Influence of inoculum and initial pH on dairy effluent biodegradation and mineralization

In luence o Inoculum and Ini ial pH on Dai y E luen Biodeg ada ion and Mine aliza ion P. Ál a ez-Ma eos, J. Pe eda-Ma ín, F. Ca a-Escoba , M. M. Du an-Ba an es, and. E. Guillén-Jiménez Chemical Enginee ing Depa men , Facul y o Chemis y, Uni e si y o Se ille C/P o eso Ga cía González s/n, 41012, Se ille (Spain) E-mail: [email p o ec ed] Dai y e luen s a e cons i u ed mainly by milk, alkaline washing was es, and wa- e . In acco dance oo he cha ac e is ics o his ype o indus ial was ewa e , a biolog- ical ea men was applied in a ba ch eac o . The p ocess chosen was ni i ica ion o educe he nu ien con en . In o de o enhance ammonium oxida ion, he medium was inocula ed wi h municipal was ewa e om a seconda y cla i ica ion ank o he Ranilla ea men plan (Se ille, Spain). Municipal was ewa e o empe a u e a abou 26 °C du ing he wa me season (Sep embe ) showed highe concen a ions o ni i e and ni a e. Di e en ini ial pH alues (8.5, 10.5, 11.0, 11.5 and 12.0) we e es ed, and he o ganic ma e con en , exp essed as Chemical Oxygen Demand (COD), was main- ained cons an a 1000 mg. The municipal was ewa e was aken in wo di e en sea- sons, win e (Feb ua y) and summe (Sep embe ), o know he e ec o season on he ni i ica ion. The esul s indica e a di e ence in he a e o he p ocess bu no in he beha iou . The educ ion in COD is nea ly he same in bo h cases, and he di e ence depends on he ini ial pH alue a he han on he season o he inoculum. Key wo ds: Dai y indus ies, Ni i ica ion, Biological ea men , Ae obic ea men , Ba ch eac o . In oduc ion Dai y e luen s ha e an alkaline pH alue (6.612.2) and a e ich in o ganic ma e 2. This ype o indus ial was ewa e con ains milk and, he e o e, ammonium ( om he amino acids) and phospha e ( om he caseins). Consequen ly, hese componen s a e a ac o in eu ophica ion o na u- al esou ces. Some scien is s3belie e ha he p esence o ni ogen could inc ease he educ ion o phospho us in esh wa e . This la e elemen encou ages he g ow h o algae which consume ni- ogen. When he phospho us is emo ed, algal g ow h does no ake place, and he esidual ni o- gen emains in he wa e . Ne e heless, his nu- ien would each he sea, whe e i is a ailable o o he algae, and he eu ophica ion would ake place he e. A he same ime, i has been ound4 ha was ewa e ea men plan s a e esponsible o eu ophica ion o na u al esou ces. Consequen ly, he ammonium con en o e luen s om was e- wa e ea men plan s will ha e o be educed in he nex ew yea s. A solu ion o he p oblems, a ising om he wo ypes o was ewa e , could be sough by es ing a mix o hem. The aim o his pape is o s udy he educ ion o ammonium con en applying ni i ica ion in dai y e luen s in wo di e en seasons, in win e and in summe . Municipal was ewa e , om he seconda y cla i ica ion ank o a was ewa e ea men plan , was used o inocula e he dai y e luen s. Ni i ica- ion was accomplished by en iching he medium wi h o he ypes o mic oo ganism. Ma e ials and me hods Dai y was ewa e s con ain mainly milk ( e- sponsible o hei oxygen demand), as well as emain o cleaning agen s ( esponsible o hei high alkalini y). The expe imen was ca ied ou using milk and wa e mix u es om he second- a y sedimen a ion ank o a was ewa e ea men plan (WWTP), o a oid hick solids and o ob ain a municipal was ewa e