INTERNATIONAL DOCTORAL
SCHOOL OF THE USC
Da id Manuel
Kennes Veiga
PhD Thesis
Unde s anding he enzyma ic
and come abolic
bio ans o ma ion o o ganic
mic opollu an s unde ae obic
he e o ophic condi ions
San iago de Compos ela, 2022
Doc o al P og amme in Chemical and En i onmen al Enginee ing
TESIS DE DOCTORADO
Unde s anding he enzyma ic and
come abolic bio ans o ma ion o o ganic
mic opollu an s unde ae obic he e o ophic
condi ions
Da id Manuel Kennes Veiga
ESCUELA DE DOCTORADO INTERNACIONAL DE LA UNIVERSIDAD DE SANTIAGO DE COMPOSTELA
PROGRAMA DE DOCTORADO EN INGENIERÍA QUÍMICA Y AMBIENTAL
SANTIAGO DE COMPOSTELA
2022
DECLARACIÓN DEL AUTOR/A DE LA TESIS
D./Dña.
Da id M. Kennes Veiga
Tí ulo da ese:
Unde s anding he enzyma ic and come abolic bio ans o ma ion o o ganic
mic opollu an s unde ae obic he e o ophic condi ions
P esen o mi esis, siguiendo el p ocedimien o adecuado al Reglamen o y decla o que:
1) La esis aba ca los esul ados de la elabo ación de mi abajo.
2) De se el caso, en la esis se hace e e encia a las colabo aciones que u o es e abajo.
3) Con i mo que la esis no incu e en ningún ipo de plagio de o os au o es ni de abajos
p esen ados po mí pa a la ob ención de o os í ulos.
4) La esis es la e sión de ini i a p esen ada pa a su de ensa y coincide la e sión imp esa con la
p esen ada en o ma o elec ónico.
Y me comp ome o a p esen a el Comp omiso Documen al de Supe isión en el caso que el o iginal no
es é deposi ado en la Escuela.
En San iago de Compos ela, 21 de diciemb e de 2021.
Fi ma elec ónica
AUTORIZACIÓN
DE LOS
DIRECTORES
/
TUTORES DE LA TESIS
Unde s anding he enzyma ic and come abolic
bio ans o ma ion o o ganic mic opollu an s unde
ae obic he e o ophic condi ions
D. Juan M. Lema Rodicio, Ca ed á ico de Ingenie ía Química
Dña. Ma a Ca balla A cos, P o eso a Con a ada Doc o a
INFORMAN:
Que la p esen e esis, se co esponde con el abajo ealizado po D. Da id M. Kennes Veiga, bajo nues a
di ección/ u o ización, y a
u o izamos
su
p esen ación, conside ando
que eúne l os
equisi os
exigidos
en el R
eglamen o
de Es udios de
Doc o ado de la USC,
y
que
como di ec o es de es a no incu e
en las causas de
abs ención es ablecidas
en la Ley 40/2015.
De acue do con lo indicado en el Reglamen o de Es udios de Doc o ado, decla amos ambién que la
p esen e esis doc o al es idónea pa a se de endida en base a la modalidad de Monog á ica con
ep oducción de publicaciones, en los que la pa icipación del doc o ando ue decisi a pa a su elabo ación
y las publicaciones se ajus an al Plan de In es igación.
En San iago de Compos ela, 21 de diciemb e de 2021
AGRADECIMIENTOS
La p esen e esis se ha podido lle a a cabo g acias a los p oyec os de in es igación
COMETT (CTQ2016-80847-R) y ANTARES (PID2019-110346RB-C21) y a la ayuda
de apoyo a la e apa p edoc o al de la Xun a de Galicia (ED481A-2018/113).
En p ime luga , me gus a ía ag adece a mis di ec o es de esis, Juan y Ma a, po
habe me dado la opo unidad de ealiza es e doc o ado. Vues os consejos, apoyo y
con ianza han sido undamen ales pa a desa olla es a esis y supe a los con a iempos
que se han ido p esen ando du an e el camino. He ap endido y e olucionado mucho
du an e es os años, an o a ni el pe sonal como p o esional, y en g an pa e se debe a
ues a supe isión. Muchas g acias.
I would also like o hank Ka h in Fenne o accep ing me o a doc o al s ay a he
EAWAG, in he Depa men o En i onmen al Chemis y. I eally enjoyed hose 6
mon hs, I lea n a lo and I could unde s and a li le be e he bio ans o ma ion
mechanisms o he mic opollu an s. I am also e y g a e ul o he momen s I sha ed wi h
he people o he Depa men , especially wi h Be nade e and Anas asia, who g ea ly
helped me in he adap a ion p ocess o a new g oup and coun y.
Me sien o muy a o unado po habe podido compa i es e doc o ado con gen e
ma a illosa del Biog oup, que me ayudó y acompañó en es a a en u a. G acias a Lo ena
po sus enseñanzas y colabo ación du an e el doc o ado; a Sabela y Alba po aden a me
en el boni o mundo de la mic obiología; y a Ma , Mónica y Rosa po acili a
eno memen e el desa ollo de es a esis. Quie o ag adece ambién a C is ina su excelen e
labo como men o a, o eciéndome siemp e sus consejos y apoyo. Además, ha sido un
place y una sue e pode compa i es os años con Miguel, And és, Paula, Chechu, Lucía,
Ma ías, Alba, Ricky… Mis mejo es y más aleg es ecue dos han enido de ues a mano
en los innume ables ca és, cha las, comidas, iajes…
Finalmen e, muchas g acias a mis pad es po habe me inculcado el amo po la
ciencia y la in es igación y po habe me animado y acompañado du an e el doc o ado; y
a Ma ía, mi he mana, po se la aleg ía de la casa y ae siemp e los momen os más
di e idos. Y, po supues o, ambién a Sa a, po sus incondicionales ánimos, paciencia,
comp ensión y ca iño que han sido an impo an es pa a llega has a aquí.
Sin odos oso os, la culminación de es a esis hab ía sido mucho más di ícil, po lo
que el éxi o de es e abajo ambién es ues o. ¡Muchas g acias!
