Nitrification characteristics of nitrobacteria immobilized in waterborne polyurethane in wastewater

Availableonlineatwww.sciencedirect.comScienceDirectSCIENCESJourmal of Environmental Sciences 2012, 24(6)999-1005w jesc.accnteLLNitrification characteristics of nitrobacteria immobilized in waterbornepolyurethane in wastewater of corn-based ethanol fuel productionYamei dong,2, Zhenjia Zhang, Yongwei Jin', Jian Lu, Xuehang ChengJun Li, Yan-yan Deng, Ya-nan Feng, Dongning Chen1.SchoolofEmironmentalScienceandEngineering,ShanghaiJiaoTongUniversiry,Shanghai200240,China.E-mall:yMDong515@gmalL.com2. National Engineering Research Center for Nanotechnology. Shanghai 200241,China3. Indian River Research and Education Center Universiry of Florida. Fort Pierce, FL 34945, US4. Division of Emvironmental Science and Engineering. Yamaguchi University Tokiwadai 2-16-1, Yamaguchi 755-8611, Japan5. School of Municipal and Emvirurmental Engineering Shengyang Jianzhu University, Shenyang 110168, ChinaReceived 09 August 2011: revised OI December 2011: accepted 1I December 2011bstractA technology to achieve stable and high ammonia nitrogen removal rates for com distillery wastewater (ethanol fuel production)treatment has been designed. The characteristics of nitrifying bacteria entrapped in a waterborne polyurethane(wPU) gel camerwere evaluated after acclimation. In the acclimation period, nitrification rates of wPU-immobilized nitrobacteria were monitoredand polymerase chain reaction(PCR)was also camied out to investigate the change in ammonium-oxidizing bacteria. The resultsshowed that the pellet nitrification rates increased from 21 to 228 mg-N/Lpellet-hr)and the quantity of the ammonia oxidationbacteria increased substantially during the acclimation. a continuous ammonia removal experiment with the anaerobic pond effluent ofa distillery wastewater system was conducted with immobilized nitrifying bacteria for 30 days using an 80 L airlift reactor with pelletsat a fill ratio of 15%(V/V). Under the conditions of 75 mg/L influent ammonia, hydraulic retention time(HRT)of 3.7-5. 6 hr, anddissolved oxygen(DO)of 4 mg/L, the effluent ammonia concentration was lower than 10 mg/L and the ammonia removal efficiency90%. While the highest ammonia removal rate, 162 mg-N/(L-pellet-hr), was observed when the HRT was 1.3 hr.Key words: immobilized nitrobacteria; com distillery wastewater; ethanol fuel production; nitrification rate; waterborneDOI:101016/S1001074211)608930Introductionconventional processes, the use of immobilized bacteriahas the following advantages: the compact physical strucDistillery wastewater is the main byproduct originating in ture of the carrier pellets, high biomass retention, anddistilleries,and its volume is approximately 10 times that premium tolerance with respect to variations in load andof the ethanol produced(Krzywonos et al., 2009). Uti- pollution levels(Sumino et al., 1992). At the same time, alization of the distillery wastewater has become a serious screen is used to separate the entrapped bacteria from theissue. The distillery wastewater is relatively non-toxic, but wastewater to prevent the washout of bacteria(Asano etit usually contains a high concentration of complex organic al., 1992; Dong ct al, 2011). Thus, gel entrapment is ablematter and suspended solids. The treatment methods for to prolong the biomass retention times even under shortdistillery wastewater can be classified into two major hydraulic retention times(HrT). However, little effortgroups. One includes the processes of recycling and uti- has been made to evaluate the nitrification activity of gellizing valuable solids from corn distillery wastewater, and carriers of microorganisms for distillery wastewater.the other comprises both aerobic and anaerobic processesIn this research, nitrification by immobilized bacteriaby which the distillery wastewater can be biodegraded ( Li pellets was studied to design a novel advanced treatmentet al., 2006: Rajeshwari et al, 2000). It was reported that technology for the anaerobic pond effiuent of a distilleryan anaerobic-aerobic process had superior performance in wastewater system. Pellets of waterborne polyurethanethe degradation of high