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-
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D., Spani, C., Willey, B., S a up and seasonal
swings in Ni i e. Wa e En i onmen and Technol-
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5. Al a ez-Ma eos, P., Leon-Mo illo, A. Guillén-Jime-
nez, E., Tecnologia del Agua, 174 (1998) 6168
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lác eos en égimen discon inuo: in luencia de las
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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
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