p ocesses
A icle
Role o Mic oalgae in he Reco e y o Nu ien s om
Pig Manu e
Ana Sánchez-Zu ano 1,* , Ma ina Cia di 1, Tomás La a ga 1, JoséMa ía Fe nández-Se illa 1, Rupe o Be mejo 2
and Emilio Molina-G ima 1


Ci a ion: Sánchez-Zu ano, A.; Cia di,
M.; La a ga, T.; Fe nández-Se illa,
J.M.; Be mejo, R.; Molina-G ima, E.
Role o Mic oalgae in he Reco e y o
Nu ien s om Pig Manu e. P ocesses
2021,9, 203. h ps://doi.o g/
10.3390/p 9020203
Recei ed: 30 Decembe 2020
Accep ed: 19 Janua y 2021
Published: 21 Janua y 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Depa men o Chemical Enginee ing, Uni e si y o Alme ía, 04120 Alme ía, Spain;
[email p o ec ed] (M.C.); omasla a [email p o ec ed] (T.L.); [email p o ec ed] (J.M.F.-S.);
[email p o ec ed] (E.M.-G.)
2
Depa men o Physical and Analy ical Chemis y, Uni e si y o Jaén, 23700 Lina es, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed]
Abs ac :
Animal p oduc ion ine i ably causes he emission o g eenhouse gases and he gene a ion
o la ge amoun s o slu y, bo h ep esen ing a se ious en i onmen al p oblem. Pho osyn he ic
mic oo ganisms such as mic oalgae and cyanobac e ia ha e been p oposed as al e na i e s a egies
o bio emedia e ag icul u al was e while consuming ca bon dioxide and p oducing aluable biomass.
The cu en s udy assessed he po en ial o he mic oalga Scenedesmus sp. o emo e nu ien s om
pigge y was ewa e (PWW) and he in luence o he mic oalga on he mic obial conso ia. Maximum
N-NH
4+
consump ion was 55.3
±
3.7 mg
·
L
−1·
day
−1
while P-PO
43−
emo al a es we e in he ange
0.1–1.9 mg
·
L
−1·
day
−1
. N-NH
4+
emo al was pa ially caused by he ac ion o ni i ying bac e ia,
which led o he p oduc ion o N-NO
3−
. N-NO
3−
p oduc ion alues whe e lowe when mic oalgae
we e mo e ac i e. This wo k demons a ed ha he pho osyn he ic ac i i y o mic oalgae allows
us o inc ease nu ien emo al a es om PWW and o educe he coli o m bac e ial load o he
e luen , minimising bo h hei en i onmen al impac and heal h isks. Mic oalgae assimila ed pa
o he N-NH
4+
p esen in he media o p oduce biomass and did no o con e i in o N-NO
3−
as in
adi ional p ocesses.
Keywo ds: Scenedesmus; was e ea men ; bio echnology; pho osyn hesis; espi ome y; biomass
1. In oduc ion
Animal p oduc ion will inc ease because o an inc easing popula ion, expec ed o
each 9–10 billion people by 2050 [
1
]. Mea p oduc ion is one o he main causes o
g eenhouse gas emissions [
2
] and ine i ably causes la ge amoun s o slu y, which is a
se ious en i onmen al conce n [
3
]. Pig manu e has been adi ionally used as a e ilise in
u al a eas. Cu en ly, Spanish egula ions limi he u ilisa ion o pig manu e as an o ganic
e ilize o up o 170 kgN
·
ha
−1·
yea
−1
(Di ec i e 91/676/CEE) and his causes was e
managemen p oblems in egions whe e ag icul u al lands a e sca ce and high amoun s o
manu e a e p oduced.
Mic oalgae-bac e ia conso ia ha e been p oposed as a s a egy o p ocess was ewa e
and pig manu e because o hei abili y o ecycle o ganic ma e and nu ien s [
4
]. Indeed,
mic oalgae a e capable o consuming 25 nN
·
ha
−1·
yea
−1
and 2.5 nP
·
ha
−1·
yea
−1
and
simul aneously p oduce up o 200 n
·
yea
−1
o aluable biomass, which could be u he
used o p oduce bio e ilize s and bios imulan s o ag icul u e [
5
]. An added ad an age o
mic oalgae is ha hey ix a mosphe ic ca bon dioxide, one o he main p oblems associa ed
wi h ag icul u e and ood p oduc ion. Howe e , wo impo an issues mus be conside ed
when mic oalgae a e used o pigge y was ewa e (PWW) ea men : (i) high ammonium
concen a ions, such as hose p esen in PWW, can lead o ammonia oxici y [
6
], and (ii)
mic oalgae can a ec he mic obial communi y s uc u e ha appea s na u ally in PWW [
7
].
P ocesses 2021,9, 203. h ps://doi.o g/10.3390/p 9020203 h ps://www.mdpi.com/jou nal/p ocesses
P ocesses 2021,9, 203 2 o 11
The la e is o key impo ance as he composi ion o he mic oalgae-bac e ia conso ia is
key o an e icien nu ien emo al.
Du ing he day, mic oalgae consume ino ganic ca bon, ni ogen, and phospho us
(as well as o he compounds) o p oduce biomass while simul aneously eleasing oxygen.
Oxygen p oduced by mic oalgae is used by he e o ophic bac e ia o oxidise o ganic
ma e in o ino ganic compounds [
8
], p oducing ca bon dioxide ha is consumed by
mic oalgal cells [
9
]. Howe e , he eali y o hese in e ac ions is a mo e complex, wi h
di e en mic oalgal and bac e ial popula ions aking place a he same ime, including
he ae obic g ow h o he e o ophic biomass, deni i ica ion by he anoxic g ow h o
he e o ophic biomass, and ni i ica ion by he ae obic g ow h o ni i ying bac e ia (AOB
and NOB) [
10
]. Di e en in e ac ions occu be ween mic oalgae and ni i ie s in e ms o
N-NH
4+
a ailabili y. These in e ac ions a e no ye ully unde s ood and con adic o y
esul s ha e been epo ed [
11
,
12
]. Thus, u he s udies a e needed o iden i y how he
u ilisa ion o mic oalgae a ec s he bac e ial communi y ha appea s na u ally in PWW
and, he e o e, he e iciency o he in eg a ed p ocess.