2.3.4. Dissol ed oxygen, empe a u e and pH .................................................................................. 55
2.3.5. O ganic Mic opollu an s.......................................................................................................... 55
2.3.6. T ans o ma ion p oduc analysis and s uc u e elucida ion .................................................... 57
2.3.7. DNA ........................................................................................................................................ 58
2.3.8. P o eomics............................................................................................................................... 59
2.3.8.1. P o eome ex ac ion......................................................................................................... 59
2.3.8.2. P o ein de ec ion.............................................................................................................. 59
2.3.8.3. P o ein da a analysis........................................................................................................ 60
2.4. CALCULATIONS............................................................................................................60
2.4.1. Calcula ions o eac o pe o mance ....................................................................................... 60
2.4.2. OMPs mass balance................................................................................................................. 61
CHAPTER 3. The o ganic loading a e a ec s o ganic mic opollu an s’ come abolic
bio ans o ma ion kine ics unde he e o ophic condi ions in ac i a ed sludge.....65
3.1. INTRODUCTION............................................................................................................66
3.2. MATERIALS AND METHODS .....................................................................................67
3.3. RESULTS AND DISCUSSION.......................................................................................68
3.3.1. Bio ans o ma ion yield........................................................................................................... 68
3.3.2. Speci ic bio ans o ma ion a e ............................................................................................... 70
3.3.3. In luence o he e o ophic ac i i y on he OMPs bio ans o ma ion cons an ........................ 73
3.4. CONCLUSION ................................................................................................................80
CHAPTER 4. He e o ophic enzyma ic bio ans o ma ions o o ganic
mic opollu an s in ac i a ed sludge .............................................................................81
4.1. INTRODUCTION............................................................................................................82
4.2. MATERIALS AND METHODS .....................................................................................82
4.3. RESULTS AND DISCUSSION.......................................................................................83
4.3.1. Reac o pe o mance ............................................................................................................... 83
4.3.2. He e o ophic OMPs bio ans o ma ion in ac i a ed sludge sys ems ..................................... 86
4.3.3. Iden i ica ion o enzyma ic bio ans o ma ions o OMPs in ae obic he e o ophic condi ions
........................................................................................................................................................... 89
4.4. CONCLUSION ................................................................................................................96
CHAPTER 5. Sul ame hoxazole igge s speci ic enzyma ic ac i i ies unde ae obic
he e o ophic condi ions: A me ap o eomic app oach ..............................................97
5.1. INTRODUCTION............................................................................................................98
5.2. MATERIALS AND METHODS .....................................................................................98
5.3. RESULTS AND DISCUSSION.......................................................................................99
5.3.1. SMX bio ans o ma ion unde ae obic he e o ophic condi ions............................................ 99
5.3.2. TPs iden i ica ion unde ae obic he e o ophic condi ions.................................................... 101
5.3.3. Impac o SMX on mic obial communi y s uc u e .............................................................. 102
5.3.4. Impac o SMX on he me ap o eome................................................................................... 105
5.4. CONCLUSION ..............................................................................................................110
CHAPTER 6. GENERAL DISCUSSION AND CONCLUSIONS .........................111
6.1. MAIN OUTCOMES OF THE THESIS.........................................................................112
6.1.1. A e ae obic he e o ophs e ec i e o bio ans o m OMPs? ................................................. 113
6.1.2. T ans o ma ion p oduc s as a ool o iden i y key enzyma ic ac i i ies................................ 114
6.1.3. Omic echniques as a ool o iden i y key mic obial and enzyma ic playe s........................ 115
6.2. MAIN GAPS AND FUTURE PERSPECTIVES...........................................................117
REFERENCES ............................................................................................................121
LIST OF PUBLICATIONS........................................................................................145
1
LIST OF ACRONYMS
ADBI: Celes olide
AEBSF: 4-benzenesul onyl luo ide hyd ochlo ide
AHTN: Tonalide
AMO: Ammonia monooxygenase
AOA: Ammonia oxidizing a chaea
AOB: Ammonia oxidizing bac e ia
ATU: Allyl hiou ea
BES: 2-b omoe hanesul ona e
BPA: Bisphenol A
CBZ: Ca bamazepine
COD: Chemical oxygen demand
DCF: Diclo enac
DZP: Diazepam
E1: Es one
E2: 17E-es adiol
EE2: 17D-e hinyles adiol
ERY: E y h omycin
FAS: Fe ous ammonium sulpha e
FLX: Fluoxe ine
HHCB: Galaxolide
HRT: Hyd aulic e en ion ime
IBP: Ibup o en
LOQ: Limi o quan i ica ion
NED: N-(1-naph hyl)e hylenediamine
NOB: Ni i e-oxidizing bac e ia
NP: 4-nonylphenol
NPX: Nap oxen
OCT: Oc yne
OLR: O ganic loading a e
OMP: O ganic mic opollu an
OP: 4-oc ylphenol
ROX: Roxi h omycin
2
SPE: Solid phase ex ac ion
SMX: Sul ame hoxazole
SRT: Sludge e en ion ime
TCA: T ica boxylic acid
TCS: T iclosan
TMP: T ime hop im
TP: T ans o ma ion p oduc
TSS: To al suspended solids
USE: Ul asonic sol en ex ac ion
VSS: Vola ile suspended solids
WWTP: Was ewa e ea men plan
RESUMO
RESUMO
Resumo
4
Capí ulo 1. In odución e con ex o
Os mic ocon aminan es o gánicos (MCOs) ep esen an unha ex ensa gama de
sus ancias de consumo c ecen e en odo o mundo que inclúe p odu os a macéu icos, de
uso pe soal e ho monas, en e ou os. Es es compos os, a pesa de es a p esen es en
concen acións moi baixas, no ango de ng a μg L-1, supoñen un isco an o pa a o
medioambien e como pa a a saúde humana a causa da súa pe sis encia, bioacumulación
e ac i idade biolóxica. Ademais, poden acen ua enómenos de oxicidade,
mu axenicidade, esis encia an ibió ica e es es oxida i o, así como causa deso des dos
sis emas ne ioso, ho monal e ep odu i o.