ich in mic oo ganisms. Two municipal was ewa e samplings we e cho- sen, ha ing he di e en wea he cha ac e is ics o win e and summe , espec i ely. The ield s udies we e pe o med in Feb ua y and Sep em- be 1996. The a e age alues o he empe a u e pa ame e in hese mon hs we e 12 and 26 °C, e- spec i ely. The subs a e o he mic oo ganisms was milk and wa e mixes, adjus ed o 1000 mg l1 COD. The inoculum has a concen a ion o 500mgVSS/440mgCOD1. This inoculum was a mix u e o 1:1 cul u e p epa ed wi h a dai y e lu- en incuba ed a 30 °C o i e days, and a sludge om WWTP seconda y cla i ie . The same ope a ional condi ions we e applied o he wo expe imen al se ies, di e ing only in he season, when he municipal was ewa e inoculum was aken. Fi e ini ial pH alues (8.5, 10.5, 11.0, 11.5 and 12.0), we e s udied. These al- P. ÁLVAREZ-MATEOS e al., In luence o Inoculum, Chem. Biochem. Eng. Q. 14 (3) 101106 (2000) 101 O iginal scien i ic pape Recei ed: Accep ed: ues we e chosen because he dai y e luen s a e al- kaline due o he p esence o washing p oduc s2. The ope a ional empe a u e was main ained a 30 °C, he op imal o ni i ica ion o dai y e luen s5. The expe imen s we e ca ied ou du - ing 30 days, o ob ein o ganic ma e mine aliza- ion in ae obic condi ions. Con ol o a iables The changes in o ganic con en o he sam- ples can be ollowed adequa ely h ough he chem- ical oxygen demand (COD) a each momen , while he a ia ions caused by eac ions in he medium a e moni o ed h ough pH. The mine aliza ion o he o ganic ma e was ollowed by analysis o am- monium, ni i es, and ni a es. All analy ical me hods ollowed he p ocedu es ou lined by S anda d Me hods6, analyses we e pe o med o pH (pH-me e, 423), ammonium-N (Nessle iza- ion, 417 A and 417 B), ni i e-N (Spec o o o- me y, 419), ni a e-N (Epec o o ome y, 418), and COD (dich oma e e lux me hod, 508). The esul s a e gi en o N-NH4+, N-NO2and N-NO3as mg l1 N2and mg l1 O2 o COD and o uni s o pH. Equipmen The assembly consis ed o a he mos a and i e glass eac o s, ins alled in a me allic suppo ancho ed o he loo . Bo osilica e glass eac o s o 12 li e we e used (Figu e 1), he mos a ed, and s i ed by ai injec ion. Ai was supplied h ough a memb ane pump, i has go a il e o p e en he inco po a ion o pa icles om he en i onmen . Each eac o was ins alled wi h such pump. The eac o had h ee ou le s a he bo om; an ae a- ion mechanisms was placed in each o he wo side ou le s and he samples we e collec ed h ough he cen al one. The co e o he eac o was p o ided wi h an opening, illed wi h wa- e - epellan co on wool, o pe mi he escape o he gases esul ing om he me abolic eac ions aking place in he medium. The eac o was co - e ed wi h aluminium oil o p e en he inhibi ion o ni i ying mic oo ganisms caused by ligh . Ex- pe imen s we e moni o ed by daily sampling. Resul s and discussion E ec o he ini ial pH alue The mine aliza ion o o ganic ma e akes place h ough h ee s eps: 1. Ammoni ica ion o o ganic ma e which pe mi s he elease o ammonium 2. Con e sion o ammonium in o ni i es by means o ni i ying o ganisms (Ni osomo- nas) 3. Con e sion o ni i es in o ni a es by means o ni i ying o ganisms (Ni ibac e s) Depending on he ini ial pH alue, some di - e ences a e obse ed in he desamina ion o ni- ogen compounds (1s s ep) o each ype o ino- culum. He e o oph mic oo ganisms, esponsible o his p ocess, ha e