concentrations of organic matter.PUIHowever, the traditional aerobic process is ineffective for accl中国煤化工 f immobilwastewater containing high concentrations of ammonia.CNMHGg electron micro-Therefore, immobilized bacteria are being considered scopelalyccu uy puiyiucrase chain reactionor application in nitrification processes. Compared with (PCR)during the acclimation process. Characteristics ofCorresponding author. E-mail: zjzhangesjtu.edu.cnthe nitrification processes with WPU-immobilized nitrifyJournal of Environmental Sciences 2012, 24(6999-1005/ Yamei Dong et aL.ig bacteria pellets were investigated for the development 1.5 Experimental setupof a corn-based ethanol fuel production wastewater treat-ment system.Biological nitrification was carried out in an up-flow innercirculation aerated reactor, as shown in Fig. 1. The reactor1 Materials and methods(with 80 L operating volume)made of acrylic glass wasfed with ammonia wastewater. The filling ratio of immo-lI Reagentsbilized pellets was 15%(V/V). Air was supplied through asintered glass ball fixed at the column bottom. The pelletsPolyether polyol, isophoronedisocyanate(IPDD), and rose up to the top by aeration and dropped down to the2-hydroxylethyl methacrylate(HEMA)were purchased bottom of the reactor by gravity. Thus, inner circulationfrom Aldrich(USA). Reagents such as NHCL, NaHCO,, was formed, which ensured complete mixing and contactNa2HPO4, and H3BO4 were purchased from Sinopharm between wastewater and immobilized bacteria. At the topChemical Reagent Co. China). All chemicals used were of the reactor, pellets and wastewater were separated byof analytically pure gradea diagonal filter mesh. The wastewater was pumped by1.2 Synthesis of waterborne polyurethane(WPU)variable speed peristaltic pumps. The HRT was controlledby adjusting the feed flow rate.Polyether polyol IPDI, and HEMA (with a molar ratio 1.6 Acclimation of immobilized nitrobacteria in WPUof 1: 2: 2)were mixed with catalyst(Dibutyltin dilaurate.DBTL in a 500 mL flask and allowed to react in vacuumto the synthetic wastewaterat 60-70 for 5 hr. The WPU prepolymer was dripped An activity recovery experiment of the nitrifying bacinto a vessel containing distilled water at the stage of teria immobilized in WPU was conducted for over 90reaction completion, which took another 12 hr. The final days in the reactor. The compositions of the syntheticsolution was stirred vigorously for I hr so that a stable ammonia wastewater are shown in Table 1. The influentWPU emulsion was obtainedammonia concentration was increased with operating time1.3 Immobilization of nitrobacteria in WPUto evaluate the performance of the immobilized bacteriafrom stages I to V, and analysis was carried out evIn this research, the nitrifying bacteria(seed sludge) ery day. At the end of each acclimation stage, severalwere obtained from the aeration tank of the minhang pellets were taken from the reactor and refrigerated atMunicipal Wastewater Treatment Plant, Shanghai, China. 4C until bacterial DNA extraction and PCR amplificaAfter 2 months of acclimation, suspensions of nitrifying tion experiments were carried out. Biomass subsamplesbacteria were concentrated to the density of 20 g/L by were freeze-thawed three times and DNa was extractedcentrifugation and the concentrate was entrapped in thewpu gel carrier. The entrapped concentrate was thenmixed with a wPu prepolymer emulsion and a promoterEffluent(N N,N, N'-tetramethylenediamine) To initiate polymer-ization, potassium persulfate, an initiator, was added tothe beaker. The mixture was allowed to stand for 5 minat a room temperature of 25C. Consequently, WPUimmobilized nitrifying bacteria in the form of an elasticgel was obtained. The resulting polymerized gel camierwas cut into 3 mm cubes and then washed thoroughly withdistilled water1.4 Analvtical methodsoxygen(dO)and pHtemperature of the reactor was monitored using an onlineprobe, Ammonium nitrogen(NH4*-N), nitrate-nitrogenNO3-N), nitrite-nitrogen(NO2"-N), and mixed liquorupended solids(MLsS)were determined according to Flg.1 Schematic diagram of the up-flow inner circulation aeratedstandard methods(APHA, 1999).Table 1 Compositions of the synthetic ammonia