Fo many yea s, espi ome y has been conside ed as a apid app oach o assess
me abolic ac i i ies in an economic and eliable way. Respi ome y-based me hods ha e
been applied in con ec ional was ewa e ea men o cha ac e ise he e o ophic and au-
o ophic biomass unde di e en ope a ional and en i onmen al condi ions [
13
–
16
]. This
s a egy has also been applied o quan i y pho osyn hesis and espi a ion a es o cul u es
o pho o ophic o ganisms such as mic oalgae and cyanobac e ia [
17
–
19
]. Mo e ecen ly,
echniques based on espi ome y o ac i a ed was ewa e ea men and pho o ophic
axenic cul u es ha e been adap ed o he mic oalgae-bac e ia conso ia ha appea in
was ewa e [8,20,21].
The main goals o he cu en s udy we e o p o ide a be e unde s anding o he
mic oalgae-bac e ia in e ac ions ha occu in he mic oalgae-based PWW ea men p o-
cesses and o assess he nu ien emo al e iciency o he mic oalga Scenedesmus sp., widely
s udied because o i s esis ance o a wide ange o en i onmen al condi ions.
2. Ma e ials and Me hods
2.1. Mic oalgae and Cul u e Condi ions
Scenedesmus sp. has been widely s udied o ou doo mic oalgae p oduc ion and
was ewa e ea men . This s ain was p e iously isola ed om eshwa e used in g een-
house e iga ion by ou esea ch g oup and is, he e o e, adap ed o he local clima e. The
selec ed s ain can g ow well a pH, empe a u e, and salini y alues anging be ween 7–10,
26–40
◦
C, and 0–5 g NaCl
·
L
−1
[
22
]. S ock cul u es we e main ained pho o-au o ophically
in 1.0 L capaci y pho obio eac o s using an A non medium [
23
]. Cul u es we e con in-
uously bubbled wi h ai —1.0% CO
2
mix u e o con ol he pH a 8.0
±
0.2. The cul u e
empe a u e was kep cons an a 22
±
1
◦
C by egula ing he ai empe a u e in he cham-
be . The cul u e was a i icially illumina ed in a 12:12 h ligh :da k cycle using ou Philips
PL-32W/840/4p whi e-ligh lamps, p o iding an i adiance o 750
µ
E
·
m
−2·
s
−1
on he
pho obio eac o s su ace. The a e age composi ion o he con ol medium and he pigge y
was ewa e used is lis ed in Table 1.
2.2. Pho obio eac o s
Expe imen s we e ca ied ou in 1.0 L capaci y lab-scale s i ed- ank pho obio eac o s
made wi h polyme hylme hac yla e (0.08 m in diame e and 0.20 m heigh ). To acili a e
he up-scaling o he p ocess, eac o s we e ope a ed simula ing ou doo aceway bio e-
ac o s. Two se o expe imen s we e pe o med in iplica e (Figu e 1). In he i s se o
expe imen s, pho obio eac o s we e ope a ed unde ei he ligh o da k condi ions and
we e ed 5- old dilu ed PWW. Cul u es p oduced in ligh o da k condi ions we e e med
L-5 and D-5, espec i ely. The p ocedu e was epea ed bu using 25- old dilu ed PWW as
he cul u e medium. In his case, cul u es p oduced in ligh o da k condi ions we e e med
L-25 and D-25, espec i ely. In bo h cases, he cul u es we e inocula ed wi h Scenedesmus
P ocesses 2021,9, 203 3 o 11
sp. a an ini ial concen a ion o 0.5 g
·
L
−1
and we e ope a ed in ba ch mode o 6 days
ollowed by ope a ion in con inuous mode by eplacing daily 20% o he cul u es olume
wi h esh PWW o 10 days, when he s eady s a e was eached. Dissol ed oxygen (DO)
was con olled below 200%Sa by on demand ai supply. The pH was con olled a 8.0
±
0.2
by on-demand injec ion o CO2.
Table 1.
A e age composi ion o he cul u e medium and pigge y was ewa e used as he in luen in
he bio eac o s. Concen a ions exp essed as mg·L−1.
Pa ame e s Pigge y Was ewa e A non
pH 8.1 ±0.3 7.5 ±0.2
COD 2181.7 ±100.9 16.0 ±1.2
Ni ogen-Ni a e 56.4 ±2.7 140.0 ±4.5
Chlo ide 2060.2 ±23.5 78.9 ±2.1
Po assium 1800 ±1.6 325.1 ±6.3
Calcium 350.1 ±0.2 364.9 ±5.5
Magnesium 108.2 ±14.1 12.2 ±0.6
Phospho us-Phospha e 119.2 ±5.1 39.3 ±3.1
Ni ogen-Ammonium 1485.6 ±17.7 0.0 ±0.1
I on 4.8 ±0.01 5.0 ±0.3
Coppe 1.1 ±0.1 0.02 ±0.00
Manganese 2.6 ±0.0 0.5 ±0.02
Zinc 20.1 ±0.2 0.06 ±0.01
Bo on 5.3 ±0.1 0.4 ±0.0
P ocesses 2021, 9, 203 3 o 11
Calcium
350.1 ± 0.2
364.9 ± 5.5
Magnesium
108.2 ± 14.1
12.2 ± 0.6
Phospho us-Phospha e
119.2 ± 5.1
39.3 ± 3.1
Ni ogen-Ammonium
1485.6 ± 17.7
0.0 ± 0.1
I on
4.8 ± 0.01
5.0 ± 0.3
Coppe
1.1 ± 0.1
0.02 ± 0.00
Manganese
2.6 ± 0.0
0.5 ± 0.02
Zinc
20.1 ± 0.2
0.06 ± 0.01
Bo on
5.3 ± 0.1
0.4 ± 0.0
2.2. Pho obio eac o s
Expe imen s we e ca ied ou in 1.0 L capaci y lab-scale s i ed- ank pho obio eac-
o s made wi h polyme hylme hac yla e (0.08 m in diame e and 0.20 m heigh ). To acil-
i a e he up-scaling o he p ocess, eac o s we e ope a ed simula ing ou doo aceway
bio eac o s. Two se o expe imen s we e pe o med in iplica e (Figu e 1). In he i s se
o expe imen s, pho obio eac o s we e ope a ed unde ei he ligh o da k condi ions and
we e ed 5- old dilu ed PWW. Cul u es p oduced in ligh o da k condi ions we e e med
L-5 and D-5, espec i ely. The p ocedu e was epea ed bu using 25- old dilu ed PWW as
he cul u e medium. In his case, cul u es p oduced in ligh o da k condi ions we e
e med L-25 and D-25, espec i ely. In bo h cases, he cul u es we e inocula ed wi h
Scenedesmus sp. a an ini ial concen a ion o 0.5 g·L−1 and we e ope a ed in ba ch mode
o 6 days ollowed by ope a ion in con inuous mode by eplacing daily 20% o he cul-
u es olume wi h esh PWW o 10 days, when he s eady s a e was eached. Dissol ed
oxygen (DO) was con olled below 200%Sa by on demand ai supply. The pH was con-
olled a 8.0 ± 0.2 by on-demand injec ion o CO2.