o adap o he medium, a de eloping sys ems o bu e pH, which is he op- e a ional pa ame e un a ou able o hei g ow h. In he i s s ep, gene aly, mic oo ganisms a e a ec ed by he season o he yea and pH. Howe e , simila esul s we e ob ained in he dasamina ion o o ganic ma e s wi h bo h munic- ipal was ewa e s when he ini ial pH alue we e 8.5, 11.0 and 11.5 (Figu e 2). I seems ha , in his case, pH has a highe e ec on he medium han does he season o he inoculum, and he esul s he e o e a e almos he same. The i s one is he bes o his ype o mic oo ganisms7, meanwhile an alkaline hyd olysis o o ganic ma e s akes place in he o he s wo, which a ous he elease o ammonium8. A ini ial pH alues o 10.5 (Figu e 2), he desamina ion o o ganis ma e s akes place mo e quickly in he expe imen s ca ied ou wi h he was ewa e aken in Sep embe . 102 P. ÁLVAREZ-MATEOS e al., In luence o Inoculum, Chem. Biochem. Eng. Q. 14 (3) 101106 (2000) Fig. 1  Diag am o he expe imen al de ice In he case o high pH alues, such as 12.0, he expe imen ca ied ou wi h municipal was e- wa e aken in Feb ua y showed a as e esponse o he medium condi ions (Figu e 2). I seems ha he e o oph mic oo ganisms adap ed o he un a- ou able condi ions, such as lowe empe a u es, mean while he Sep embe inoculum, and hei adap a ion mechanisms, a e consequen ly mo e de eloped. E ec o he ype o he inoculum on he ni i ica ion Signi ican di e ences a e ob ained in deg a- da ion o ni ogen compounds when he municipal was ewa e is aken om he plan a di e en seasons o he yea (Table 1). Because a biological eac o con ains a wide a ie y o micoo ganisms, i was belie ed ha he a ia ion in he composi ion o mic oo ganisms was no signi ican h ough he yea . Howe e , he esul s showed some di e ences be ween win- e and summe samples. These seasons a e cha - ac e ised by di e en wea he condi ions, and empe a u e was chosen as ope a ional pa ame e because o i s e ec s on a municipal was ewa e ea men plan 9. Some o hese e ec s a e he ol- lowing:  The chemical eac ion a es a e highly de- penden on empe a u e. The Van Ho A he- nius exp ession10,11 shows his dependency7172: g1=g2·QT1T2 (1) whe e: g1is he eac ion a e a he empe a u e T1/°C g2is he eac ion a e a he e e ence em- pe a u e o T2/°C qis he empe a u e ac o . The ac o qmay be s ima ed by plo ing ln g e sus T, gi ing a slope equal o ln q.qis dimen- sionless. On he o he hand, ln qis app oxima ely 0.0015 u, whe e uis he empe a u e ac o .  Some ope a ional pa ame e s, such as gas ans e a e and sedimen a ion cha ac e is ics o he sludge, a e esponsible o he good ope a ion o he plan , and hey a e also dependen on em- pe a u e. The exp ession (1) explains why he ni ogen compound deg ada ion a e was highe in samples aken in Sep embe han in hose aken in Feb u- a y, and whose mean empe a u es we e 26 °C and 12 °C, espec i ely. The p esence o mic o- o ganisms, and he e ec o he empe a u e on hem, mus also be aken in o accoun . Mic obial g ow h is s imula ed a empe a u e as high as 26 °C. The mos -known ni i ying bac e ia a e he Ni osomonas and Ni obac e , wi h op imum g ow h empe a u e a 30 ºC and 28 °C, espec- i ely12. The e o e, i was expec ed ha he mu- P. ÁLVAREZ-MATEOS e al., In luence o Inoculum, Chem. Biochem. Eng. Q. 14 (3) 101106 (2000) 103 Fig. 2 Ammonium concen a aion in he expe i- men s ca ied ou wi h inoculum o Feb u- a y and Sep