Os MCOs ema an nas es acións depu ado as de augas esiduais (EDAR), que os
eciben a a és de di e en es u as, como nas augas esiduais u banas, hospi ala ias e
indus iais. Non obs an e, a pesa de se capaces de educi as concen acións de moi os
compos os, as EDAR non o on deseñadas pa a elimina os MCOs, que ecuen emen e
son emi idos ao medioambien e sen a a . Ademais, as aplicacións, p opiedades ísico-
químicas e pe sis encia dos MCOs é moi ampla e as condicións medioambien ais e de
p oceso aplicadas (como empe a u a, pH, comunidade mic obiana ou po encial edox,
en e ou os) son undamen ais pa a educi a súa concen ación, o que da luga a g andes
di e enzas na súa p esenza e e iciencia de eliminación nas EDAR.
A eliminación dos MCOs nos p ocesos biolóxicos das EDAR oco e p incipalmen e
po (i) ola ilización, (ii) so ción ao lodo e (iii) bio ans o mación, aínda que pa a algúns
compos os amén se poden p oduci p ocesos de ans o mación abió ica. A e apa de
bio ans o mación es á conside ada como o mecanismo p incipal xa que a ola ilización
e a so ción soamen e son ele an es pa a compos os que p esen an al os alo es da
cons an e da lei de Hen y e do coe icien e de pa ición sólido-líquido, espec i amen e.
Di a bio ans o mación pode p oduci se me abólicamen e se a ene xía xe ada a pa i
da eacción de ans o mación dos MCOs é su icien e pa a p omo e o c ecemen o e
man emen o celula . Sen emba go, a causa das baixas concen acións ás que es án
p esen es os MCOs nas EDAR, a bio ans o mación xe almen e se p oduce
come abólicamen e. Du an e o come abolismo, os mic oo ganismos ans o man
compos os que non son de c ecemen o e non poden se u ilizados como única on e de
nu ien es e ene xía na p esencia ob igada dun subs a o p ima io. Es e úl imo a ópase a
maio es concen acións no medio, se e como doado de elec óns e pode man e a
Resumo
5
poboación mic obiana e induci a p odución de enzimas e co ac o es que son capaces de
bio ans o ma os MCOs g azas a súa e sá il ac i idade ca alí ica e a simili udes en e a
es u u a química dos MCOs e a dos subs a os na u ais da enzima.
Os p ocesos de bio ans o mación ecuen emen e dan luga á mine alización
incomple a dos MCOs, que son con e idos en p odu os de ans o mación (PT) que
poden posuí os mesmos ou incluso maio es e ec os ad e sos pa a o ecosis ema que os
seus compos os de o ixe. Des e xei o, pa a educi os iscos da desca ga dos MCOs ao
medioambien e e pa a maximiza a súa eliminación, é undamen al comp ende os
mecanismos de bio ans o mación que oco en nos compa imen os biolóxicos das
EDAR. Es e es udo debe p es a especial a ención aos PTs o mados e busca iden i ica
os mic oo ganismos e ac i idades enzimá icas que xogan un ol p incipal no p oceso de
bio ans o mación.
O a amen o biolóxico máis común nas EDAR baséase nun sis ema de lodos
ac i ados, que no malmen e implica ac i idades he e ó o as e au ó o as ni i ican es.
Men es que a segunda oi amplamen e es udada a causa do ele an e ol xogado pola
enzima amonio monooxixenasa, o papel dos mic oo ganismos ae obios he e ó o os na
bio ans o mación dos MCOs aínda non es á cla o a causa da complexidade e a iedade
das súas ac i idades me abólicas. O es udo dos mecanismos he e ó o os de
bio ans o mación é impo an e pola ele ancia des a poboación pa a elimina a ma e ia
o gánica e polo seu c ecen e in e ese en no as concepcións de EDAR ene xé icamen e
máis e icien es, onde as e apas A, que aballan a al as axas de ca ga o gánica e cu os
empos de e ención de sólidos, ope an basicamen e en condicións he e ó o as.
Polo an o, es a ese busca ob e una mello imaxe dos mecanismos que dan luga a
bio ans o mación come abólica dos MCOs baixo condicións ae obias he e ó o as,
in en ando esponde as seguin es cues ións:
xSon capaces os o ganismos he e ó o os de bio ans o ma amplamen e
MCOs con di e sas p opiedades ísico-químicas?
xP odúcese come abólicamen e di a bio ans o mación?
xUnha maio ac i idade he e ó o a a ec a a e iciencia de
bio ans o mación dos MCOs? E a súa ciné ica ou cons an e ciné ica de
bio ans o mación?
Resumo
6
xCales son as p incipais eaccións de bio ans o mación que le an a cabo
os o ganismos he e ó o os?
xExis e algunha poboación mic obiana he e ó o a que xogue un papel
des acado du an e a bio ans o mación dos MCOs?
xCales son as ac i idades enzimá icas máis ele an es du an e a
bio ans o mación come abólica dos MCOs en condicións ae obias he e ó o as?
Capí ulo 2. Ma e iais, mé odos e equipos
Nes e capí ulo desc íbese a alimen ación u ilizada pa a o desen ol emen o dos
expe imen os le ados a cabo nos dis in os ipos de eac o (en con inuo, secuenciais en
ba ch e en ba ch). Ademais, p esén anse os MCOs es udados ao longo da ese e os
mé odos analí icos u ilizados pa a a súa de e minación e a dos PTs o mados.
Adicionalmen e, p opo cionase unha desc ición de allada dos mé odos analí icos
emp egados pa a moni o iza o es ado dos expe imen os (demanda química de oxíxeno
(DQO), especies de ni óxeno, concen ación de sólidos, oxíxeno disol o, pH e
empe a u a) e pa a ob e in o mación mic obiolóxica (ADN e p o eínas), que se iu pa a
iden i ica os mic oo ganismos e enzimas cla e in oluc ados na bio ans o mación dos
MCOs. Finalmen e, p esén anse os cálculos emp egados pa a analiza os da os ob idos
nos dis in os capí ulos.
Capí ulo 3. A elocidade de ca ga o gánica a ec a a ciné ica de
bio ans o mación come abólica baixo condicións he e ó o as en
sis emas de lodos ac i os
O ol dos mic oo ganismos ae obios he e ó o os du an e a bio ans o mación dos
MCOs nos sis emas de lodos ac i os aínda non es á cla o. Nes e capí ulo, búscase da
espos a a súa capacidade pa a bio ans o ma come abólicamen e un g upo de 15 MCOs
con di e sas p opiedades ísico-químicas engadidos en concen acións en e 1 e 40 μg L-
1, así como coñece a in luenza da ac i idade he e ó o a na ciné ica de bio ans o mación
come abólica.