wastewaterAcclimationNH,CINazHPO4NaClMgSO4stages(mgL)gL)(mg/L)(mgL)522中国煤化工3444044CNMHG105481376.2417618441474·150 L influent tankNo 6Nitrification characteristics of nitrobacteria immobilized in waterbome polyurethane in wastewater(Ausubel et al, 1990). To evaluate the activity and quantity 2 Results and discussionof the ammonia-oxidizing bacteria, the DNA fragmentsencoding the amoA gene were amplified using the forward 2.1 Mechanisms of WPU immobilization nitrobacteriamer A189(GGNGACTGGGACT'TCIGG)and reverseA682(GAASGCNGAGAAGAASGC) (Zhou et al. 2007). Due to their flexto their flexibility and non-biodegradability, syntheticThe whole volume was made up to 20 HL, and PCR was gels are promising for the application of entrapping nitri-DNA 2.0 10XPCR buffer 2.0, MgCl2 1.2, dNTPs 0. 5. tying cells for wastewater treatment systems (Leenen erPCR primers 2.0, Taq dna polymerase 0.5 and sterilewater 11.8. After being mixed on a vibrating mixer. PCR microorganism immobilization, polyurethane is standoutamplification was carried out using a dNa thermal cycler due to its good properties, such as mechanical, physical,at 96 C for 4 min, 95.C for 45 sec, 58C for 45 sec, and and chemical stability(Sumino et al., 1992). A new mi-72C for 90 sec for 29 cycles(Holmes et al. 1995).A and gelation of wPU has been developed. The WPUpositive control was carried out to ensure the accuracy ofresults. The preparation of amplified samples for gel elec- gels provide greater mechanical strength and have theadvantage of easy preparation without repetitive freezingtrophoresis was carried out with gel concentration 1.5% and thawing, such as is necessary for PVA( Chen and Linand electrophoresis voltage 100 V, and the electrophoresimaging was performed under ultraviolet light.1994). wPU with carboxylate groups can be dispersed inwater. The dispersed WPU emulsion is generally unstable1.7 Conversion of synthetic wastewater to distillery and could flocculate when it mixes with the suspendedcells with the addition of the promoter and initiator That is,the wPU emulsion would be crosslinked to form a hard gelDistillery wastewater was substituted for the synthetic block. The proposed chemical structure is shown in Fig. 2wastewater as the influent after the acclimation period. The WPU gels were cut into 3-mm cubes and Fig 3a showsMost of the organic matter and some of the nitroge the shape of beads prepared using this method As shownin the distillery wastewater were removed by anaerobictreatment. The nitrification process of the anaerobic porin Fig. 3b, each bead was filled with cellseffluent adopted the WPU-immobilized nitrifying bacteria 2.2 Acclimation of nitrobacteria immobilized in WPUpellets in place of the traditional biological treatment.to the synthetic wastewaterThe characteristics of the effluent from the anaerobicpond containing the distillery wastewater were as follows: Because of the thermal efficiency of polymerization andNH4+N30.1-156.,2mgL,NO2N0.04553mg/Lne mass transfer resistance to species such as oxygenNO3-N.20-6. 88 mg/L, COD 47-374 mg/L and pHand ammonia ion, the nitrifying bacteria activity decreasedof immobilize6.94-7.99. A continuous ammonia removal experimentmation process was necessary for the activity recoverywith the immobilized nitrifying bacteria was carried out of the immobilized nitrifying bacteria. Figure 4a showsin the aerobic reactor with pellets with fill ratio 15%by volume. The process continued for 45 days and wasdivided into two stages. During the first stage, the ammonia in wPU pellets as determined by the ammonia removalprocessing capacity of the WPU-immobilized nitrifying rate. The immobilized nitrifying bacteria were acclimatedbacteria pellets with distillery wastewater was tested for 30 tion reactor. The volume of the immobilized pellets wasdays. The ammonia nitrogen removal load under differentHRT was investigated for 15 days in the second stage.15% of the working volume of the reactor. Continuousexperiments were conducted with temperature of 25C,HRT of 8 hr, and do of 3-5 mg/L. The initial ammoniaF-CHz-c-PU-C-CH-f一C-0CH2-CH2H C- CH2-rV凵中国煤化工CNMHGCHr-N-C-0wO-C-N-CHFlg. 