embe nicipal was ewa e aken in Sep embe would be iche in ni i ying bac e ia han hose aken in Feb ua y. Consequen ly, he oxida ion o ammo- nium o ni i e and o his o ni a e was p oduced some days ea lie in he Sep embe inoculum. The concen a ions o he oxida ion p oduc s we e highe wi h his inoculum. An example is he ex- pe imen ca ied ou a he ini ial pH alue o 10.5 (Figu es 3). I is also obse ed (Figu e 2) ha he elease slopes o ammonium, ha is, he deamina ion o he o ganic ma e , a e pa allel o each season. The expe imen ca ied ou wi h municipal was ewa e aken in Feb ua y showed a i e day delay. This could indica e ha he di e ence be- ween hese expe imen s is no due o he pa ici- pa ion o di e en indi iduals, cha ac e ised by di e en g ow h a es, bu o he numbe o indi- iduals. As a esul o an ea lie o ganic ma e deamina ion, ni i ica ion akes place soone , be- cause he p esence o ammonium and simple o - ganic ma e helps ni i ying bac e ia o g ow. On he o he hand, in he es s ca ied ou wi h mu- nicipal was ewa e aken in Feb ua y, he ni i i- ca ion has ha dly begun (Figu es 3). I could be said ha he summe season helps he g ow h o ni i ying mic oo ganisms and, con- sequen ly, be e esul s a e ob ained o his p o- cess in ha pa o he yea . Some ea men plan s, such as Bu e Wo h Was ewa e Plan (New Je sey), ake ad an age o his ci cums ance and ca y ou a seasonal ni i ica ion in he mon hs om May o Oc obe 13. O he companies, such as Hooke s Poin Ad anced Was ewa e T ea men Plan (Flo ida), despi e low solids de- en ion ime ob ained a conside able ni a es mass concen a ion (45 mg l1) which was a ib- u ed o he empe a u e o he municipal was e- wa e (2130 °C)14. These examples show ha he season o he yea con ibu es o imp o ing he p ocesses which ake place in a was ewa e ea - men plan , such as, in his case, ni i ica ion. 104 P. ÁLVAREZ-MATEOS e al., In luence o Inoculum, Chem. Biochem. Eng. Q. 14 (3) 101106 (2000) Table 1 To al Kjeldal Ni ogen (NKT) ini ial and Maximum concen a ions (mg/li e) o ammonium, ni i e and ni a e, and ime equi ed wi h a Sep embe was ewa e inoculum. Seasons pHi8.5 /d 10.5 /d 11.0 /d 11.5 /d 12.0 /d NKT (mg/L) 25 25 25 25 25 Sep em- be N-NH4+ N-NO2 N-NO3 14.40 7.75 4.07 10 33 31 12.25 6.95 4.41 8 22 33 11.75 7.29 4.10 12 23 31 9.01 5.49 2.90 8 33 31 7.40 3.34 2.16 10 33 29 Feb ua y N-NH4+ N-NO2 N-NO3 15.00 6.17 3.26 10 30 30 15.49 0.09 0.46 14 29 29 14.12 5.69 3.33 10 21 20 9.50 2.67 1.83 15 29 30 9.25 0.06 0.71 11 30 30 Fig. 3 E olu ion o Ammonium, Ni i e and Ni- a e in he expe imen s ca ied ou a he ini ial pH 10.5 The beha iou in he es ca ied ou a he ini ial pH alue o 10.5 was simila o ha in he es o he expe imen s es ed a o he ini ial pH alues. Howe e , some di e ences we e known, as commen ed below. The he e o oph mic oo ganisms, in con as o he au o ophs, we e no a ec ed by he sea- son in which he municipal was ewa e was aken. The deg ada ion o he o ganic ma e (exp essed as COD) was dependen on he ope a ional condi- ions, such as pH alue, a he han by he season o he yea . Table 2 shows he o ganic ma e e- mo al, in pe cen age, a e en days o unning. In gene al, i can be obse ed ha he o ganic ma - e is be e deg aded a Sep embe empe a u e han a he Feb ua y empe a u e. In e ac ion be ween he ype o inoculum and ini ial pH alue in ni i ica ion As i has been shown, ha he municipal was ewa e