high
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NH4+-N wastewater discharged. NH4+-N is usually oxidized to nitrate and nitrite, the nitrate and nitrite usually may cause serious eutrophication, and they are hazardous to humans. So NH4+-N wastewater treatment is essential for preventing eutrophication and human harm. High NH4+-N
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wastewaters, such as piggery wastewater and landfill leachate contain not only high concentrations
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of nitrogen compounds (up to 1000-3000 mg·L-1) but also high concentrations of organic matter. In high NH4+-N wastewaters, the C/N ration is often quite low and the ammonia concentration is
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50-100 times higher than that in municipal wastewater [1-3]. All these contributed to the difficulty of
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conventional biological treatment of high NH4+-N wastewaters.
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Generally, the most common and cost-efficient approach for ammonia wastewater treatment is biological treatment [4, 5]. However, traditional nitrification process is not effective in such high
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ammonia environment with such a low C/N. One reason is that autotrophic bacteria are vulnerable to high concentrations of wastewater with C/N ratio of 0-2 [6]. Another reason is that excessive high ammonia exerts a toxic and inhibitory effect on microbial activity [7, 8]. Thus, traditional nitrification can be used only after pretreating the wastewater to increase C/N ratio [9] or decrease ammonia concentration of the wastewater [10, 11], which greatly increases the operation cost. Hence, the utilization of an ammonia-tolerant organism in biological treatment units seems to be a reasonable approach for high NH4+-N wastewater treatment. Application of photosynthetic bacteria (PSB) might be employed in degrading pollutants in high NH4+-N wastewater treatment. PSB are widely distributed in the ocean, lakes, soil and activated sludge also in 3
ACCEPTED MANUSCRIPT high-temperature, low-temperature, high-ammonia or low-ammonia environment. They grow in both aerobic and anaerobic light conditions. They are metabolically the most versatile among all
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prokaryotes [12]. PSB have been used to treat a variety of wastewaters such as cadmium wastewater,
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olive mill wastewater and sulfide containing wastewater [15-19]. And using PSB for wastewater treatment has been proven to be a cost effective method. This is because PSB do not only remove pollutants in wastewater, but also accumulate useful materials such as single-cell protein,
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biopolymers, antimicrobial agents, carotene, pantothenic acid and therapeutic compounds [13, 14].
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The biomass can be recycled as useful raw materials in food, medical and agriculture industries. In this work, a new process was investigated, a strain of PSB isolated from aqua-farm,
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demonstrated an amazing tolerance to high NH4+-N. The aim of the paper was to study the ability of
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PSB to treat high NH4+-N wastewater in together with biomass recovery.
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MATERIALS AND METHODS
Isolation and identification of ammonia-tolerant microorganism
Aqua-farm water
provided the medium for PSB isolation and selection. The bacteria strain was isolated with the medium [12] consisting of DL-malate: 4 g·L-1, MgSO4: 0.12 g·L-1, (NH4)2SO4: 1 g·L-1, CaCl2: 0.075 g·L-1, KH2PO3: 0.3 g·L-1, Na2EDTA: 0.020 g·L-1, VB1: 0.001 g·L-1. Nicotinic acid: 0.001 g·L-1, Biotin: 0.015 g·L-1. Trace element solution, 1 ml. The trace element solution contained ZnSO4•7H2O: 20 mg·L-1, MnCl2•4H2O: 6 mg·L-1, H3BO3: 60 mg·L-1, CoCl2•6H2O: 40 mg·L-1, CuCl2•2H2O: 2 mg·L-1. pH was 7.0. And the culture was anaerobically at 28-35 ºC and light intensity of 2000 lux conditions for 96 hours. 4
ACCEPTED MANUSCRIPT The mixed culture were examined microscopically, and they were purified and identified according to the biochemical reactions such as catalase test, tolerance to 3% NaCl, gram test, nitrate
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reduction test, indole test, H2S reaction and spectrum scanning. All experiments were carried out
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according to Bergey’s manual [20].
Artificial high NH4+-N wastewater was used for PSB
Wastewater and reactor operation
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treatment. And its characteristics were as follows: COD, NH4+-N and total phosphorus (TP) were
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2300, 2300, and 45 mg·L-1. The initial pH was around 7.0. The carbon to nitrogen ratio (C/N) was 1, which was much lower than that in the traditional wastewater treatment.
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The bioreactors were 500 mL glass flasks. These flasks were sterilized NH4+-N at 121 oC for 30
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min before use. Each time, 400 mL artificial high NH4+-N wastewater was added to the bioreactor.