Con es e obxec i o, ope ouse un eac o con inuo de anque axi ado (CSTR) a ca o
elocidades de ca ga o gánica (VCO) di e en es (0.2, 0.4, 0.6 e 0.8 g DQO L-1 d-1) a a és
do uso de concen acións c ecen es de subs a o p ima io (ace a o) e man endo cons an e
Resumo
7
o empo de e ención hid áulico (24 h). Adicionalmen e, pa a asegu a unicamen e a
p esenza de ac i idade he e ó o a, engadiuse alil iou ea, un inhibido ni i ican e, e
man í ose o empo de e ención celula ao edo de 5 días. Ademais, usando como inoculo
os espec i os eac o es CSTR, amén se le a on a cabo ensaios en ba ch con
concen acións iniciais de subs a o p ima io (ace a o) de 0.2, 0.4, 0.6 e 0.8 g DQO L-1.
O obxec i o des es ensaios oi obse a se a iacións na ac i idade he e ó o a
mic obiana, de e minada como a ac i idade especí ica máxima de DQO, a ec aban a
ciné ica de bio ans o mación dos MCOs e, pa icula men e, a cons an e de
bio ans o mación ciné ica (kbiol).
En p imei o luga , os esul ados dos CSTR amosa on que a e icacia de
bio ans o mación oi supe io ao 80 % pa a a maio ía dos MCOs, coa excepción de
ca bamazepina e diazepam (in e io a 20 % a causa da súa na u eza ecalci an e),
ime op im (35 %) e e i omicina (70 %), e que os alo es se man i e on cons an es ás 4
VCOs es udadas. Es es da os con i man as al as capacidades dos mic oo ganismos
he e ó o os pa a bio ans o ma os MCOs. Ademais, obse ouse que a biodeg adación
da on e de ca bono p ima ia e a bio ans o mación dos MCOs oco ía de modo
simul áneo e que maio es VCOs de on luga a maio es elocidades especí icas de
bio ans o mación, des acando ao come abolismo como mecanismo p incipal de
bio ans o mación baixo as condicións es udadas.
Po ou a banda, os ensaios en ba ch pe mi i on obse a que a maio asa de
bio ans o mación especí ica de e minada nos eac o es CSTR a maio es ac i idades
he e ó o as oco e a a és de a iacións en kbiol. Es e esul ado pe mi e a i ma que a
kbiol dun de e minado compos o non depende soamen e das súas p opiedades
ísicoquímicas e das condicións ambien ais, senon amén da ac i idade especí ica do lodo
causada polo subs a o p ima io. A co elación posi i a en e kbiol e a ac i idade
especí ica máxima de DQO oi obse ada pa a odos os MCOs non ecalci an es, con
excepción de dúas agancias. Non obs an e, a in luenza da ac i idade he e ó o a oi
desigual en e os compos os es udados, indicando que a súa acción é especí ica pa a cada
MCO.
En conclusión, nes e capí ulo amósase que maio es VCOs conducen a maio es asas
de bio ans o mación come abólica dos MCOs a a és do aumen o dos seus espec i os
alo es de kbiol. Non obs an e, is o non implica necesa iamen e unha mello a no
endemen o de bio ans o mación dos compos os semp e que se apo e un empo de
CHAPTER 1
CHAPTER 1. INTRODUCTION AND CONTEXT
SUMMARY
Bio ans o ma ion o ace-le el o ganic mic opollu an s (OMPs) by complex
mic obial communi ies in was ewa e ea men acili ies is a key p ocess o hei
de oxi ica ion and en i onmen al impac educ ion. The e o e, unde s anding he
me abolic ac i i ies and mechanisms ha con ibu e o hei bio ans o ma ion is essen ial
when de eloping app oaches aiming o minimize hei discha ge. This chap e add esses
he ele ance o come abolic bio ans o ma ion and discusses he main enzyma ic
ac i i ies cu en ly known o ake pa in he ae obic emo al o OMPs in was ewa e
ea men plan s. Fu he mo e, he mos common me hodologies o deciphe such
enzymes a e discussed.
This chap e , unde he consen o he publishe (Else ie ), was ed a ed a e :
Kennes-Veiga, Gonzalez-Gil, L., Ca balla, M., Lema, J. M. (2021). Enzyma ic
come abolic bio ans o ma ion o o ganic mic opollu an s in was ewa e ea men plan s:
A e iew. Bio esou ce Technology, 344B, 126291. ISSN: 0960-8524.
Fu he in o ma ion can be ound in he “Lis o publica ions” (page 145).
Chap e 1
16
1.1. ORGANIC MICROPOLLUTANTS IN WASTEWATER TREATMENT
PLANTS
O ganic mic opollu an s (OMPs) co e a wide ange o subs ances o wo ldwide
inc easing consump ion, including pha maceu icals, pe sonal ca e p oduc s, ho mones
and indus ial chemicals (Luo e al., 2014). Despi e being p esen a e y low
concen a ions, in he ng o Pg L-1 ange, hey pose he po en ial o ha m he en i onmen
due o hei pe sis ence, bioaccumula ion and biological ac i i y (Ma go e al., 2015).
Besides, hey can os e oxici y, mu agenici y, an ibio ic esis ance and oxida i e s ess,
as well as causing diso de s o he ne ous, ho monal and ep oduc i e sys em (Bilal e
al., 2019; Tiwa i e al., 2017). The applica ions, physicochemical p ope ies and
pe sis ence o OMPs a e e y b oad, causing hei p esence and emo al e iciency o
a y g ea ly in was ewa e ea men plan s (WWTPs), which, al hough hey ha e p o en
o be capable o educing hei concen a ions, we e no designed o emo e hem (Ma go
e al., 2015; Pe ie e al., 2014). The ex en o which OMPs a e emo ed also depends on
en i onmen al and p ocess condi ions, including empe a u e, pH, was ewa e
composi ion, mic obial communi y and edox po en ial, among o he s (Al a ino e al.,
2018a; Cao e al., 2020; Gulde e al., 2014; K uglo a e al., 2016; Rios-Miguel e al.,
2021).
