Batch culture enrichment of ANAMMOX populations from anaerobic and aerobic seed culturesS.Suneethi a ,*,Kurian Joseph ba Centre for Environmental Studies,Anna University,Chennai 600025,IndiabEnvironmental Engineering,Centre for Environmental Studies,Anna University,Chennai 600025,Indiaa r t i c l e i n f o Article history:Received 27March 2010Received in revised form 30July 2010Accepted 31July 2010Available online xxxx Keywords:ANAMMOX EnrichmentNitrogen removal Aerobic seed culture Anaerobic seed culturea b s t r a c tDischarge of nitrate and ammonia rich wastewaters into the natural waters encourage eutrophication,and contribute to aquatic toxicity.Anaerobic ammonium oxidation process (ANAMMOX)is a novel bio-logical nitrogen removal alternative to nitrification–denitrification,that removes ammonia using nitrite as the electron acceptor.The feasibility of enriching the ANAMMOX bacteria from the anaerobic digester sludge of a biomethanation plant treating vegetable waste and aerobic sludge from an activated sludge process treating domestic sewage is reported in this paper.ANAMMOX bacterial activity was monitored and established in terms of nitrogen transformations to ammonia,nitrite and nitrate along with forma-tion of hydrazine and hydroxylamine.Ó2010Elsevier Ltd.All rights reserved.1.IntroductionAmmoniacal nitrogen rich wastewaters arise from municipal solid waste landfills (500–3000mg/L),starch production (800–1100mg/L),domestic sewage (100mg/L),swine wastewater (115–175mg/L),sludge liquor (100–2000mg/L),yeast effluent (180–450mg/L),fertilizer manufacture,and agricultural activities (500–1000mg/L)(Berge et al.,2005;Schmidt et al.,2003).Discharge of nitrogen from wastewater into surface water bodies results in increased algal biomass (eutrophication),emissions of nitrous oxide to atmosphere during oxidation of ammonia,and toxicity to aquatic invertebrate and vertebrate species (Philips et al.,2002).For example,the tolerance limit for Salomonid fish is 0.5mg NH 3-N/L,and LC 50(96h)for Tiger prawn and Australian Crayfish is 14and 26mg NO 2À/L,respectively (Paredes et al.,2007;Philips et al.,2002).Nitrite nitrogen let out into water bodies with-out treatment may cause methaemoglobinemia and gastric cancer among human populations (Philips et al.,2002;Guo and Qi,2006).Removal of nitrogen from wastewaters by biological methods involving autotrophic nitrification and heterotrophic denitrifica-tion requires separate oxic and anoxic units for treatment and depends on external addition of carbon source (methanol –3kg/kgN)(Jin et al.,2008).It results in high production of sludge (1kgVSS/kgN)with substantial requirement of resources in termsof energy (2.8kWh/kgN)and space (Ganigue et al.,2008).Pro-cesses such as partial nitrification,ANAMMOX (ANaerobic AMMo-nium OXidation),SHARON (single reactor system for high activity ammonia removal over nitrite),CANON (complete autotrophic nitrogen removal over nitrite)and OLAND (oxygen limited autotrophic nitrification and denitrification)have the potential to overcome the shortcomings of conventional treatments (Ganigue et al.,2008).These processes operate in a single reactor unit,with reduced aeration (1kWh/kgN)and organic load requirements,with 90%savings in operational costs (Wang et al.,2009).Development of ANAMMOX bacteria is needed,when scaling up larger and complicated reactor configurations like membrane bio-reactor (MBR)to achieve better acclimatization favoring high nitrogen removal performance,especially under ammonia shock loads (Wang et al.,2009).Enrichment of ANAMMOX bacteria had been carried out by monitoring chemical nitrogen transformations (Yamamoto et al.,2008)and/or by studying the microbial eco-physiology through molecular biology techniques (Cho et al.,2010;Wang et al.,2009).This investigation describes the success-ful enrichment in batch cultures of ANAMMOX bacteria from seed cultures of anaerobic and aerobic origin by observing the chemical changes in