2 Chemical structure of crosslinked WPUJournal of Environmental Sciences 2012, 24(6)999-1005/ Yamei Dong et alFig 3 Appearance of WPU-immobilized nitrobacteria pellets (a)and SEM micrograph of immobilized pellet surtace(b)Influent5o-EffluentTime(day)Acclimation stagesMarker IV BlankcFig 4 Nitrification characteristics of WPU-immobilized nitrobacteria pellets with increasing ammonium loading(synthetic wastewater)(a), ammoniumremoval rate of WPU-immobilized nitrobacteria pellets at different acclimation stages(b), and gel picture of PCR amplifications(c). Experimentalcondition: 25C, HRT 8 hr, Do 3-5 mg/L, initial ammonia nitrogen 40-360 mg/L, filling ratio of immobilized pellets 15%6.nitrogen concentration was 40 mg/L, which was gradu- increased to 118 and 123 mg-N/ L-pellet- hr) at the thirdally increased to 360 mg/L within 95 days. As shown and fo中国煤化工邶 The nitrificationin Fig. 4b, at the first stage of acclimation, the initial activtime. Howeverammonia oxidation rate of the immobilized pellets was it sheCNMH Ached a maximumonly 45 mg- N/(L-pellet-hr), which then increased to 72 of 236 mg-N/L-pellet hr)in stage V. As the ammoniamg-N/(L-pellet-hr) at the second acclimation stage, then nitrogen removal rate reached to above 98% at the endnitrifiacteristics of nitrobacteria immobilized in waterbome polyurethane m wastewater"of stage V, this indicated that stage V was the endof acclimation. The results also suggested that about 3months were required for the recovery of activity of theimmobilized nitrifying bacteria after the formation of thepellets. To estimate the amplified product base length bythe strip on the Marker, as shown in Fig. 4c, the boxmarked regiothe target bands, which was the gene(about 500 bps). The intensity of the gel image light 4hfragment length of amplified ammonia oxidizingbacteriarelated to the amplified sample volume of the amoA genefragment, that is, the stronger the band intensity was, the|→D0o-Removal efficiencymore ammonia-oxidizing bacteria there were For samplescollected from stage I to Iv, the light intensity graduallygrew stronger. These results confirmed that the quantityof the ammonia-oxidizing bacteria increased substantiallyTime(day)during the acclimation. This phenomenon demonstratesFIg 6 Effect of Do on ammonia nitrogen removal efficiencythat the immobilized pellets began to acclimate to thesynthetic wastewater and could recover their activity and was less than 20%. The ammonia nitrogen removal rateammonia oxidation ability(wijHfels and Tramper, 1995)increased gradually as DO rose from 2 to 4 mg/L. WhenDO was 4 mg/L, the effluent ammonia concentration was3 Conversion of synthetic wastewater to the distillery lower than 10 mg/L and ammonia removal efficiency waswastewater system90%, When do was higher than 4 mg/L, a low ammoniumAfter the acclimation, immobilized pellets were applied nitrogen concentration was detected in the effluent water;to the treatment of distillery wastewater in the up-flow but the removal rate was stable at g0% when the Do furtherinner circulation reactor. Nitrification characteristics in the rose to 5-7 mg/L. The optimum Do concentration was 4treatment of distillery wastewater were evaluated. Figure 5 mg/L, taking account of energy consumption in the actualshows the time course of the concentrations of nitrogenous engineenng project.compounds in the influent and effluent in all the reactors.econd, an incomplete nitrification process occurred inThe influent ammonia concentration varied greatly with the reactor. Most of the ammonium was oxidized to nitrite,an average concentration of 75 mg/L, and the hrt of not to nitrate. As shown in Fig. 5. the average effluentthe reactor was kept at 3.7-5.6 hr. DO in the experiment nitrite concentration was 32 mg/L, which was much largerwas 1-3 mg/L from day 1-13, and 5-7 mg/L from day than that of nitrate, 12 mg/L. According to the previousnitrification were confirmedsupplied (Garrido et al., 1997; Tokutomi, 2004: Antileo etFirst, Do was an important factor in ammonia nitrogen al., 2006). However, inour experiment, this situation couldremoval. As shown in Fig. 6. sufficient Do in the water not be changed when a higher Do level (5-7 my/L) wastreatment system can improve the nitrification efficiency applied in the later