Progress in Research of Biological Denitrification of AA/O Process and the Feasibility of Enhancing

International Journal of Energy and Power (IJEP) Volume 4, 2015 www.ijep.org doi: 10.14355/ijep.2015.04.008

Progress in Research of Biological Denitrification of AA/O Process and the Feasibility of Enhancing Electricity Generation and Denitrification of MFC‐AA/O Reactor Lige Yang1, Beizhen Xie2, Yuming Fu3, Hong Liu*,4 School of Biological Science and Medical Engineering, Institution of Environmental Biology and Life Support Technology, International Joint Research Center of Aerospace Biotechnology & Medical Engineering, Beihang University, Beijing 100191, China 1270814818@qq.com; 2 kakashowxbz@163.com; 3fuyuming@buaa.edu.cn; *4lh64@buaa.edu.cn

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Abstract In the anaerobic/anoxic/oxic process (AA/O), the ammonia nitrogen can be oxidized to form nitrates, and nitrates are transformed to nitrogen gas through denitrification, then nitrogen removal efficiencies. However, lack of carbon source in the AA/O process, it is difficult to enhance denitrification, meanwhile, realize phosphorus removal effectively. The present study finds that a microbial fuel cell (MFC) embedded in the AA/O process can improve the efficiencies of removal of nitrogen and phosphorus, along with electricity production. In MFC‐AA/O process, the biocathode denitrification is the key to further increase its nitrogen removal efficiency, but also affects the output voltage of MFC. So it is necessary to study the advantage denitrification bacteria in MFC‐AA/O process. The research achievements on biological denitrification of AA/O process were comprehensively summarized, and the feasibility of enhancing electricity generation and denitrification of MFC‐AA/O reactor was discussed in this paper.

Keywords AA/O Process; MFC; MFC‐AA/O Reactor; Biological Nitrogen Removal; Denitrification Bacteria

Introduction More and more countries and regions have established strict discharge standards of nitrogen and phosphorus for the quickness of environmental pollution and water eutrophication. The efficiency of biological nitrogen and phosphorus removal process is essential to avoid eutrophication [1‐3]. The anaerobic/anoxic/oxic (AA/O) process is a widely used wastewater treatment process for nitrogen and phosphorus removal. However, due to various reasons (competition of carbon sources among different microbes), it is difficult to achieve high removal efficiency of different pollutants simultaneously[4]. In our previous research, in order to improve the denitrification efficiency of AA/O process, a continuous flowing membraneless Microbial Fuel Cell (MFC) was embeded into an AA/O wastewater treatment process. Anaerobic tank of the AA/O process acted as the anode chamber of MFC while anoxic tank was cathode chamber [5]. Based on the above structure, AA/O wastewater treatment process had its pollutant removal enhanced obviously along with producing electric energy.

In MFC‐AA/O technology, the biocathode denitrification is the key of reactor operation. Hence, studies on the influence and the function of the advantage denitrification bacteria in MFC‐AA/O process play a significant role in sewage treatment in the future. In this context, the research achievements from home and abroad on biological denitrification of AA/O process were comprehensively reviewed. The feasibility of enhancing electricity generation and denitrification of MFC‐AA/O reactor was discussed, and the method of enhancing the performance of MFC‐ AA/O was analyzed.

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The Research Status on Biological Denitrification of AA/O Process The most prevalent forms of nitrogen in wastewaters are organic, ammonium, nitrite and nitrate nitrogen. In the sewage, organic nitrogen and ammonia nitrogen, account for 40%~60%, 50%~60%, respectively. Nitrate nitrogen form only accounts for about 0~5% of the nitrogen. Therefore, sewage biological denitrification process generally includes: bacterial decomposition of proteinaceous matter hydrolysis of urea transform organic nitrogen to ammonium form, then ammonia nitrogen can be oxidized to form nitrates by two groups of microoganisms in the presence of oxygen. Finally, nitrates are transformed to nitrogen gas through a process called denitrification. In addition, part of the ammonia nitrogen can be used for cells synthesis and be removed from the wasted biological sludge. The complete biological denitrification process includes three froms (ammoniation, nitrification and denitrification), which denitrifying microorganism in the whole process of biological nitrogen removal plays a key role [6].