Figu e 1. G aphical desc ip ion o he expe imen s pe o med unde ligh and da k condi ions.
Pho obio eac o s we e a i icially illumina ed using eigh 28 W luo escen ubes
(Philips Dayligh T5), p og ammed o mimic ou doo condi ions: 12 h da k, 12 h ligh wi h
a p og essi e inc ease in ligh in ensi y om 08:00 o 14:00 h. The maximum i adiance
(PAR) inside he eac o s in he absence o cells was 1000 μE·m−2·s −1, measu ed using an
Figu e 1. G aphical desc ip ion o he expe imen s pe o med unde ligh and da k condi ions.
Pho obio eac o s we e a i icially illumina ed using eigh 28 W luo escen ubes
(Philips Dayligh T5), p og ammed o mimic ou doo condi ions: 12 h da k, 12 h ligh wi h
a p og essi e inc ease in ligh in ensi y om 08:00 o 14:00 h. The maximum i adiance
(PAR) inside he eac o s in he absence o cells was 1000
µ
E
·
m
−2·
s
−1
, measu ed using an
SQS-100 sphe ical quan um senso (Walz GmbH, E el ich, Ge many). Tempe a u e was
kep cons an a 25.0 ±1.0 ◦C.
P ocesses 2021,9, 203 4 o 11
2.3. Pho osyn hesis and Respi a ion
A pho o- espi ome e was used o ob ain he mic oalgal ne pho osyn he ic a e and
he bac e ial espi a ion a es in he pho obio eac o s unde di e en ope a ional condi ions.
The equipmen consis ed o an 80 mL jacke ed anspa en cylind ical glass lask, which was
magne ically s i ed and a i icially illumina ed using LED lamps. The pho o- espi ome e
was also equipped wi h senso s o i adiance (QSL-1000, Walz, Ge many), empe a u e (PT-
100, C ison Ins umen s, Ba celona, Spain), pH (C ison 5343; C ison Ins umen s, Ba celona,
Spain), and dissol ed oxygen (C ison 5002; C ison Ins umen s, Ba celona, Spain), as well as
a di use ha allowed o con ol he low a e o gases (ai , O2, N2, and CO2).
The p o ocol and me hodology applied allowed us o dis inguish be ween he
me abolisms o he h ee main popula ions ha appea in mic oalgae-bac e ia was ew-
a e : mic oalgae, he e o ophic bac e ia, and ni i ying bac e ia [
8
]. In he i s place,
mic oalgae-bac e ia cul u es we e subjec ed o nu ien s a a ion (con inuous ligh o 200
µ
E
·
m
−2·
s
−1
and an ae a ion a e o 0.2
·
−1·
min
−1
) du ing 24 h o emo e he o ganic
ma e and he ammonium p esen in he media. Then, cul u e samples we e placed inside
he pho o- espi ome e and subjec ed o ou ligh –da k pe iods o 4 min each while he
a ia ion in DO unde di e en condi ions was measu ed and egis e ed. Du ing he ligh
phases, pho osyn he ic mic oalgae gene a ed oxygen, which was u he consumed by
endogenous espi a ion du ing da kness pe iods. The mic oalgae ne pho osyn hesis a e
was calcula ed as he di e ence be ween he slope o oxygen p oduc ion du ing he ligh
pe iod minus he slope o oxygen consump ion du ing he da k pe iod. In he second
place, cul u e samples we e used o de e mine he he e o ophic espi a ion a e. Fo his
pu pose, 0.8 mL o sodium ace a e (30.0 g
·
L
−1
) we e added o he cul u es be o e being
subjec ed o ou ligh –da k cycles o 4 min each. The espi a ion a e o he he e o ophic
bac e ia was calcula ed as he slope o oxygen consump ion wi h sodium ace a e minus
he slope o he oxygen consump ion du ing he da k pe iod in he endogenous cul u e.
Mo eo e , o de e mine ni i ying ac i i y, 0.8 mL o ammonium chlo ide (3.0 g
·
L
−1
) we e
used as a subs a e. As ammonium chlo ide can be consumed by bo h ni i ying bac e ia
and mic oalgae, wo sepa a e oxygen consump ion a es we e measu ed. The i s one a e
addi ion o ammonium chlo ide alone, and he second one a e addi ion o ammonium
chlo ide and an allyl hiou ea solu ion (ATU), which was used as an ammonia-oxidizing
bac e ia inhibi o . ATU (1.0 g
·
L
−1
) was added un il a concen a ion o 10 mg
·
L
−1
and he
ni i ying espi a ion a e was calcula ed as he di e ence be ween he o al ammonium
chlo ide espi a ion wi hou ATU and he mic oalgae ammonium chlo ide espi a ion a e.
Finally, o co ec he in luence o oxygen deso p ion on he analy ical de e mina ions,
he oxygen mass ans e coe icien was calcula ed using equa ion:
dCO2
d =KLa(C∗
O2−CO2), (1)
whe e
dCO2
d
is oxygen accumula ion exp essed as he de i a e o CO
2
(mg
·
L
−1
) o e ime,
KLa
is he global oxygen mass ans e coe icien (h
−1
), and
C∗
O2
is he oxygen sa u a ion
concen a ion in he cul u e [8].
2.4. Bac e ial Coun s
He e o ophic mic obio a was calcula ed by pla e coun using Nu i i e Aga in he
s eady s a e. An incuba ion ime o 48 h a 30
◦
C was used o es ima e he mesophilic ae obic
mic obio a [
24
]. To al coli o ms and Esche ichia coli in he s eady s a e we e quan i ied.
Samples we e dilu ed in phospha e bu e ed saline solu ion (PBS) o he decimal scale
10
−4
. Each dilu ion was inocula ed in iplica e in o s e ile and disposable Pe i dishes.
Cul u e medium C omocul
®
Coli o m Aga (Me ck KGaA, Ge nsheim, Ge many) was
used. The Pe i dishes we e hen incuba ed unde con olled condi ions a 36
◦
C o 24 h in
he da k. Resul s we e exp essed as CFU
·
mL
−1
. The p esence o Salmonella was e alua ed
by inocula ing 10 mL o each sample in o a lask wi h 50 mL Bu e ed Pep one Wa e (BPW)
o p e-en ichmen a 37
◦
C o 24 h. An aliquo o 0.1 mL was subsequen ly en iched in
P ocesses 2021,9, 203 5 o 11
10 mL o Rappapo Vassiliadis (RV) b o h (Condalab, Mad id, Spain) a 42
◦
C du ing 48 h.