stage, indicating that a high Do levelof immobilized cell pellets. When the Do concentration could not sharply increase the nitrate concentration in thewas less than 2 mg/L, the ammonia nitrogen removal rate effiuent. This result indicated that nitrite oxidizers lost theiractivity possibly due to the following factors: free ammoni-● Influent NH-N-+ Effluent NO,Num inhibition, the high alkalinity of distillery wastewater,-o-EfMluent NH-N -Effluent NO: - Nand oxygen transfer resistance Nitrite accumulation is oneof the characteristics of this process and it also offers thepossibility to achieve shortcut nitrification-denitrificationDO 5-7 mE/LThe concept of shortcut nitrogen removal is denitrifyingnitrite to nitrogen gas before it oxidizes to nitrate. withthis, up to 25% of the oxygen and approximately 40% ofthe carbon source can be saved in the biological nitrogenremoval process(Bae et al., 2002). In practical applicationof this process, an anoxic denitrification tank would beplaced in the final system to remove total nitrogenMoreover, the average COd of the influent and effluentwere both about 150 mg/L (data not shown), Pelletson th中国煤化工 this phenomenonCNMHGbacteria had beenTime(day)Fig 5 Concentrations of nitrogenous compounds in the influent anwastewater and the majority of pores of the pellets wereeffluents of com distillery wastewater. Experimental condition: HRT3. 7- filled with nitrifying bacteria, with no space for het-Joumal of Environmental Sciences 2012, 24(6)999-1005/ Yamei Dong ct alerotrophic bacteria(Qiao et al., 2008)WPU-immobilized nitrifying bacteria pellets using ourAfter stabilization, the optimum HRT was obtained experimental setup. The highest ammonia removal rate,by increasing the flow rate. The operation was initiated 162 mg-N/(L-pellet-hr), was observed when the HRT waswith anlia concentration of 40 mg/L, Do of 4 1.3 hr. Based on the developed nitrification process, themg/L and hRT of 1. 2-3.5 hr. Considering HRT and the process was simple, economical and can be applied atamount of ammonia removed, the ammonia removal rate the pilot-scale for distillery wastewater treatment systems.and ammonia removal efficiency were calculated and are However, further studies on the composition of the nitrify-shown in Fig. 7. When HRT was between 1.2 and 2.5 hr, ing bacteria are required. Factors such as the concentrationthe ammonia nitrogen removal rate was unstable and the of dissolved oxygen, free ammonia concentration and pheffluent ammonia nitrogen could not be stabilized below value may affect accumulation of nitrite by immobilized15 mg/L. A short HRT led to a short nitritation time, pellets. In the future total nitrogen treatment, shortcutwhich subsequently led to the instability of the bioreactor. nitrification can be realized by controlling the factors toWhen hRt was between 2.5 to 3.5 hr, the ammonon in the whole process.nitrogen removal efficiency was above 85% and efiuentammonia nitrogen was below 15 mg/Lnia Acknowledgmentsremoval efficiency of the system operating with 2.3 hr of This work was supported by the National Water Pollu-HRT was 86%, dropping to 60% for 1.2 hr of HRT. The tion Control and Management Technology Major Projectsremoval rate reached its highest(162 mg-N/(L-pellet- hr)(No 2008ZX07101-010-03), the National Natural Sciencewhen the hrT was 1.3 hr. However, the removal rate Foundation of China(No 50708058), the National Highdecreased with a further decrease in the hrT. a decrease Technology Research and Development Program(863)in the hRT to less than 1.3 hrcaused the removal efficiency of China(No. 2012AA062703), the Shanghai Municipaldecrease drastically, indicating the limitations of exter- Science and Technology Commission Major Project(Nonal diffusion and wash-out of ammonia. Considering the 04DZ12030-2)and the Shanghai Committee of Sciencelimits of concentration of ammonia nitrogen emission and and Technology (No. 10231201800)astewater treatment system instability, it was appropriateto set the optimum HRT at 2.0-3.0 hr. When HRT was2.0-3.0 hr, the effluent ammonia concentration was lowerReferencesthan 10 mg/L and the ammonia removal efficiency rate was85%. 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