OMPs emo al in biological p ocesses o con en ional WWTPs occu s mos ly due
o (i) ola iliza ion, (ii) so p ion o he sludge and (iii) bio ans o ma ion (Pomiès e al.,
2013; Tiwa i e al., 2017), al hough abio ic ans o ma ion p ocesses may also ake place
o some compounds (Yu e al., 2018). Bio ans o ma ion is conside ed he main
esponsible mechanism since ola iliza ion and so p ion a e only ele an o compounds
showing high alues o he Hen y’s law cons an o solid-liquid pa i ion coe icien ,
espec i ely (Nguyen e al., 2021; Su e al., 2021). OMPs bio ans o ma ion could occu
me abolically i he ene gy gene a ed om such eac ion is su icien o p omo e biomass
g ow h and main enance. Di e en ly, come abolic bio ans o ma ion is he abili y o
mic oo ganisms o deg ade non-g ow h subs a es ha canno be used as he sole nu ien
and ene gy sou ce in he obliga e p esence o a p ima y subs a e. The la e is a ailable
a highe concen a ions, se es as an elec on dono and can main ain he mic obial
popula ion and induce he p oduc ion o enzymes and co ac o s which a e capable o
bio ans o ming he OMPs hanks o hei e sa ile ca aly ic ac i i y (Fe nandez-Fon aina
e al., 2014; Fische and Majewsky, 2014; K ah e al., 2016). Among he scien i ic
In oduc ion and con ex
17
communi y, i is widely assumed ha he main bio ans o ma ion mechanism in eal
en i onmen al condi ions is come abolism (Fische and Majewsky, 2014; Lema and
Sua ez, 2017; T an e al., 2013).
Ei he me abolically o come abolically, bio ans o ma ion p ocesses can lead o
incomple e mine aliza ion o OMPs, hus being con e ed in o ans o ma ion p oduc s
(TPs) ha may pose e en inc eased ad e se e ec s o he aqua ic ecosys ems and human
heal h (Gulde e al., 2016). Consequen ly, a ho ough analysis o he a e and
ans o ma ion eac ions o he OMPs in he biological compa men s o WWTPs is o
i al impo ance o p ope ly assess he en i onmen al impac s (Men e al., 2017). Fo
such eason, in ecen imes esea ch has e ol ed om (i) ying o de elop and op imize
analy ical me hods o de e mine OMPs concen a ions in di e en ma ixes in o (ii)
assessing hei occu ence in s eams and compa men s and de e mining he emo al
e iciencies o a ying echnologies and ope a ional condi ions and inally in o (iii)
a emp ing o comp ehend he unde lying emo al mechanisms, me abolic pa hways and
enzyma ic ac i i ies occu ing in WWTPs du ing so p ion and bio ans o ma ion
p ocesses (Al a ino e al., 2018a), which would g ea ly help o mi iga e OMPs in
WWTPs.
1.2. COMETABOLIC BIOTRANSFORMATION OF OMPS IN WWTPs
P e ious s udies ha e shown ha many OMPs can be bio ans o med me abolically
o come abolically (Fig. 1.1) depending on hei physicochemical p ope ies and he
en i onmen al condi ions (T an e al., 2013). Making a dis inc ion be ween me abolism
and come abolism is impo an o ha e a be e unde s a ing o he mechanisms in ol ing
OMPs bio ans o ma ion and o op imize hei o e all emo al. Howe e , mul iple
s udies do no disc imina e be ween hem because di e en ia ion can be di icul ,
pa icula ly when wo king wi h mixed mic obial communi ies such as ac i a ed sludge.
Indeed, me abolism and come abolism may coexis (Manda ić e al., 2018); some imes is
unclea i ene gy de i es om OMPs biodeg ada ion o dead cells (T an e al., 2013); and
me abolic and come abolic s eps can be in e ela ed and subs i u able since hey a e pa
o a me abolic ne wo k e ol ed as a whole by he mic obial communi y (Fische and
Majewsky, 2014). In his sense, mic oo ganisms a e con inuously de eloping new
ca abolic pa hways o subs a es o access ene gy and nu ien s o o de oxi y
compounds, and can de elop new me abolic ools which may allow o u n come abolic
Chap e 1
18
p ocesses in o me abolic ones (Fische and Majewsky, 2014; Kol enbach e al., 2014). In
ac , p omiscuous enzymes, which can ca alyze eac ions wi h mul iple subs a es, ha e
been associa ed wi h bo h p ocesses ha occu in en ionally (me abolism) and
o ui ously (come abolism) (Fische and Majewsky, 2014).
The me abolism o OMPs is challenging because g ow h-linked deg ada ion
eac ions can only p oceed when eac ion he modynamics and kine ics a e a o able. To
da e, only he e o ophic mic oo ganisms ha e p o en o be capable o ca ying ou such
p ocess and, al hough he OMPs concen a ion h eshold equi ed o me abolic ac i i ies
emains unclea , i appea s o be a le els conside ably highe han hose ypically
de ec ed in en i onmen al samples and WWTPs (Nsenga Kumwimba and Meng, 2019;
T an e al., 2013). Thus, OMPs biodeg ada ion may no be ene ge ically a o able o
mic oo ganisms, equi ing p omiscuous enzymes o bio ans o m hem hanks o
s uc u al simila i ies wi h p ima y subs a es, e en i such eac ions end o p oceed a
much lowe a es (Kol enbach e al., 2014; Lema and Sua ez, 2017; Nguyen e al., 2021).
As a consequence, in WWTPs, i is assumed ha OMPs a e bio ans o med
come abolically as a side e ec , linked o he elimina ion o mac opollu an s such as
o ganic ca bon, ni ogen and phospho ous (Fische and Majewsky, 2014) and, pe haps,
o endogenous decay when g ow h subs a es a e deple ed (M. H. Kim e al., 2020).
Du ing come abolism, he ini ial g ow h o non-g ow h subs a e concen a ion a io is
impo an since high alues may help mic oo ganisms o eco e om oxici y caused by
OMPs and TPs and because ini ial subs a e concen a ions de e mine whe he su icien
educing powe could be di e ed o non-g ow h subs a e bio ans o ma ion and cell
g ow h and main enance (M. H. Kim e al., 2020; Rios-Miguel e al., 2021). Acco dingly,
highe come abolic bio ans o ma ion e iciencies could be expec ed wi h inc eased
p ima y subs a e loading a es, as obse ed in ni i ying and he e o ophic ac i a ed
sludge (Fe nandez-Fon aina e al., 2012; Helbling e al., 2012; Kennes-Veiga e al., 2020).