nitrogen profile.ANAMMOX (ANaerobic AMMonium OXidation)is an innovative technological advancement in removal of ammonia nitrogen from wastewater,carried out by microbial oxidation of ammonia with nitrite occurring under anoxic conditions (Wang et al.,2009;Schmidt et al.,2003;Shivaraman and Shivaraman,2003).ANAM-MOX process combines ammonia and nitrite directly to dinitrogen0960-8524/$-see front matter Ó2010Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2010.07.121*Corresponding author.Tel.:+914422301283;fax:+914422354717/22201480.E-mail addresses:sundar.suneethi@ (S.Suneethi),kuttiani@ (K.Joseph).gas(Shivaraman and Shivaraman,2003).Start up of ANAMMOX process is considered difficult owing to the slow growth rate(dou-bling time–11days)of ANAMMOX bacteria(Chamchoi and Nitis-oravut,2007).The ANAMMOX reaction is carried out by autotrophic Plancto-mycetes,such as‘Candidatus Brocadia anammoxidans’and‘Candid-atus Kuenenia Stuttgartiensis’,which makes use of ammonium as the electron donor(energy source)and nitrite as the electron acceptor as indicated in Eq.(1).NHþ4þ1:32NOÀ2þ0:066HCOÀ3þ0:13Hþ!1:02N2þ0:26NOÀ3þ0:066CH2O0:5N0:15þ2:03H2Oð1ÞIn the ANAMMOX process N2gas is the primary reaction prod-uct along with a small amount of nitrate.They utilize dissolvedcarbon dioxide or bicarbonate for the cell biosynthesis resultingin hydrazine and hydroxylamine as intermediary metabolites.Presence of hydrazine and hydroxylamine in traces adds proof ofANAMMOX process(Shivaraman and Shivaraman,2003).ANAM-MOX process is generally favorable in environments with longsludge retention time(30–50days),stable operation,presence ofnitrite(<70mg/L),and the absence of electron donors(such asammonium ion)that would cause nitrite reduction via denitrifica-tion are absent(Berge et al.,2005).Optimum temperature of32–35°C and neutral pH works well for ANAMMOX activity.TheTable1Configurations of ANAMMOX enrichment units.S.no.Source of seedculture ReactorcapacityReactorlabelsFood/seed ratio(%)Seed Amm-N concentration(mg/L)SamplingfrequencyStudy period1Anaerobic 2.6L R160/406750Once in10days200days(tilldate) 2R260/4071003R360/4025,0004A160/40700070days(till date) 5A260/4020156100mL B160/402900Once in a day30days 7B240/6016808B360/4011209B440/60112010B550/50196011Aerobic5L C150/50199Once in10days70days(till date) 12C250/50enrichment units(A)2.6and2S.Suneethi,K.Joseph/Bioresource Technology xxx(2010)xxx–xxxprerequisite of ANAMMOX process is the preceding partial nitrifi-cation step,which converts ammonium to nitrite.To obtain high removal efficiency,ammonium and nitrite need to be fed to the reactor in the ratio of 1:1.3(Trigo et al.,2006).The desired ammonium/nitrite mixture is obtained because after 50%of the ammonium is oxidized,the decrease in pH to 6.7prevents the oxi-dation of the remnant ammonium.Sludge retention is important owing to the slow growth rates of the bacteria (0.003h À1;0.072/days at 32°C)and low biomass yield (0.13g dry weight/g Amm-N oxidized)(Chamchoi and Nitisoravut,2007;Trigo et al.,2006;Third et al.,2005).2.Methods2.1.Experimental setupEnrichment of ANAMMOX bacteria from the anaerobic and aer-obic seeds were carried out in batch culture.Biosludge from the anaerobic digester of a vegetable waste biomethanation plant and from activated sludge process were used as seed cultures.Twelve ANAMMOX enrichment units (B1,B2,B3,B4,B5,R1,R2,R3,A1,A2,C1,C2)filled with different ratios of seed and enrichment med-ium as food,along with NH 4Cl/NaNO 2as supplement,as presented in Table 1,were prepared using 100mL,2.6and 5L capacity jars sealed with rubber cork.The experimental setup used for R1–R3,A1–A2,C1–C2is presented in Fig.1(A)and that of B1–B5is pre-sented in Fig.1(B).Varying dilutions of seed culture were adopted based on its initial characteristics.The composition of the enrich-ment medium used was as specifically described by van de Graaf et al.