Finally, o assess he p esence o Salmonella-suspec ed colonies, each RV b o h cul u e was
pla ed on o Xylose Lysine Desoxychola e (XLD) (PanReac AppliChem, Ba celona, Spain)
aga and incuba ed a 37 ◦C o 24 h.
2.5. S a is ical Analysis
Resul s a e he a e age o h ee independen expe imen s and a e exp essed as
mean
±
s anda d de ia ion (SD). Di e ences be ween samples we e analysed using analy-
sis o a iance (ANOVA) wi h JMP 13 (SAS Ins i u e Inc., Ca y, NC, USA). A Tukey pai wise
compa ison o he means was conduc ed o iden i y whe e sample di e ences occu ed.
The c i e ion o s a is ical signi icance was p< 0.05.
3. Resul s
3.1. Nu ien Remo al
Mass balances we e conduc ed on he main nu ien s (N-NH
4+
, N-NO
3−
, P-PO
43−
,
and COD) p esen in he eac o s’ inle s and ou le s. The inle concen a ion o N-NH
4+
a ied om 40–50 mg
·
L
−1
in L-25 and D-25 o 290–300 mg
·
L
−1
in L-5 and D-5, espec i ely
(p< 0.05; Figu e 2A). In he s eady-s a e, he N-NH
4+
concen a ions in he ou le o he
eac o s we e 3.4
±
2.5, 3.6
±
1.4, 94.6
±
2.6 mg
·
L
−1
, and 21.1
±
1.4 in L-25, D-25, L-5, and
D-5, espec i ely. N-NH
4+
emo al e iciency was signi ican ly a ec ed by bo h nu ien
concen a ion (p< 0.05) and absence o p esence o ligh (p< 0.05). The depu a ion e iciency
o he N-NH
4+
p esen in he mos dilu ed cul u e media, L-25 and D-25, was g ea e han
92%. The cul u es’ N-NH
4+
consump ion was 8.5
±
0.5 and 8.4
±
0.3 mg
·
L
−1·
day
−1
in
L-25 and D-25, espec i ely. These alues we e lowe han hose ob ained o L-5 and D-5,
which we e 40.5
±
1.1 and 55.3
±
3.7 mg
·
L
−1·
day
−1
, espec i ely (p< 0.05). The highes
N-NH4+ emo al was ob ained in D-5 (p< 0.05).
P ocesses 2021, 9, 203 5 o 11
Cul u e medium C omocul ® Coli o m Aga (Me ck KGaA, Ge nsheim, Ge many) was
used. The Pe i dishes we e hen incuba ed unde con olled condi ions a 36 °C o 24 h
in he da k. Resul s we e exp essed as CFU·mL−1. The p esence o Salmonella was e alu-
a ed by inocula ing 10 mL o each sample in o a lask wi h 50 mL Bu e ed Pep one Wa e
(BPW) o p e-en ichmen a 37 °C o 24 h. An aliquo o 0.1 mL was subsequen ly en-
iched in 10 mL o Rappapo Vassiliadis (RV) b o h (Condalab, Mad id, Spain) a 42 °C
du ing 48 h. Finally, o assess he p esence o Salmonella-suspec ed colonies, each RV b o h
cul u e was pla ed on o Xylose Lysine Desoxychola e (XLD) (PanReac AppliChem, Ba -
celona, Spain) aga and incuba ed a 37 °C o 24 h.
2.5. S a is ical Analysis
Resul s a e he a e age o h ee independen expe imen s and a e exp essed as mean
± s anda d de ia ion (SD). Di e ences be ween samples we e analysed using analysis o
a iance (ANOVA) wi h JMP 13 (SAS Ins i u e Inc., Ca y, NC, USA). A Tukey pai wise
compa ison o he means was conduc ed o iden i y whe e sample di e ences occu ed.
The c i e ion o s a is ical signi icance was p < 0.05.
3. Resul s
3.1. Nu ien Remo al
Mass balances we e conduc ed on he main nu ien s (N-NH4+, N-NO3−, P-PO43−, and
COD) p esen in he eac o s’ inle s and ou le s. The inle concen a ion o N-NH4+ a ied
om 40–50 mg·L−1 in L-25 and D-25 o 290–300 mg·L−1 in L-5 and D-5, espec i ely (p <
0.05; Figu e 2A). In he s eady-s a e, he N-NH4+ concen a ions in he ou le o he eac o s
we e 3.4 ± 2.5, 3.6 ± 1.4, 94.6 ± 2.6 mg·L−1, and 21.1 ± 1.4 in L-25, D-25, L-5, and D-5, espec-
i ely. N-NH4+ emo al e iciency was signi ican ly a ec ed by bo h nu ien concen a-
ion (p < 0.05) and absence o p esence o ligh (p < 0.05). The depu a ion e iciency o he
N-NH4+ p esen in he mos dilu ed cul u e media, L-25 and D-25, was g ea e han 92%.
The cul u es’ N-NH4+ consump ion was 8.5 ± 0.5 and 8.4 ± 0.3 mg·L−1·day−1 in L-25 and D-
25, espec i ely. These alues we e lowe han hose ob ained o L-5 and D-5, which we e
40.5 ± 1.1 and 55.3 ± 3.7 mg·L−1·day−1, espec i ely (p < 0.05). The highes N-NH4+ emo al
was ob ained in D-5 (p < 0.05).
Figu e 2. Inle and ou le concen a ion and emo al/p oduc ion o (A) N-NH4+, (B) N-NO3−, and
(C) P-PO43− in L-25, D-25, L-5, and D-5. Di e en le e s indica e signi ican di e ences.
Figu e 2.
Inle and ou le concen a ion and emo al/p oduc ion o (
A
) N-NH
4+
, (
B
) N-NO
3−
, and
(C) P-PO43−in L-25, D-25, L-5, and D-5. Di e en le e s indica e signi ican di e ences.