Howe e , i is no always he case since he e is an uppe (sa u a ion limi ) and lowe
(ac i a ion limi ) h eshold o p ima y subs a e loading a es in luencing come abolism
(Ca nei o e al., 2020; Gonzalez-Gil e al., 2018b; Sheng e al., 2021). Loading a es
below ha h eshold lead o insu icien educing powe supply o come abolism and
alues abo e he h eshold each a bio ans o ma ion limi due o a come abolic
decoupling be ween OMPs and p ima y subs a es (Sheng e al., 2021) o because o
he modynamic cons ain s such as chemical equilib ium o enzyma ic e e sibili y
In oduc ion and con ex
19
(Gonzalez-Gil e al., 2019a, 2018a, 2018b). In e es ingly, some s udies ha e also
obse ed ha unde s a a ion condi ions o low a ailabili y o easily deg adable ca bon,
bio ans o ma ion may imp o e hanks o he s imula ion o mul iple mic oo ganisms and
an inc ease in he abundance o di e si y o some enzymes (Ache mann e al., 2018b;
Nguyen e al., 2021). The speci ici y cons an a io, o kine ic e iciency, o non-g ow h
o g ow h subs a es o a mic obial cul u e, which cha ac e izes he inhe en compe i ion,
is also key du ing come abolism since compe i ion o he enzyma ic ac i e si e may
occu , leading o hinde ed bio ans o ma ion o some OMPs due o he highe
concen a ions a which g ow h subs a es a e p esen (Gonzalez-Gil e al., 2021; M. H.
Kim e al., 2020; Plósz e al., 2010). Non-compe i i e inhibi ion may also occu when
OMPs and p ima y subs a es a e no s uc u ally analogous and bind o di e en ac i e
si es, esul ing in educed o e all oxida ion a es (Lema and Sua ez, 2017; Su e al.,
2015). Fu he mo e, he p esence o speci ic moie ies in OMPs may acili a e o hinde
come abolism and in luence ini ial bio ans o ma ion eac ions ha condi ion he
likelihood o subsequen eac ion s eps and he o e all bio ans o ma ion a e and
e iciency (Helbling e al., 2012; Kol enbach e al., 2014; Nguyen e al., 2021; T an e
al., 2013).
OMPs come abolism has been obse ed in mul iple en i onmen s. Fo ins ance,
unde ni i ying condi ions, i was obse ed he come abolic bio ans o ma ion o se e al
OMPs along wi h ammonium oxida ion (Gonzalez-Gil e al., 2021; Han e al., 2019;
Sheng e al., 2021; Wang e al., 2019). Ammonia oxidizing bac e ia (AOB) and a chaea
(AOA), he no el commamox popula ion (Han e al., 2019) and slow-g owing
he e o ophs (Gonzalez-Gil e al., 2021) ha e been epo ed o pa icipa e in
come abolism, while o he popula ions, such as ni i e-oxidizing bac e ia (NOB), ha e
shown a less ele an con ibu ion (Yu e al., 2018). The e is also e idence o
come abolism unde he e o ophic condi ions (Fe nandez-Fon aina e al., 2016; Fische
and Majewsky, 2014; Gonzalez-Gil e al., 2021; Kennes-Veiga e al., 2020), al hough he
ca bon sou ce o he p ima y subs a e has a conside able in luence on he
bio ans o ma ion a e and ex en (La che and Ya geau, 2011; Oli ei a e al., 2019;
To esi e al., 2016a). The combina ion o ni i ying and he e o ophic ac i i ies has been
shown o imp o e OMPs bio ans o ma ion and o educe TPs accumula ion hanks o he
highe mic obial di e si y and a b oade exp ession o non-speci ic enzymes (Fe nandez-
Fon aina e al., 2016; Khunja e al., 2011). In con as , in o ma ion abou come abolism
Chap e 1
unde anoxic condi ions is sca ce despi e e idence o hei equal o e en highe
capabili ies o bio ans o m some OMPs (Falås e al., 2013). None heless, To esi e al.
(2017) p o ed come abolism o se e al pha maceu icals in deni i ying eac o s and
Ma ínez-Quin ela e al. (2021), unde he ecen ly disco e ed N-Damo p ocess, which
consis s o anae obic me hane oxida ion wi h ni i e, also obse ed come abolic
bio ans o ma ion o mul iple compounds. Simila ly, anae obic sys ems ha e also p o en
hei capaci y o educe OMPs en i onmen al impac in was ewa e e luen s (A ias e al.,
2018; Phan e al., 2018) and i is belie ed ha hey could enhance he emo al o some
compounds poo ly bio ans o med unde ae obic condi ions (Ha b e al., 2019; Lin e al.,
2020). Gonzalez-Gil e al. (2017a, 2018b) and Ca nei o e al. (2020) p o ed
come abolism o mul iple compounds du ing anae obic diges ion; Al a ino e al. (2014)
obse ed a linea ela ionship be ween he bio ans o ma ion a e o sul ame hoxazole,
ime hop im and nap oxen and he me hanogenic ac i i y in an up low anae obic sludge
blanke eac o and Oli ei a e al. (2019) de e mined sul ame hazine come abolism wi h
anae obic sludge using se e al ca bon sou ces. Finally, OMPs come abolism has also
been obse ed in o he biological p ocesses, as in mic oalgae-based sys ems (Liu e al.,
2021; Vo e al., 2020), in eas - amine condi ions (Tang e al., 2021) and along wi h
polyhyd oxyalkanoa es me abolism in a bio eac o se up o phospho ous emo al
(To esi e al., 2019).
20
In oduc ion and con ex
21
Fig. 1.1. Rep esen a ion o he me abolic and come abolic bio ans o ma ion o
OMPs.