(1996).It contains (mg/L):KH 2PO 425,CaCl 2ÁH 2O 300,FeSO 412,EDTA 7,NaHCO 31050,MgCl 2165,and trace metals 1.25mL/L.The trace element solution was (mg/L):EDTA 15,000,ZnSO 4Á7H 2O 430,CoCl 2Á6H 2O 240,MnCl 2Á4H 2O 990,CuSO 4Á5H 20250,NiCl 2Á2H 2O 190,NaSeO 4Á10H 2O 320,H 3BO 414,and NaMO 4Á2H 2O 220.2.2.Strategy of operationThe batch cultures were maintained at neutral pH using 1M HCl/NaOH solution.Mixing was done manually once a day.Light interference was avoided by covering the enrichment units in dark cloth and aluminum foil.Nitrogen compounds such as ammonia,nitrite and nitrate were analyzed every 10days and whenever ammonium or nitrite was found consumed,the Ammonia and ni-trite ratio was maintained at 1:1by external supplements of NH 4Cl/NaNO 2along with the enrichment medium.During the start up period NaNO 3(10ppm)was added along with the medium to favor the elimination of denitrifiers and to prevent the generation of H 2S by sulfur reducing bacteria (Wang et al.,2009).Periodical replenishment of the medium was performed to avoid issues re-lated to lack of nutrients.Batch cultures R1–R3,A1–A2,C1–C2sys-tems were operated for 200days in fed batch mode.Anoxic condition was maintained by suffocation (i.e.cutting the supply of oxygen)method and gas generation was monitored by water displacement method.The ANAMMOX enrichment units B1–B5of 100mL capacity were operated with regular medium addition every day,but with no supplement.So sampling frequency was reduced to once a day and nitrogen compound analysis such as ammonia,nitrite and nitrate were performed.These systems were operated in fed batch mode for a period of 30days.2.3.Sampling and analysisThe primary characterization of the seed culture was carried out after double filtration using muslin cloth,followed by centrifuga-tion at 5000rpm for 15min 100mL samples were collected every 10days,from the sampling port after complete shaking from R1–R3,A1–A2,and C1–C2.Likewise 1mL samples were collected every day from B1–B5using syringe from the common sampling/medium addition port.The samples were filtered through 0.45l filter paper (Whatman).Nitrogen transformations were studied from the anal-yses of NO À2and NO À3performed by spectrophotometric method and Amm-N by Buchi distillation apparatus.ANAMMOX biomass development was determined from the metabolites namely hydra-zine and hydroxylamine (Watt and Chrisp,1952;Frear and Burrell,1955)and indirectly by the TSS,MLVSS and MLSS estimations,which was carried out as per standard methods (APHA,1998).Table 2Initial characteristics of seed cultures.S.No.ParametersValuesAnaerobic seedAerobic seed 1pH7.37.02Total suspended solids 34,27013,8503Nitrate 60544Nitrite254BDL 5Ammoniacal nitrogen 25,0402806COD 36,40013607MLSS 34,29014,5008MLVSS 18,60090009Hydrazine2BDL 10Hydroxylamine244All values are expressed as mg/L except pH.Table 3Performance of ANAMMOX enrichment units from anaerobic seed.S.no.Enrichment units Time (days)Nitrate (mg/L)Nitrite (mg/L)Ammmoniacal nitrogen (mg/L)1A1111281472674521024153682146532041432599100043088268219154045352316565093107143760595319887035131509A21168958201510101170240016601120632914579521230822685590134065460251145020BDL 81156010BDL 16516702BDL189Table 4Performance of ANAMMOX enrichment units from aerobic seed.S.no.Enrichment units Time (days)Nitrate (mg/L)Nitrite (mg/L)Ammmoniacal nitrogen (mg/L)1C11448711992107226711573209568961004301300652135407564524565037073967601485219087049132699C212043200101065631415911204579638012301154784813407535079145040125111156028851541670616200S.Suneethi,K.Joseph /Bioresource Technology xxx (2010)xxx–xxx33.Results and discussion 3.1.Characteristics of seed cultureThe initial characteristics of seed culture used from both anaer-obic and aerobic systems are presented in Table 2.The anaerobicseed used was highly thick,viscous and dark green in color,whereas the aerobic seed was grayish with significant settleable biomass.pH was in the range of 7–anic load was in the range of 30,400–42,400mg/L (as COD)for anaerobic seed and 1290–1434mg/L