The second main ni ogen o m in PWW was N-NO
3−
(Figu e 2B). The inle concen-
a ion o N-NO
3−
in he eac o s a ied om 3.3 mg
·
L
−1
in L-25 and D-25 o 11.3 mg
·
L
−1
in L-5 and D-5, espec i ely (p< 0.05; Figu e 2B). The concen a ion o N-NO
3−
was highe
in he ou le han in he inle (p< 0.05). N-NO
3−
concen a ion in he ou le o he pho o-
bio eac o s was 19.2
±
0.5, 30.2
±
2.5, 23.5
±
3.0, and 29.1
±
2.0 mg
·
L
−1
in L-5, D-5, L-25,

P ocesses 2021,9, 203 6 o 11
and D-25, espec i ely. These alues ep esen a N-NO
3−
p oduc ion o 1.6
±
0.1, 3.8
±
0.5,
1.8
±
1.3, and 1.9
±
0.5 mg
·
L
−1·
day
−1
, espec i ely. N-NO
3−
p oduc ion was especially
highe in D-5 (p< 0.05).
The cu en s udy also de e mined P-PO
43−
in he inle and ou le o he eac o s.
Resul s a e shown in Figu e 2C. Signi ican di e ence in he inle s we e obse ed, being
23.8 mg
·
L
−1
in L-5 and D-5, and 5.0 mg
·
L
−1
in L-25 and D-25, espec i ely (p< 0.05). The P-
PO
43−
emo al a e was calcula ed as 1.9
±
0.1, 0.7
±
0.2, 0.2
±
0.1, and 0.1 mg
·
L
−1·
day
−1
in L-5, D-5, L-25, and D-25, espec i ely. P-PO
43−
concen a ions in he ou le s we e
14.2
±
0.4, 20.3
±
0.9, 4.1
±
0.9, and 5.0
±
0.1 mg
·
L
−1
, espec i ely. In addi ion, P-PO
43−
emo al a es co esponded o consump ion e iciencies o 40 and 15% o L-5 and D-5 and
o 18 and 0% o L25 and D-25, espec i ely.
Finally, he COD concen a ion o he eac o s was also assessed (Figu e 3). L-5 and
D-5 eac o s we e ed wi h 436.3 mg
·
L
−1
while a signi ican ly lowe concen a ion was ed
o L-25 and D-25 eac o s, measu ed as 83.2 mg
·
L
−1
(p< 0.05). COD alues in he ou le s
whe e 352.5
±
14.8, 487.3
±
0.2, 142.1
±
5.6, and 133.5
±
13.4 mg
·
L
−1
in L-5, D-5, L-25, and
D-25. COD consump ion was 16.7
±
3.1 mg
·
L
−1·
day
−1
o L-5 and no COD emo al was
obse ed in D-5, L-25, and D-25. Indeed, o hese eac o s, he ou le COD concen a ion
was highe han in he inle (p< 0.05).
P ocesses 2021, 9, 203 6 o 11
The second main ni ogen o m in PWW was N-NO3− (Figu e 2B). The inle concen-
a ion o N-NO3− in he eac o s a ied om 3.3 mg·L−1 in L-25 and D-25 o 11.3 mg·L−1 in
L-5 and D-5, espec i ely (p < 0.05; Figu e 2B). The concen a ion o N-NO3− was highe in
he ou le han in he inle (p < 0.05). N-NO3− concen a ion in he ou le o he pho obio e-
ac o s was 19.2 ± 0.5, 30.2 ± 2.5, 23.5 ± 3.0, and 29.1 ± 2.0 mg·L−1 in L-5, D-5, L-25, and D-25,
espec i ely. These alues ep esen a N-NO3− p oduc ion o 1.6 ± 0.1, 3.8 ± 0.5, 1.8 ± 1.3,
and 1.9 ± 0.5 mg·L−1·day−1, espec i ely. N-NO3− p oduc ion was especially highe in D-5
(p < 0.05).
The cu en s udy also de e mined P-PO43− in he inle and ou le o he eac o s. Re-
sul s a e shown in Figu e 2C. Signi ican di e ence in he inle s we e obse ed, being 23.8
mg·L−1 in L-5 and D-5, and 5.0 mg·L−1 in L-25 and D-25, espec i ely (p < 0.05). The P-PO43−
emo al a e was calcula ed as 1.9 ± 0.1, 0.7 ± 0.2, 0.2 ± 0.1, and 0.1 mg·L−1·day−1 in L-5, D-
5, L-25, and D-25, espec i ely. P-PO43− concen a ions in he ou le s we e 14.2 ± 0.4, 20.3 ±
0.9, 4.1 ± 0.9, and 5.0 ± 0.1 mg·L−1, espec i ely. In addi ion, P-PO43− emo al a es co e-
sponded o consump ion e iciencies o 40 and 15% o L-5 and D-5 and o 18 and 0% o
L25 and D-25, espec i ely.
Finally, he COD concen a ion o he eac o s was also assessed (Figu e 3). L-5 and
D-5 eac o s we e ed wi h 436.3 mg·L−1 while a signi ican ly lowe concen a ion was ed
o L-25 and D-25 eac o s, measu ed as 83.2 mg·L−1 (p < 0.05). COD alues in he ou le s
whe e 352.5 ± 14.8, 487.3 ± 0.2, 142.1 ± 5.6, and 133.5 ± 13.4 mg·L−1 in L-5, D-5, L-25, and D-
25. COD consump ion was 16.7 ± 3.1 mg·L−1·day−1 o L-5 and no COD emo al was ob-
se ed in D-5, L-25, and D-25. Indeed, o hese eac o s, he ou le COD concen a ion
was highe han in he inle (p < 0.05).
Figu e 3. Inle and ou le concen a ion and emo al/p oduc ion o COD in L-25, D-25, L-5, and D-5.
3.2. Respi ome ic Analysis
The ne pho osyn he ic a e was 15.3 ± 0.7, 1.1 ± 0.5, 6.7 ± 0.8, and 0.3 ± 0.2 mg·L−1·h −1
in L-5, D-5, L-25, and D-25, espec i ely. Ne pho osyn hesis was signi ican ly a ec ed by
bo h nu ien concen a ion (p < 0.05) and absence o p esence o ligh (p < 0.05). Bo h e-
ac o s ope a ing unde ligh condi ions showed a highe pho osyn he ic a e, being highe
in L-5 han in L-25, despi e o a simila biomass concen a ion (Figu e 4A). The he e o-
ophic bac e ia espi a ion a e was 1.35 ± 0.11, 1.54 ± 0.21, 0.26 ± 0.12, and 0.33 ± 0.13
mg·L−1·h −1 in L-5, D-5, L-25, and D-25 (Figu e 4B). He e o ophic ac i i y in L-25 was 5-
old lowe han in L-5 (p < 0.05). The espi a ion a e o ni i ying bac e ia was 1.4 ± 0.2,
2.5 ± 0.1, 0.5 ± 0.1, and 0.4 ± 0.1 mg·L−1·h −1 in L-5, D-5, L-25, and D-25. Highe oxygen con-
sump ions we e obse ed o samples dilu ed 5- old when compa ed o 25- old, being
highe in D-5 han in L-5 (p < 0.05).