AA/O Process Overview An AA/O process consists of traditional activated sludge process, biological nitrification and denitrification, and biological phosphorus removal process. Under different microbial flora, the organic matter, nitrogen and phosphorus in wastewater are effectively removed while the wastewater flows through three different functional partition of AA/O. In the process, phosphorus removing bacteria(PAOs) in the anaerobic stage release phosphorus, denitrifying bacteria and denitrifying phosphorus removing bacteria(DPB) in anoxic phosphorus reduce nitrate and nitrite to nitrogen gas, while ammonification and nitrification occur in the aerobic tank, which achieves the removal of nitrogen and phosphorus in wastewater.

Denitrification and Microorganism The nitrate reduction contains assimilation of nitrate reduction and alienation of nitrate reduction. Alienation of nitrate reduction includes fermentable nitrate reduction and respiratory nitrate reduction. The process of microorganism reducing nitrate or nitrite into gaseous nitrogen is called denitrification.

Traditional theory holds that oxygen is known to repressing the denitrification reductases. Thus, the denitrification process is under the environment of anaerobic or micro oxygen. In the 1980s, the researchers first isolated aerobic denitrifying bacteria. They found that periplasmic nitrate reductase also could realize denitrification [7, 8]. Zumft found that the environment revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases [9]. In 2002, Philippot defined the denitrification as that denitrification is a microbial process in which oxidized nitrogen compounds are used as alternative electron acceptors for energy production [10].

Taking organic compounds as electron donors and NO3‐ or NO2‐ as electron acceptors, heterotrophic denitrification discharge N2 to generate energy by anaerobic respiration .The nitrate reductase, nitrite reductase, nitrogen oxide reduction enzymes and nitrous oxide reduction enzymes are used in the process [10, 11].

Studies have shown that the earliest discovered denitrification microbes are bacteria, including more than 50 genera and more than 130 kinds, such as Alcaligenes, Pseudomonaceae, Achromobacter, Aerobacter et al [12‐14]. In addition to these denitrifying bacteria, some fungi and actinomycetes have the same function, such as Fusarium, Saccharomyces, Streptomyces etc [15, 16]. So far, aerobic denitrification has been only found in bacteria and no press reports fungi and actinomycetes have the same way [17].

Denitrification of AA/O Reactor Denitrification of AA/O reactor mainly acts in anaerobic tank. Because the microbes of denitrification are buried into the flocculation of activated sludge, it is difficult to separate all the microbes in the flocculation. The types and numbers of bacteria in activated sludge are very different. In the sewage treatment system, Proteobacteria is the largest number and the most abundant specie, which accounting for more than 50% of total bacteria. Bacteroidetes, Firmicutes and Actinobacteria and other general categories of bacteria account for 5% ~ 20% [18, 19].

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By using fluorescence in situ hybridization (FISH), Thomsen et al found that in 18 wastewater treatment plants which have the function of denitrification, the vast majority of denitrifying bacteria belongs to the following categories: Aquaspirillum, Azoarcus, Thauera and Rhodocyclus, accounting for 20%‐49% of total bacteria and playing an important role in wastewater treatment [20]. Albertsen et al also found that Thauera could use nitrate as electron acceptor to degrade organic compounds [21]. Liu et al confirmed that Thauera could be used to denitrify through rapidcomplete onset (RCO) and progressive onset (PO) [22].

Saha et al firstly found that bacteria of Emticicia of the Bacteroidetes could get reduction of nitrate [23]. Liu et al also discovered a new specie of the genus Emticicia in soil which can get the reduction of nitrate and nitrite [24].

Kuba et al observed that the condition of the alternation between anaerobic and anoxic runs easily to enrich the anaerobic microbe of possessing both denitrification and phosphorus removal effect [25, 26]. The microbes could use O2 or NO3‐ as electron acceptor. Researchers of Dutch Delft University of technology and the University of Tokyo confirmed the dephosphorization effect, and called it denitrifying phosphorus removing bacteria (DPB) [27].

DPB is applied to wastewater treatment and can realize continuous process of denitrification and phosphorus removal [28].

The Feasibility of Enhancing Electricity Generation and Denitrification of MFC-AA/O Reactor Because, competition for carbon source in the AA/O process leads to its low denitrification and phosphorus removal efficiency, some new techniques, such as JHB (Johannesburg) technology, the inversion of AA/O process etc., are formed. But the modification of process increases the operating costs. Along with the low economic benefits, the advantage position of the main microbes in the water treatment is difficult to keep. Under room temperature, MFC can make use of the catalysis of enzymes in the microorganism to convert chemical energy in organic to electrical energy, and its efficiency of power conversion is high [29, 30]. Biocathode MFC is based on biological denitrification of nitrate as electron acceptor. Gregory et al found that the cathode can be used as the sole electron donor of nitrate reduction, also verified the feasibility of denitrification as MFC cathode reactions [31].