for aerobic seed.Ammoniacal nitrogen was 25times higher than nitrite concentration in anaerobic seed,while nitrite4S.Suneethi,K.Joseph /Bioresource Technology xxx (2010)xxx–xxxlevels were very low in aerobic seed.The anaerobic seed culture used had MLSS and MLVSS of about 34,000and 18,000mg/L respectively,while that of aerobic seed had MLSS and MLVSS of 14,500and 9000mg/L.Hydrazine and hydroxylamine were also detected in the seed cultures.The type of biomass inside the ANAMMOX enrichment unit was a suspension of free cells during the trial period.3.2.ANAMMOX enrichment performance using both anaerobic and aerobic seedsThe enrichment units of A1–A2and C1–C2were performed to study if the source of seed culture plays a role in nitrogen removalperformance.As earlier indicated in Table 1,the seeds were taken from anaerobic and aerobic origins.The levels of ammonia varied from 6700–2000mg/L in A1–A2,while in C1–C2it was around 200mg/L.Over the duration of study the nitrite levels reduced to less than 50mg/L,whereas significant levels of nitrate were main-tained throughout even when nitrite levels went insignificant.The changes in nitrogen species observed are presented in Tables 3and 4.It is inferred that the changes in nitrogen levels in the form of ammonia,nitrite and nitrate varies depending upon the source of seed.In this case,anaerobic seed achieved the highest ammonium removal of 98%(A1)and 90%(A2)on 70th day,when compared to the aerobic seed,which was 94%(C1)and 76%(C2)by 30th day itself.This rapid efficiency of the aerobic seed did not sustain throughout,S.Suneethi,K.Joseph /Bioresource Technology xxx (2010)xxx–xxx 5leading to instability of the system.The levels of hydrazine and hydroxylamine during the study period in A1–A2and C1–C2were around 0.01–0.002mg/L and 0.08–4.64mg/L respectively,confirm-ing the ANAMMOX activity in the system.Hence with an improved sustained stabilization to higher nitrogen loads,further studies on anaerobic seed in different scales of reactor were adopted.3.3.ANAMMOX enrichment from anaerobic seedNitrogen transformations in three configurations of ANAMMOX enrichment units using anaerobic seed vis-à-vis R1,R2and R3at the end of 200days trial,are depicted in Fig.2.For better under-standing,the experimental period is divided into three stages:Stage 1(day 1–70),Stage 2(day 71–140)and Stage 3(day 140–200).In the first 70days (Stage 1)denitrifying activity was the pre-ferred process in R1and R2,in presence of nitrite and lack of oxy-gen.No ANAMMOX activity was noticed as evident from high levels of nitrite and ammonia concentration in R1(6750and 11,480mg/L)and in R2(11,379and 15,960mg/L),which is indica-tive of denitrification.This could be mainly attributed to formation of ammonium ion,at low pH (3–5).Similar trends were also re-ported by Dapena-Mora et al.(2004)and Third et al.(2005).High level of ammonia (87,500mg/L)with no trace of nitrite was ob-served in R3.Breakdown of organic nitrogen in biomass by cell ly-sis would have resulted in the accumulation of ammonia (Wang et al.,2009).The COD of 3048mg/L was used as carbon source and electron donor by denitrifiers,with nitrite in influent as elec-tron acceptor.Since the denitrifiers (heterotrophs)grow faster than ANAMMOX (autotrophs),denitrifying bacteria might predom-inate in the 1st stage.ANAMMOX activity was initiated in Stage 2,this period,with both ammonia and nitrite getting removed simultaneously.The ammonia levels decreased to 1680mg/L in R3.The ammonia con-centration was 560and 1960mg/L in R1and R2,respectively.It was also noticed that nitrite concentrations soared (R1–89,057mg/L;R2–61,800mg/L;R3–14,429mg/L)showing decreasing activity of denitrifying bacteria.Thus it is conjectured that ANAMMOX bacteria may have carried out all ammonia re-moval,while the nitrite presence could be due to the activity of aerobic ammonia oxidizers.The possibility of nitrite oxidizing bac-teria (NOB)and ammonia oxidizing bacteria (AOB)activities