aken in Sep embe had he bes e- sul s in he ni i ica ion p ocess (Table 1). The au o oph bac e ia esponsible o he oxida ion o he ammonium and ni i e (Ni osomonas and Ni obac e , espec i ely) do no seem o be a - ec ed by he ini ial pH alue o he medium. When au o ophs can g ow, he pH alue o he medium is abou 8.5, because i has been bu e ed h oughou he o ganic ma e mine aliza ion by he he e o oph mic oo ganisms. This pH alue is op imum o ni i ying bac e ia g ow h, which akes place a a highe a e. A he ini ial pH alue o 12.0, he e is a delay o abou en days be o e au o oph mic oo gan- isms begin ni i ying. In his case, he pH is bu - e ed o abou 9.0 and he ni i ying bac e ia con- sequen ly need a pe iod o adjus men o he me- dium. The maximum ni i e and ni a e concen a- ions eached, depend on he ini ial pH alue o he medium. A highe ini ial pH alue in ol es a g ea e ni i ying bac e ia selec ion. Table 1 shows he maximum concen a ions o ammonium, ni i e, and ni a es eached in he expe imen s ca ied ou wi h he municipal was e- wa e o Sep embe and Feb ua y, a di e en ini- ial pH alues. I is obse ed ha he maximum concen a ions o ni i e and ni a e do no a y ma kedly when ini ial pH alue is in he ange 8.5 o 11.0. Howe e , he maximum concen a ions dec ease a highe ini ial pH alues (11.5 and 12.0). The e a e no many mic oo ganisms ha can su i e unde hese condi ions, and conse- quen ly, he selec ion is e y s ong. Taking in o accoun ha he ammonium can be eleased in he a mosphe e as ammonia (depending on he pH), he one emaining in he medium, is he sub- s a e o he ni i ying bac e ia. Table 1 shows ha when ini ial pH is highe he maximun am- monium concen a ion is lowe , o each ype o inoculum. These esul s ag ee wi h hose o Balmelle15: he di e ences in he ni i ica ion p ocess can be a ibu ed o chemical di e ences o he medium, such as pH. I is belie ed ha his is he main ac- o esponsible o he di e en esul s because he ope a ional empe a u e was kep cons an h oughou he es s. In he expe imen s ca ied ou wi h munici- pal was ewa e aken in Feb ua y, concen a ions o ni i e and ni a e we e close o hose ob ained wi h he Sep embe ones. A 8.5 and 11.0 ini ial pH, he only di e ence be ween hem was he de- lay o en days in Feb ua y was ewa e due o he seasonal empe a u e. A he o he ini ial pH al- ues, ni i ica ion does no ake place when he was ewa e is aken in Feb ua y. O he ni i ying mic oo ganisms, Ni obac e is he mos sensi i e o empe a u e, so i comp ises he limi ing s ep in he ni i ica ion p ocess. The mic oo ganisms o he municipal was ewa e o Feb ua y g ew a 12 °C (mean empe a u e o he mon h), bu in he expe imen s ni i ica ion s ill ook place. E en hough empe a u e is an inhibi o y ac o , i has been con i med ha he inhibi ion is e e s- ible16,17. Conclusions F om his wo k, some gene al ideas o he e - ec ipo de inóculo de la WWTP on he ni i- iac ion a e es ablished.  The ni a es om ammonium, in a munici- pal was ewa e ea men plan , a e highe a he en i onmen al empe a u e o 30 °C (o 26 °C, mean empe a u e o he Sep embe inoculum).  A lowe empe a u es, such as 12 °C, ni i- ica ion in dai y was ewa e also akes place, bu a lowe a e. Subjec ing he inoculum aken in Feb ua y o he empe a u e o 30 °C, ni i ica ion akes place ea lie , al hough he using o munici- pal was ewa e o Sep embe was as e .  