OMPs
Was e p oduc s
(CO
2
, H
2
O, TPs )
Ene gy
(mobili y, hea )
Mac omolecules
(Cell componen s,
g ow h, s o age)
P ecu so
molecules
Nu ien s
Anabolism
(syn hesis eac ions)
ADP
Ene gy
ATP
Ca abolism
(deg ada i e eac ions)
P ima y subs a e
Was e p oduc s
(CO
2
, H
2
O)
Ene gy
(mobili y, hea )
Mac omolecules
(Cell componen s,
g ow h, s o age)
P ecu so
molecules
Nu ien s
ADP
Ene gy
ATP
Ca abolism
(deg ada i e eac ions)
OMPs
TPs
OMPs
TPs
Anabolism
(syn hesis eac ions)
ns
)
Me abolism Come abolism
Chap e 1
22
1.3. ENZYMATIC ACTIVITIES INVOLVED IN THE BIOTRANSFORMATION
OF OMPs UNDER AEROBIC CONDITIONS
The me abolic capabili ies and enzyma ic ne wo k o he mic obial communi ies
p esen in WWTPs de e mine hei bio ans o ma ion po en ial (Fische and Majewsky,
2014; Helbling e al., 2012). Howe e , he complexi y o biological sys ems and he
limi ed knowledge abou enzyma ic mechanisms has esul ed in sca ce in o ma ion abou
he ole o mic obial s ains and enzymes in ol ed in bio ans o ma ion, which is c ucial
o de elop isk-assessmen ools ha help p edic OMPs pa hways and hal -li es in
WWTPs (Ache mann e al., 2020).
Come abolic bio ans o ma ion consis s o a b oad sequence o indi idual eac ions
and se e al OMPs may unde go a ious emo al s a egies concu en ly, leading o a
huge a ay o candida e enzyma ic ac i i ies ha may pa icipa e in bio ans o ma ion
(Helbling e al., 2010a; Kol enbach e al., 2014). Such enzymes a e equen ly belie ed
o be loca ed in acellula ly, ca ying ou mos bio ans o ma ion eac ions hanks o hei
high me abolic e sa ili y. Howe e , ex acellula enzymes a e pa icula ly equi ed o
he b eak-down o la ge and ionized OMPs ha pose di icul ies o cellula up ake and
limi bio ans o ma ion a es; and hey may be highly ele an o educe he selec ion
p essu e o an ibio ic- esis an genes (K ah e al., 2016; Zums ein and Helbling, 2019).
Simila ly, i is gene ally assumed ha bio ans o ma ion occu s hanks o ca abolic
enzymes, bu anabolic enzymes also seem o play a key ole, highligh ing he challenge
o deciphe ing hem (Ache mann e al., 2018a; S adle e al., 2018).
The mos common biological ea men in WWTPs is based on an ac i a ed sludge
sys em whe e au o ophic ni i ie s and he e o ophs ep esen he mos impo an
mic obial popula ions, al hough hei ela i e con ibu ion o OMPs bio ans o ma ion
emains unclea (Lema and Sua ez, 2017; Nguyen e al., 2021; Polesel e al., 2017).
Ni i ying mic oo ganisms can be classi ied in o: AOB and AOA, in ol ed in he
oxida ion o ammonia o ni i e, and NOB, capable o oxidizing ni i e o ni a e.
Nume ous s udies pe o med in ecen yea s ha e shown he key ole played by ni i ying
enzymes du ing bio ans o ma ion (Nsenga Kumwimba and Meng, 2019; Su e al., 2021)
and, al hough u he s udies a e needed o elucida e he ole o p omising enzymes such
as hyd oxylamine and ni i e oxido educ ases, enzymes p esen in AOB and AOA seem
o g ea ly con ibu e o he p ocess, while hose p esen in NOB may play a mino ole
(Helbling e al., 2012; Su e al., 2021; Yu e al., 2018). Ammonia monooxygenase (AMO;
In oduc ion and con ex
23
EC 1.14.99.39), p esen in AOB and AOA, is conside ed esponsible o he
bio ans o ma ion o mul iple compounds (Fe nandez-Fon aina e al., 2016; Men e al.,
2017; Su e al., 2021; Wang e al., 2019; Yu e al., 2018), equen ly h ough oxygen
inse ions esul ing in hyd oxyla ion eac ions and some imes in dehyd ogena ion o
educ i e dehalogena ion (Helbling e al., 2012; Su e al., 2021). Howe e , some s udies
ha e shown ha he con ibu ion o bio ans o ma ion o ni i ie s and AMO in ac i a ed
sludge sys ems may o en be o e -es ima ed and beli le he ole o o he he e o ophic
and ni i ying enzymes (Helbling e al., 2012; Men e al., 2017). In ac , he
bio ans o ma ion o ce ain compounds in ni i ying ac i a ed sludge eac o s could
happen hanks o slow-g owing he e o ophs (Ache mann e al., 2018b; Gonzalez-Gil e
al., 2021), sugges ing ha a en ion should also be paid o side sludge mic obial ac i i ies.
In his sense, high sludge e en ion imes (SRT) a e conside ed o imp o e OMPs
bio ans o ma ion hanks o a b oade mic obial and unc ional di e si y and a la ge
ne wo k o enzyma ic ac i i ies p oduced by he addi ional p esence o slow-g owing
mic oo ganisms (Ache mann e al., 2018b; Yuxin Wang e al., 2020). Ache mann e al.
(2018a) obse ed a cha ac e is ic end be ween he SRT and he bio ans o ma ion o
OMPs unde going oxida ion eac ions, sugges ing ha hose eac ions may be ca alyzed
by enzymes less gene ally widesp ead among bac e ia, ha may become mo e abundan
wi h he de elopmen o some g oups o mic oo ganisms. Di e en ly, hey obse ed a
weak dependence be ween he SRT and he bio ans o ma ion o OMPs unde going
subs i u ion eac ions, indica ing ha enzymes b oadly exp essed by di e en
mic oo ganisms may be ca ying hem ou , possibly because hey a e in ol ed in
mechanisms o cen al me abolism o gene al de ense. Simila ly, To esi e al. (2018) did
no ind a posi i e co ela ion be ween some OMPs bio ans o ma ion and biodi e si y,
implying ha he SRT and mic obial ichness may be only ele an when he
bio ans o ma ion s eps a e pe o med by a limi ed numbe o axa o enzymes.