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PSB were inoculated and the initial cell concentration in wastewater was 10 mg (dry weight)/L. The amount of PSB was 5% (by volume) in conducted experiments. The artificial high NH4+-N
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wastewater and PSB were rotated at 120 r·min-1 with temperature 10, 20, 30, 40 and 50 oC, respectively, in the five experimental groups. The dissolved oxygen (DO) was less than 0.5, 0.5-1.5 and 5.5-6.5 mg·L-1, respectively; the value was chosen according to previous studies. The illumination intensity were <500, 500, 1000, 2000, 4000 and 8000 lux, respectively. The dissolved oxygen in the control group (< 0.5 mg·L-1) condition was realized by micro aeration (the purity of nitrogen was 98.0%). After saturated with nitrogen, the bioreactors were sealed with sealing membrane to keep micro-oxygen or anaerobic condition. The dissolved oxygen (DO) of 0.5-1.5 mg·L-1 was realized without aeration, and the bioreactor was directly covered with oxygen enriching membranes. The dissolved oxygen of 5.5-6.5 mg·L-1 was realized with micro 5
ACCEPTED MANUSCRIPT aeration (the purity of oxygen was 98.5%).
Chicken manure wastewater from a plant was treated biologically using the
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Case study
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All experiments were repeated twice.
isolated PSB. The characteristics of the chicken manure wastewater were as below: total COD, total nitrogen (TN), NH4+-N and TP were about 3000, 8000, 7000 and 15 mg·L-1, respectively. The pH
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Analysis
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was about 9.6.
Samples were collected from bioreactors and were centrifuged at 9000 rpm for 10
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min. The supernatant was used to test the COD with APHA standard methods [22]; the collected PSB
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were used to measure the biomass (dry weight). The pH was measured using pH tester and the DO
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was measured using dissolved oxygen meter. The biomass was tested by APHA standard methods. NH4+-N levels were analyzed by the Nesslerization method at an absorbance of 420 nm using a
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TU-1900 spectrophotometer.
RESULTS AND DISCUSSIONS
Isolation and identification of the ammonia-tolerant microorganism
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ammonia-tolerant microorganism was isolated from an eutrophic aqua-farm. The identification of bacteria was based on cultural and biochemical characteristics, the results were shown in Tab. 1. The isolate strain was examined microscopically as gram-negative bacteria. After 48 h culture under anaerobic light conditions, cell suspensions were red and the absorption spectra of living cells 6
ACCEPTED MANUSCRIPT suspension showed maxima absorbance peaks at 270, 280, 375, 800 and 860 nm. The main peak at 860 nm was closely related to bacteriochlorin a, and the main peaks at 280, 375 and 800 nm was
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closely related to carotenoids. The contained pigments of the isolate were characteristics of purple
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non-sulfur photosynthetic bacteria [12]. Also the strain was able to grow in the presence of 3% NaCl. The strain had a negative indole reaction, H2S positive and catalase-positive in the test. According to Bergey's Manual of Determinative Bacteriology, the strain was identified as Pseudomonas sp.
Features Bacteriochlorin A Carotenoid Catalase reaction
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Gram straining
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Growth in 3% NaCl
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Properties and biochemical tests
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Tab. 1. Identification and biochemical tests of the strain
Nitrate reduction Indole test
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H2S reaction
Results + + + + — + — +
Utilization for different carbon sources
—
Ethyl alcohol
— — — — — +
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Sodium thiosulfate Methylcellulose Sodium oleate
Protein Powder Glycerinum Glucose
+ + + + +
Mannose Sodium acetate Mannitol Sorbitol Fructose
Feasibility study of the strain in high NH4+-N wastewater treatment 7
The isolated PSB
ACCEPTED MANUSCRIPT were inoculated to the biological wastewater treatment unit. The feasibility was studied and the results were shown in Fig.1. PSB could effectively treat high NH4+-N wastewater. 98.3% of COD
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was removed at the 96th hour. Meanwhile, biomass grew well in high NH4+-N wastewater. The final
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cell concentration was 435 mg ·L-1 at the 96th hour, which increased by 43 times. There was a decrease in PSB cell concentration during the 72th to 96th hour. This could be explained by the quick breeding of PSB in the first 72 hours, and the left organic compounds were insufficient for all cells,
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and bacteria began to enter into a stagnant stage for lack of food and energy. Thus PSB started to
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dissolve, and the corresponding cell concentration decreased. The COD reduction of the high NH4+-N wastewater followed a pseudo first order reaction with a first order rate constant (k) of
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0.042/d and R2 of 0.996.
Fig. 1. COD removal and PSB biomass production in high NH4+-N wastewater, DO=0.5-1.5 mg·L-1, 30 oC, 2000 lux. 8
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Biological treatment of high NH4+-N wastewater
Temperature is one of the most
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important factors that affect the growth of bacteria. Previous studies have shown that the optimum
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temperature for PSB nutrient medium was between 30-35 oC [21]. According to the biochemistry theory, DO and light intensity are key factors for PSB growth. In order to study the effects of these factors, the temperature varied between 10 to 50 oC, DO varied between 0.0 to 6.5 mg·L-1, and light
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intensity varied between 0 to 8000 lux were studied. The results were shown in Fig. 2.