The e ec o he ini ial pH inc eases wi h i s alue. A pH o 12.0, a conside able p opo ion o ammonium is los as ammonia, and less sub- s a e is oxidised o ni a e by he ni i ying bac e- ia. High ini ial pH alue also esul in a bac e ial selec ion and, consequen ly, he ni i ica ion a e is low. P. ÁLVAREZ-MATEOS e al., In luence o Inoculum, Chem. Biochem. Eng. Q. 14 (3) 101106 (2000) 105 Table 2 COD educ ions (%) a e en days o un- ning. pH 8.5 10.5 11.0 11.5 12.0 Feb ua y 93.7 88.1 91.7 76.3 87.2 Sep em- be 91.2 91.2 94.4 92.8 77.5  The ini ial pH alue and empe a u e in e - ac ion lead o h ee di e en beha iou s o he deamina ion o he o ganic ma e . pH alues lowe han 11.0 (excep 8.5), and municipal was e- wa e aken in Sep embe , esul in a high deami- na ion a e. On he o he hand, a pH alues highe han 11.0, mic oo ganisms need he muni- cipal was ewa e aken in Feb ua y o yield high deamina ion. Ne e heless, a pH 11.0 and 8.5, e - ec o he ype o he inoculum on he deami- na ion a e, is no obse ed. Re e ences: 1. Rome o Guzman, F., Rod iguez Gonzalez, M. J., Depu ación de los e idos lác eos en eac o agi- adi: E olucion de la p esion. V Cong eso Nacional de Quimica y Tecnologia del Agua. Vol. I. Ed. ANQUE, Mad id, 1987, p. 525539 2. González, J.A., Ga cía, A., Rome o, F., Ca ac e ís i- cas del e ido de una Cen al Leche a. Ag oquími- ca, Tecnología de alimen os, 22 (4) (1982) 501510 3. Blankenship, K., Reducing Nu ien Le els in he Chesapeake Bay. Wa e En i onmen and Technol- ogy, 1993, June, p. 1922 4. Nee hling, J.B.,Pi e i, D., Giuliano, L., Bolding, D., Spani, C., Willey, B., S a up and seasonal swings in Ni i e. Wa e En i onmen and Technol- ogy, 1997, Oc ube , p. 3440. 5. Al a ez-Ma eos, P., Leon-Mo illo, A. Guillén-Jime- nez, E., Tecnologia del Agua, 174 (1998) 6168 6. Apha, awwa, WPCP., S anda d Me hods o exami- na ion o wa e and was ewa e . 15 h Edi ion. Ame- ican Public Heal h Assoc. Washing on D.C., 1980 7. Ál a ez Ma eos, P., Depu ación ae obia de e idos lác eos en égimen discon inuo: in luencia de las condiciones de ope ación. PhD Thesis. Facul ad de Químicas. Uni e sidad de Se illa. 1993 8. Guillen-Jimenez, E., Al a ez-Ma eos, P., Rome o- -Guzman, F. Pe eda-Ma in, J., Wa e Resea ch, 34 (4) (2000) 12151224 9. Eche e ía Vallina,. Es udio de ac o es que pueden a ec a al a amien o biológico. P oceeding o Bulking Filamen ous in Ac i a ed Sludge. Ed. by Se ille ci y Council, (Spain), EMASESA, Oc ube , 1997. 10. Ramalho, R. S., T a amien o de aguas esiduales. Ed. Re e é, S.A. Ba celona, (Spain) 1993. 11. G ady, L. C. P., Daigge G. T., Biological was e- wa e ea men . Ma cel Dekke , Inc. New Yo k, 1999. 12. Hol , J.G., K ieg, N.R., Snea h, H.A., S aley, J.T., Williams, S.T., Be gey's Manual o De e mina i e Bac e iology. 9 h Edi ion. Williams and Wilkins Ed. USA, 1994 13. Pla , F. N., Plan o e comes space es ic ions. Wa e En i onmen and Technology, 1994, Sep em- be , p. 4748. 14. Wilson, T.E., Picka d, D.W., Biza i, E., A ni ogen success s o y. Wa e En i onmen Technology, 1994, Sep embe , p. 7074. 15. Balmelle, B., Nguyen, K.M., Capde ille, B., Co nie , J.C., Deguin, A., Wa e Science Technology, 26 (56) (1992) 1017 16. G oeneweg, J., Bea e, S., Tappe, W., Wa e Resea ch, 28 (1994) 2561 17. Shammas, N.Kh., J. Wa e Poll. Con ol Fed., 1 (1986) 52 106 P. ÁLVAREZ-MATEOS e al., In luence o Inoculum, Chem. Biochem. Eng. Q. 14 (3) 101106 (2000)