are also not omitted (Third et al.,2005).The ANAMMOX process stabilized in Stage 3.At the end of 200days trial,reduction and stabilization of ammonia and nitrite with rise in nitrate is a definitive proof of ANAMMOX activity.The utilization of ammonia could be due to the activities of AOBs living on the oxygen leakage into the ANAMMOX enrichment unit.In R1,R2and R3nitrate levels were 1308,1585and 62mg/L respectively,which could be attributed to ANAMMOX growth and propagation.However,a small part of denitrifiers surviving might reduce nitrate generated by ANAMMOX metabolism (Wang et al.,2009).Presence of hydrazine and hydroxylamine in traces adds proof of ANAMMOX process along with indirect support from TSS estimations.The mean levels of hydrazine in R1,R2and R3over the trial period were 0.004,0.010and 0.008mg/L,respec-tively.While the hydroxylamine levels were 1.56, 2.05and 4.59mg/L,respectively.At the end of 200days trial,the color of the biomass suspension changed from dark green to brownish green,indicating a transition of microbial populations,since the color characteristic of ANAMMOX populations is reddish brown (Trigo et al.,2006).3.3.1.ANAMMOX enrichment performance in smaller biological systemsThe performance of the 100mL ANAMMOX enrichment units can be used to comprehend better the changes occurring in thebiological system in a smaller scale.The changes in ammonia,ni-trate and nitrite during the 30days of study period is presented in the Fig.3.It is observed that the levels of ammonia reached its high be-tween 13and 19days in B2–B4.Highest ammonia concentration of 3920mg/L was achieved in B1at day 5,while the level of nitrate was 9988mg/L at day 17.Nitrite levels were too low throughout the study period in B1,likewise in B4and B5till day 10and 12,respectively.An unexpected low level of ammonia in B3after day 14was noticed.Highest level of nitrite in B2was achieved at day 18(about 7200mg/L)while that of ammonia and nitrate was 4760and 1240mg/L at day 5and 3,respectively.The lowest level of nitrate was recorded for B1at day 14with 4mg/L followed by B4at day 30with 20mg/L.The rapid changes in ammonia,nitrate and nitrite got stabilized after day 18,except in B3where only ammonia levels were stabi-lized,while the nitrate and nitrite started getting reduced only from day 20.Since there was no nitrogen supplement given except fresh medium everyday,the changes in nitrogen profile can only be attributed to microbial activity (Wang et al.,2009).The type of microbial populations involved could be a mixed group of ANAM-MOX,AOBs,NOBs and denitrifiers.The molecular ecology and diversity of microbes involved (data not included)are yet to be explored.4.ConclusionsThis study proved that anoxic conditions with a suitable ratio (1:1)of NH 4Cl/NaNO 2created an environmental stress to success-fully enrich ANAMMOX populations from anaerobic and aerobic seeds.Anaerobic seed showed better stabilization and ammonium removal efficiency than aerobic seed.During stabilization,denitri-fiers were initially dominant but gradually ANAMMOX populations were established by microbial community succession process.Small scale reactors achieved stabilization of ANAMMOX process in 18–20days,whereas large scale reactors took 130–140days.The study should be extended by including gas composition anal-ysis,molecular ecological studies for ANAMMOX activity confirma-tion and continuous operation to reduce the long start up time.AcknowledgementsThe authors thank the support given by the University Grants Commission Research Fellowship for meritorious scholars in sci-ences to carry out this study.ReferencesAPHA,1998.Standard Methods for the Examination of Water and Wastewater,20thed.United Book Press,USA.Berge,N.D.,Reinhart,D.R.,Townsend,T.G.,2005.The fate of nitrogen in bioreactorlandfills.Crit.Rev.Environ.Sci.Technol.35,365–399.Chamchoi,N.,Nitisoravut,S.,2007.ANAMMOX enrichment from 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