Park et al found that nitrate reduction using an electrode as direct electron donor in a biofilm‐electrode reactor [32].

So nitrogen and phosphorus removal efficiency could be improved by embedding a biocathode MFC in the AA/O process.

The Research Status on MFC‐AA/O Reactor In our previous research, we embedded a biocathode MFC into an AA/O wastewater treatment process: in the experiment, the anode and the cathode were placed at the bottom of anaerobic tank and anoxic tank, respectively.

The electrodes were both pre‐treated with ammonia. Then, Total nitrogen (TN), total phosphorus (TP), Chemical Oxygen Demand (COD) and mixed liquid suspended solids (MLSS) of the wastewater were measured by the standard methods. At the same time, the output voltage of the MFC was monitored with a multimeter to calculate the volumetric power density. Field validation experiments showed that by embedding an MFC in an AA/O process, COD, TN, and TP in the effluent of the experimental reactor were decreased by 15.9%, 9.3%, and 1.4%, respectively. The embedded MFC, with an external resistance of 2000, achieved an average voltage of 168.8 ± 8.2 mV and power density of 14.3 ± 1.4 mW m−3 . Subsequently, grey relational analysis(GRA) was applied to study the experimental data (output voltage, COD, TN, TP, and pH of input wastewater (WW), MLSS, and air temperature).

As a result, output voltage was most closely related to three factors: air temperature, TN and COD concentration in the influent. Furthermore, the influent TN, MLSS, and the influent pH have the greatest impact on the effluent TN [5].

Our research group also investigated the microbial community structure changes using PCR‐DGGE and IlluminaMiSeq high‐throughput sequencing after MFC embedded into AA/O process. We found that the microbial community structure had significant changes. A substantial amount of denitrification bacteria, enriched in the cathode plate, enhanced nutrient removal efficiency of the reactor and generated electricity at the same time [37].

So, purification of biocathode dominant denitrifying bacteria can further improve the efficiency of nitrogen and MFC‐AA/O.

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Denitrifying Bacteria Mainly Analysis Techniques Now, the mature microbiology analysis techniques, such as bioinformatics, scanning electron microscope (SEM), microbial purification etc., have provided a good support for the research of denitrifying bacteria. Frette et al used traditional microbial purification method isolated sixteen strains from an alternating activated sludge treatment plant, and all denitrified under both anaerobic and aerobic conditions [33]. According to the morphological, physiological and biochemical characters, as well as 16S rDNA sequence homology comparison, a novel aerobic denitrifiction bacterium was isolated and identified by Kong et al [34]. In Elsemiek Croese’ experiment, SEM imaging showed that on the graphite fibers more rod‐shaped microorganisms were observed, whereas in the spaces between the fibers clusters of typical Methanosarcina‐shaped cells were present abundantly in all the anode samples, which described the mechanism of electricity production of Methanosarcina in microbial electrolysis cell (MEC) [35]. And many other modern molecular biology methods were applied to the research of denitrifying microorganism [36].

Through the above analysis, the presence of dominant denitrification bacteria directly affect the effect of water purification, so by strengthening the denitrification process to strong the denitrification performance of MFC‐ AA/O is feasible, and the methods which can be used to study denitrifying bacteria have also been very mature.

Conclusions MFC‐AA/O reactor can improve pollutants removal ability, and produce electric energy at the same time.

Therefore, it will have broad application prospect and unique position in sewage treatment in the future. Studies proved that microbes living on the cathode catalyze denitrification by utilizing protons, electrons, and NO3− to fulfill the cathode reaction. The presence of denitrifying bacteria in an MFC‐AA/O reactor is the key to catalyze organic wastewater purification and electricity generation. Therefore, it has a great significance to further optimize the MFC‐AA/O process through isolating advantages denitrifying bacteria which enriched in the cathode plate, meanwhile, researching the characteristics of producing electricity and denitrification.

ACKNOWLEDGEMENTS

This work was financially supported by grants from International Science‐Technology Cooperation Program of China (2013DFR60250), National Nature Science Foundation of China (31200098), the Specialized Research Fund for the Doctoral Program of Higher Education of China (20131102120039) and the Funds for Central University Basic Scientific Research.

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