Figu e 3.
Inle and ou le concen a ion and emo al/p oduc ion o COD in L-25, D-25, L-5, and D-5.
3.2. Respi ome ic Analysis
The ne pho osyn he ic a e was 15.3
±
0.7, 1.1
±
0.5, 6.7
±
0.8, and 0.3
±
0.2 mg
·
L
−1·
h
−1
in L-5, D-5, L-25, and D-25, espec i ely. Ne pho osyn hesis was signi ican ly a ec ed
by bo h nu ien concen a ion (p< 0.05) and absence o p esence o ligh (p< 0.05). Bo h
eac o s ope a ing unde ligh condi ions showed a highe pho osyn he ic a e, being
highe in L-5 han in L-25, despi e o a simila biomass concen a ion
(Figu e 4A).
The
he e o ophic bac e ia espi a ion a e was 1.35
±
0.11, 1.54
±
0.21, 0.26
±
0.12, and
0.33
±
0.13 mg
·
L
−1·
h
−1
in L-5, D-5, L-25, and D-25 (Figu e 4B). He e o ophic ac i i y in
L-25 was 5- old lowe han in L-5 (p< 0.05). The espi a ion a e o ni i ying bac e ia was
1.4
±
0.2, 2.5
±
0.1, 0.5
±
0.1, and 0.4
±
0.1 mg
·
L
−1·
h
−1
in L-5, D-5, L-25, and D-25. Highe
oxygen consump ions we e obse ed o samples dilu ed 5- old when compa ed o 25- old,
being highe in D-5 han in L-5 (p< 0.05).
P ocesses 2021,9, 203 7 o 11
P ocesses 2021, 9, 203 7 o 11
Figu e 4. (A) Mic oalgae ac i i y a di e en in luen concen a ion and unde ligh /da k condi-
ions. (B) He e o ophic and ni i ying ac i i y a he di e en expe imen s. Di e en le e s indi-
ca e signi ican di e ences.
3.3. Mic obiologic Analysis
He e o ophic bac e ial coun s we e 2.35 × 105, 1.35 × 105, 2.5 × 105, and 1.75 × 104
CFU·mL−1 in L-5, D-5, L-25, and D-25, espec i ely (Table 2). Coli o ms we e 7.8 × 101 and
4.11 × 102 CFU·mL−1 in L-5 and D-5, espec i ely, and 2.6 × 101 and 2.05 × 102 CFU·mL−1 in
L-25 and D-25. Mo eo e , E. coli and Salmonella sp. we e no de ec ed (ND) in any sample.
Table 2. Mic obial popula ion coun s du ing he expe imen s. Da a a e exp essed as CFU·L−1.
L-25
D-25
L-5
D-5
He e o ophic bac e ia
2.50 × 105
1.75 × 104
2.35 × 105
1.35 × 105
Coli o ms bac e ia
2.60 × 101
2.05 × 102
7.80 × 101
4.11 × 102
E. coli
ND
ND
ND
ND
Salmonella sp.
ND
ND
ND
ND
4. Discussion
Nu ien emo al om was e s eams using mic oalgae-bac e ia conso ia has been
widely s udied du ing he las couple o decades. This app oach has been p oposed as he
key s a egy o educe mic oalgal biomass p oduc ion cos s o unde 1–2 €·kg−1 [24]. In-
es iga ions on mic oalgae-based bio emedia ion led o he unde s anding ha nu ien
emo al is caused by assimila ion, anae obic ammonia oxida ion, ni i ica ion, and deni-
i ica ion, among o he p ocesses. Howe e , li le is known abou he speci ic con ibu-
ion o mic oalgae o he p ocess and hei e ec on he sys ems pe o mance [25]. The
cu en s udy aimed a unde s anding he in luence o pho osyn he ic ac i i y on nu ien
consump ion du ing PWW ea men . Resul s, shown in Figu e 1, demons a ed ha he
mic oalgae-bac e ia conso ia allowed us o achie e high N-NH4+ emo al a es. The ma-
jo emo al a es we e obse ed in samples L-5 and D-5, a ibu ed o highe N-NH4+ con-
en in he inle . Almos a comple e ammonia emo al was obse ed in L-25 and D-25.
Howe e , pa o ni ogen in he o m o N-NH4+ was con e ed o N-NO3− by he ac ion
o ni i ying bac e ia, ob aining highe con en o N-NO3− in he ou le s han in he inle s.
In he eac o s ope a ed unde ligh condi ions, he assimila ion o N-NH4+ is caused by
bo h mic oalgae and ni i ying bac e ia. Mic oalgae use N-NH4+ o p oduce biomass while
ni i ying bac e ia use i o g ow h and o ca y ou he i s s ep o ni i ica ion. In he
cu en s udy, he ni i ying ac i i y p edomina ed in he eac o ope a ing unde da k
condi ions since he pho o ophic ac i i y is negligible. As a esul , he con en o N-NO3−
in he ou le o eac o s unde ligh condi ions is lowe when compa ed o he sys ems
ha we e main ained in da k, demons a ing a lowe N-NO3− p oduc ion. These indings
can be a ibu ed o wo main ac o s: (i) mic oalgal g ow h educes AOB popula ions,
and (ii) mic oalgae a e capable o assimila ing he N-NO3− p oduced du ing ni i ica ion.
The la e is less p obable because p e ious epo s sugges ed ha when N-NH4+ and N-
Figu e 4.
(
A
) Mic oalgae ac i i y a di e en in luen concen a ion and unde ligh /da k condi ions. (
B
) He e o ophic and
ni i ying ac i i y a he di e en expe imen s. Di e en le e s indica e signi ican di e ences.
3.3. Mic obiologic Analysis
He e o ophic bac e ial coun s we e 2.35
×
10
5
, 1.35
×
10
5
, 2.5
×
10
5
, and 1.75
×
10
4
CFU
·
mL
−1
in L-5, D-5, L-25, and D-25, espec i ely (Table 2). Coli o ms we e 7.8
×
10
1
and
4.11
×
10
2
CFU
·
mL
−1
in L-5 and D-5, espec i ely, and 2.6
×
10
1
and 2.05
×
10
2
CFU
·
mL
−1
in L-25 and D-25. Mo eo e , E. coli and Salmonella sp. we e no de ec ed (ND) in any
sample.