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As Fig. 2(a) showed, the highest COD removal and the best biomass growth were observed at 30 oC, with a COD reduction of about 98.3% and the cell concentration was 430 mg·L-1. Thus, the
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optimal temperature was 30 oC. Either higher or lower temperatures were bad for COD removal and
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cell accumulation.
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As Fig. 2(b) showed, COD removals under DO 0.5-1.5 or 5.5-6.5 mg·L-1 were good (>96%). The finding could be attributed to the fact that PSB thrives better in a higher DO environment.
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Meanwhile, the highest biomass production was obtained under DO of 0.5-1.5 mg·L-1, with a cell concentration of about 410 mg·L-1. Thus, the optimal DO was 0.5-1.5 mg·L-1. Lower dissolved oxygen was inadequate for photosynthetic bacteria growth, but higher dissolved oxygen did not help. Light intensity is an important factor that affects photophosphorylation. As Fig. 2(c) showed, the highest COD removal was 96.0% under 4000 lux. Meanwhile, the highest cell concentration was 505 mg·L-1 and it was improved by 74.1% than the group without a light intensity less than 500 lux. Note that the optimal light intensity was 4000 lux. Too much or too little light were both unfavorable for PSB growth. In summary, the optimal conditions for PSB growth and wastewater treatment were temperature 9
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of 30 oC, DO of 0.5-1.5 mg·L-1 and light intensity of 4000 lux.
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Fig. 2. Effects of temperature (A), DO (B) and light intensity (C) on the COD removal and cell
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concentration in high NH4+-N wastewater
The high NH4+-N wastewater has a
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The NH4+-N removal in PSB wastewater treatment
NH4+-N value of about 2300 mg·L-1, which was with a C/N ratio of 1. The characteristics of high NH4+-N and low C/N contributed to the difficulty in conventional biological treatment of this kind of wastewater. The COD removal, cell concentration and NH4+-N removal were the major considerations for PSB to treat this wastewater. Through the feasibility and optimization study, the COD removal was good (>96%), and the cell grew well in the wastewater. However, the NH4+-N removal was still about 20%-40%, which became the major problem for PSB to treat this wastewater. The reason could be that sodium acetate was the carbon source in the artificial wastewater, and sodium acetate was easily degraded by PSB. So when sodium acetate existed, this small molecule 11
ACCEPTED MANUSCRIPT carbon source was used prior to NH4+-N. Previous studies have shown that PSB were easy to degrade small molecular substances, but hard to degrade large molecular or complicated substances.
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So we suspected that if the carbon source was hard-biodegraded, there was a chance that NH4+-N
Case study
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might be degraded by PSB firstly.
Based on the results obtained with artificial high NH4+-N wastewater, the
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isolated PSB were used to treat chicken manure wastewater as a case study. The chicken manure
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wastewater contained recalcitrant organic compounds which was resistant to biodegradation. The total COD value was about 3000 mg·L-1, and NH4+-N concentration was 7000 mg·L-1. The results
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were shown in Fig. 3. The use of PSB proved to be very efficient in treating such a high NH4+-N and
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low C/N wastewater. The results indicated that the PSB has special adaptations for survival at high
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NH4+-N wastewater and it can degrade the COD and NH4+-N even at NH4+-N concentration of 7000 mg·L-1. The COD was degraded from 3000 to 1000 mg·L-1 (COD removal was 66.3%), NH4+-N was
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degraded from 7000 to 1000 mg·L-1 (NH4+-N removal was 83.2%). The COD reduction of the chicken manure wastewater for the first 96 h followed a pseudo first order reaction with a first order rate constant (k) of 0.015d-1 and R2 of 0.96.
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This work analyzed the potential of PSB for high NH4+-N wastewater
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Conclusions
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Fig. 3. The changes of COD and NH4+-N concentration in PSB chicken manure wastewater treatment
treatment, and evaluated the important factors: temperature, DO and light intensity. Also, a case
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study using chicken manure wastewater containing NH4+-N about 7000 mg·L-1 was treated using the isolated PSB. The results were as follows: (1) PSB demonstrated an amazing tolerance to high NH4+-N wastewater and could treat high NH4+-N wastewater at low C/N effectively. (2) For wastewater with NH4+-N of 2300 mg·L-1, COD/N=1, 98.3% of COD was removed, and biomass increased by 43 times. (3) The optimal conditions for PSB growth and wastewater treatment were temperature of 30 oC, DO of 0.5-1.5 mg·L-1 and light intensity of 4000 lux. (4) The PSB results in a significant improvement in the removal of NH4+-N in the chicken manure 13
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wastewater containing NH4+-N about 7000 mg·L-1, the NH4+-N removal was 83.2% .
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