Table 2. Mic obial popula ion coun s du ing he expe imen s. Da a a e exp essed as CFU·L−1.
L-25 D-25 L-5 D-5
He e o ophic bac e ia 2.50 ×1051.75 ×1042.35 ×1051.35 ×105
Coli o ms bac e ia 2.60 ×1012.05 ×1027.80 ×1014.11 ×102
E. coli ND ND ND ND
Salmonella sp. ND ND ND ND
4. Discussion
Nu ien emo al om was e s eams using mic oalgae-bac e ia conso ia has been
widely s udied du ing he las couple o decades. This app oach has been p oposed as
he key s a egy o educe mic oalgal biomass p oduc ion cos s o unde 1–2
€·
kg
−1
[
24
].
In es iga ions on mic oalgae-based bio emedia ion led o he unde s anding ha nu i-
en emo al is caused by assimila ion, anae obic ammonia oxida ion, ni i ica ion, and
deni i ica ion, among o he p ocesses. Howe e , li le is known abou he speci ic con i-
bu ion o mic oalgae o he p ocess and hei e ec on he sys ems pe o mance [
25
]. The
cu en s udy aimed a unde s anding he in luence o pho osyn he ic ac i i y on nu ien
consump ion du ing PWW ea men . Resul s, shown in Figu e 1, demons a ed ha he
mic oalgae-bac e ia conso ia allowed us o achie e high N-NH
4+
emo al a es. The
majo emo al a es we e obse ed in samples L-5 and D-5, a ibu ed o highe N-NH
4+
con en in he inle . Almos a comple e ammonia emo al was obse ed in L-25 and D-25.
Howe e , pa o ni ogen in he o m o N-NH
4+
was con e ed o N-NO
3−
by he ac ion
o ni i ying bac e ia, ob aining highe con en o N-NO
3−
in he ou le s han in he inle s.
In he eac o s ope a ed unde ligh condi ions, he assimila ion o N-NH
4+
is caused by
bo h mic oalgae and ni i ying bac e ia. Mic oalgae use N-NH
4+
o p oduce biomass while
ni i ying bac e ia use i o g ow h and o ca y ou he i s s ep o ni i ica ion. In he
cu en s udy, he ni i ying ac i i y p edomina ed in he eac o ope a ing unde da k
condi ions since he pho o ophic ac i i y is negligible. As a esul , he con en o N-NO
3−
in he ou le o eac o s unde ligh condi ions is lowe when compa ed o he sys ems
ha we e main ained in da k, demons a ing a lowe N-NO
3−
p oduc ion. These indings
can be a ibu ed o wo main ac o s: (i) mic oalgal g ow h educes AOB popula ions,
and (ii) mic oalgae a e capable o assimila ing he N-NO
3−
p oduced du ing ni i ica ion.
The la e is less p obable because p e ious epo s sugges ed ha when N-NH
4+
and
P ocesses 2021,9, 203 8 o 11
N-NO
3−
a e bo h p esen in he media, mic oalgae gene ally p e e he o me [
26
,
27
].
PWW ea men p ocesses allow adequa e N-NH
4+
emo al a es bu lead o an inc ease in
he p oduc ion o N-NO
3−
and, he e o e, o a loss o nu ien s. The use o mic oalgae in
was ewa e ea men p ocesses could be e y a ou able as hese nu ien s could be used
o mic oalgal biomass p oduc ion. Ope a ing unde ligh condi ions, when mic oalgal
pho o ophic ac i i y is enhanced, allowed us o a oid high nu ien losses and o ob ain
highe nu ien emo al a es [10].
The phospho us emo al a es epo ed show ha mic oalgal pho o ophic ac i i y
(L-25 and L-5) inc eased phospho ous consump ion om PWW. These alues we e lowe
when compa ed o hose epo ed in a p e ious s udy du ing he ea men o 10- and
20- old dilu ed PWW unde indoo and ou doo condi ions (81–99%) [
4
]. Howe e , in
ha s udy, he au ho s ope a ed wi h an hyd aulic e en ion ime (26 days) highe han
he one assessed in he cu en s udy (5 days), and i is accep ed ha ope a ional con-
di ions ha e a signi ican impac on biomass p oduc i i y and nu ien emo al a es,
especially p ocess du a ion [
10
]. In he cu en s udy, phospho ous consump ion in L-5
was almos wice he alue o L-25. To explain his di e ence, i is impo an o highligh
ha e alua ing phospho ous up ake in mic oalgae-bac e ia based sys ems is pa icula ly
di icul . Phospho us emo al is in luenced by mul iple en i onmen al ac o s such as
empe a u e o pho ope iod [
28
]. Indeed, highe phospho us emo al a es we e epo ed
in summe han in win e [
29
]. Mo eo e , luxu y phospho ous up ake phenomena has been
epo ed a high phospha e concen a ions in a mixed mic oalgal conso ium domina ed
by Scenedesmus [
30
]. In his case, when phospha e aqueous concen a ion inc eased om
5 o 15 mg
·
L
−1
, he mic oalgal acid soluble polyphospha e con en inc eased up o h ee
imes [
30
]. In he expe imen s p esen ed in his wo k, he en i onmen al condi ions such as
empe a u e and ligh we e kep cons an . Thus, his di e ence in phospho us consump ion
could be a ibu ed o he phenomenon o luxu y up ake since he biomass concen a ion
eached by L-25 and L-5 was simila (a ound 0.6 g
·
L
−1
). The e o e, phospho us emo al
in mic oalgae-bac e ia conso ia in ol e phenomena including he assimila ion by bo h
mic oalgae and bac e ia o o m biomass and in acellula polyphospha e compounds and
also phospho ous p ecipi a ion a high pH alues (i i is no con olled) [
31
]. Phospho ous
assimila ion in o algal-bac e ial biomass was likely he main emo al mechanism based on
he adequa e con olled pH alues p e ailing in he pho obio eac o s (pH = 8.0), which
a oided phospha e p ecipi a ion [32].
The COD emo al ob ained in he expe imen s was pa icula ly low. COD emo al
was only obse ed in L-5, allowing a emo al a e o 20%. In his con ex , he ac ion o
eadily biodeg adable o ganic ca bon in PWW in luenced he COD emo al, and di icul ed
he in e -s udies compa ison. Mo eo e , he biodeg adabili y ange om 0% o 80% in
PWW due o a m swine manu e managemen p ac ices such as shed cleansing o was e
s o age condi ions [
33
]. In he cu en s udy, he PWW used was kep in a s o o e a
yea , and he e o e, mos o he o ganic ma e p esen could be no eadily biodeg adable.
A espi ome ic me hodology was used o assess he main mic obial me abolisms
ha appea ed in mic oalgae-bac e ia cul u es unde di e en PWW concen a ions and
ligh /da k condi ions: mic oalgae, he e o ophic bac e ia, and ni i ying bac e ia. Resul s
showed ha mic oalgae ac i i y unde da k condi ions was especially low, esul ing in a
minimal pho osyn he ic ac i i y due o he esidual mic oalgal cells in he pho obio eac o s.
Al hough a highe ac i i y was expec ed in L-25 han in L-5, since ammonium concen a-
ions abo e 100 mgN
·
L
−1
ha e been epo ed as inhibi o y o mic oalgae cul u es [
34
],
esul s showed he opposi e e ec . The ne pho osyn hesis in he eac o s unde ligh
condi ions di e ed signi ican ly be ween 5 and 25 imes dilu ed PWW. The g ea e alue
was obse ed o L-5, despi e a simila biomass concen a ion being achie ed in bo h assays.
The obse ed dec ease in pho osyn he ic ac i i y could ha e been caused by a limi a ion o
mic onu ien s, which we e p esen in e y low concen a ions in L-25 and D-25. P e ious
au ho s desc ibed ha mic o-elemen s (such as i on and manganese) ha e an impo an
ole on he g ow h and pho osyn he ic elec on anspo o mic oalgae [
35
,
36
]. I on is
P ocesses 2021,9, 203 9 o 11
an essen ial elemen o pho osyn hesis and espi a ion in mic oalgae, whose g ow h is
o en limi ed due o he poo i on solubili y [
37
]. In na u al en i onmen s, many he -
e o ophic bac e ia p oduce side opho es, small o ganic molecules ha igh ly bind o i on
and he eby inc ease i s solubili y. The e o e, he e o ophic bac e ia can solubilize i on,
which could be a ailable o mic oalgae because, o da e, mic oalgae we e no epo ed
as side opho e p oduce s [
38
]. Thus, he low pho osyn he ic ac i i y in L-25 could ha e
been caused by a low he e o ophic ac i i y. He e o ophic ac i i y in L-25 was i e imes
lowe han he he e o ophic espi a ion measu ed in L-5. In u n, he low he e o ophic
ac i i y de ec ed in L-25 could be explained by he limi ed biodeg adable o ganic ma e
measu ed in he samples. Rela ed o he espi a ion a e o ni i ying bac e ia measu ed by
espi ome ic echniques, esul s show ha a es unde ligh and da k condi ions did no
di e signi ican ly when PWW dilu ed 25 imes was used, which is in line wi h p e ious
epo s [
39
]. Howe e , ni i ying ac i i y a ied be ween ligh and da k condi ions when
he s i ed- ank eac o s we e ed wi h PWW dilu ed 5 imes. This a iabili y may be
he esul o he high mic oalgae ac i i y measu ed in L-5, which could compe e o he
ammonium p esen in he medium wi h ammonium oxidizing bac e ia [11].
He e o ophic bac e ia include all bac e ia ha use o ganic nu ien s o g ow h.
These bac e ia a e na u al inhabi an s o ood, ai , animal/human body, and all ypes o
wa e . Wi hin his g oup, bo h bac e ial pa hogens and coli o ms (Esche ichia,Klebsiella,
En e obac e ,Ci obac e , and Se a ia) a e included [
40
]. He e o ophic pla e coun (HPC)
can be used o de ec ion o all bac e ia ha consume o ganic compounds, bu canno
be used as indica o s o pa hogenic condi ions. In he samples, he e o ophic bac e ia,
coli o ms bac e ia, E. coli, and Salmonella sp. we e measu ed in he ou le s, a e emo ing
he mic oalgae-bac e ia biomass. Resul s sugges ed ha he mic oalgae-bac e ia cul u es
unde ligh condi ions, when mic oalgae pho o ophic ac i i y was enhanced, p esen ed a
g ea e numbe o he e o ophic bac e ia. This di e ence can be due o di e en ac o s. On
he one hand, he use o mic oalgae in was ewa e ea men in ol es many associa ions
wi h o he mic oo ganisms p esen in was ewa e . These associa ions ha e been desc ibed
in he phycosphe e, he mic oscale a ea su ounding mic oalgae cells whe e me aboli es
a e exchanged be ween mic oalgae and bac e ia [
41
]. The phycosphe e is equi alen o
an “oasis” o he e o ophic bac e ia, whe e high concen a ions o ixed o ganic ca bon is
exc e ed o consump ion [
42
]. On he o he hand, he s i ed- ank eac o s ope a ed unde
ligh condi ions achie ed alues o dissol ed oxygen up o 200%, which can be consumed
by he e o ophic bac e ia. The e o e, inc easing o pho o ophic ac i i y could ha e led
o an inc ease in he e o ophic bac e ia, because hey o m conso ia ha a ou nu ien
emo al and biomass p oduc ion. Mo eo e , esul s sugges ed ha mic oalgae ac i i y
allowed o he educ ion o he con en o coli o m bac e ia as lowe coli o m bac e ia
we e ound in he eac o s ope a ed unde ligh condi ions. This was in line wi h p e ious
publica ions ha desc ibed ha he en i onmen al ac o s ha a e a ou able o algal
g ow h a e un a ou able o he su i al o coli o ms [43].
5. Conclusions
This wo k demons a ed ha he pho osyn he ic ac i i y o mic oalgae allows us o
imp o e he nu ien emo al a es in PWW and o educe he coli o m bac e ial load
o he e luen s. This was mainly caused by mic oalgae, which allowed N-NH
4+
assim-
ila ion ins ead o con e ing i in o N-NO
3−
, which occu s in adi ional PWW due o
he oxidizing ammonium ac i i y. Mic oalgae u ilisa ion also led o a educ ion o he
phospho us p esen in he PWW due o i s assimila ion in o mic oalgal biomass. The
mic oalgae-bac e ia conso ia enhanced bo h he ac i i y o mic oalgae ha mainly con-
sumed he N and P p esen in he PWW and he ac i i y o he e o ophic bac e ia ha
consumed o ganic ma e . Fu he s udies will include he up-scaling o he p ocess ou -
doo s and a comple e cha ac e isa ion o he mic oo ganisms p esen in he conso ia using
me agenomic analyses.