The capabili y o he e o ophic cul u es o bio ans o m OMPs is e y ele an and
i has been p o en ha jus hei basal exp ession may be su icien o achie e high
emo al ex en s o mul iple xenobio ics (Fische and Majewsky, 2014). Thei
bio ans o ma ion po en ial comes om hei abili y o exp ess a huge numbe o di e en
enzyma ic ac i i ies hanks o he wide ange o ca bon sou ces ha hey can assimila e,
making i a di icul ask o de e mine hei abundance and impo ance (Kennes-Veiga e
al., 2021b). Nume ous bio ans o ma ion eac ions documen ed in ae obic expe imen s
Chap e 1
1.4. APPROACHES TO DETERMINE KEY ENZYMES
A huge amoun o enzymes pa icipa e in biological was ewa e ea men s and,
hanks o unc ional edundancy, he same me abolic p ocess can be conduc ed by se e al
mic oo ganisms and enzyma ic ou es (Sambamoo hy and Raman, 2018). The e o e,
linking he bio ans o ma ion o OMPs wi h speci ic enzyma ic ac i i ies is a complex
ask ha en ails a deep unde s anding o he biological p ocesses and he de elopmen o
app op ia e expe imen al s a egies. In his sec ion, he main app oaches employed o
iden i y he key enzymes a e desc ibed and analyzed om a c i ical poin o iew (Fig.
1.2).
30
In oduc ion and con ex
31
Fig. 1.2. F equen app oaches applied o de e mine he enzyma ic ac i i ies in ol ed in
OMPs bio ans o ma ion.
Chap e 1
32
1.4.1. In i o assays
In i o assays e e o a me hodology whe e cell- ee enzymes a e cul u ed in a
con olled and a i icial en i onmen ou side o he li ing mic oo ganism o add ess hei
ole on OMPs bio ans o ma ion. So a , wo app oaches o his echnique ha e been
ollowed: (i) use o a sole pu i ied enzyme, ei he ob ained comme cially (Gonzalez-Gil
e al., 2017) o in he labo a o y om a speci ic mic oo ganism (P io e al., 2010; Xu e
al., 2015); and (ii) use o an enzyma ic lysa e di ec ly ex ac ed om he eac o biomass
and composed o a mix u e o enzymes (Gonzalez-Gil e al., 2019a; K ah e al., 2016;
Zums ein and Helbling, 2019). The p ocedu e o bo h s a egies is summa ized in Fig.
1.3. When using sole enzymes, signi ican e o s a e needed o selec he sui able ones.
Fi s ly, he enzyme should be ep esen a i e o he biological sys em unde s udy and
show a ele an ac i i y in he bio eac o (Gonzalez-Gil e al., 2017), which equi es a
deep unde s anding o he me abolic pa hways and species in ol ed in he p ocess.
Secondly, i has o be easible o ob ain he enzyme in a pu i ied o m. In he case o using
a cell- ee lysa e wi h a cock ail o enzymes di ec ly ex ac ed om he biomass eac o ,
di e en p ocedu es can be conduc ed. I he goal is o s udy ex acellula enzymes, cell
lysis is no needed (Zums ein and Helbling, 2019), bu i should be pe o med o ex ac
in acellula enzymes (K ah e al., 2016). To eco e memb ane enzymes and disassemble
enzymes om he ex acellula polyme ic subs ance, su ac an s and ca ion exchange
esins should be added, espec i ely. Once he cell- ee lysa e is ob ained, he supply o
co ac o s and inhibi o s should be conside ed o p omo e and supp ess speci ic enzyma ic
ac i i ies, which could aid in he iden i ica ion o speci ic enzyma ic ac i i ies ela ed o
OMPs emo al (Gonzalez-Gil e al., 2019a).
The use o sole pu i ied enzymes in in i o assays allows o es and p o e speci ic
bio ans o ma ion pa hways wi hou he in e e ence o o he enzymes, which could
happen when using an enzyma ic lysa e, allowing o na ow down he p edic ion and
iden i ica ion o TPs. Success ul esul s wi h comme cially pu i ied enzymes we e
epo ed by Gonzalez-Gil e al. (2019b, 2017), who demons a ed ha ace a e kinase and
hexokinase could ans o m OMPs wi h ca boxyl and hyd oxyl g oups and mode a e
s e ic hind ance. Acco ding o Bisswange (2011), housands o enzymes exis , bu less
han 15 % a e desc ibed in de ail and only hund eds a e comme cially a ailable. E en i
so, enzyma ic subs a e selec i i y and a ini y can di e depending on he
mic oo ganism syn hesizing he enzyme. Fo ins ance, ace a e kinase om E. coli is mo e
In oduc ion and con ex
33
subs a e-speci ic han ha om M. he mophila (Bock e al., 1999), which migh esul
in a lowe a ini y o ce ain cosubs a es (i.e., OMPs) and explain why a educed BPA
bio ans o ma ion was achie ed when using he o me ins ead o he la e , which is
p esen in anae obic diges e s (Gonzalez-Gil e al., 2019b, 2017).
An al e na i e o comme cially pu i ied enzymes is o isola e mic obial species om
a bio eac o , iden i y he enzymes o in e es and pu i y hem o pe o m in i o
expe imen s. The e a e some s udies whe e cy och ome P450 enzymes o speci ic
bac e ia we e exp essed in E. coli o pe o m in i o expe imen s ha demons a ed hei
abili y o hyd oxyla e diclo enac (P io e al., 2010; Xu e al., 2015); al hough he
mic oo ganisms s udied do no belong o was ewa e biological sys ems. This
me hodology enables es ing a wide spec um o enzymes, bu i s expe imen al success
depends on complex challenges anging om isola ing and cul i a ing pu e
mic oo ganisms o exp essing and pu i ying he enzymes. In ac , he s a emen o Fische
and Majewsky (2014a) poin ing ou ha he isola ion o a ge enzymes p esen in
WWTPs has no been documen ed, is s ill alid nowadays. The closes app oach epo ed
is he ob ainmen o a cock ail o ex ac ed enzymes om biomass o ac i a ed sludge
sys ems (K ah e al., 2016; Zums ein and Helbling, 2019) and anae obic diges e s
(Gonzalez-Gil e al., 2019b), which allowed wo king wi h a wide a ie y o na i e
enzymes. This me hod, despi e being a simpli ica ion o in i o p ocesses, seems a mo e
ealis ic s a egy han wo king wi h indi idual enzymes. Ye , i migh be di icul o
asce ain which a e he speci ic enzymes esponsible o OMPs bio ans o ma ion. In ac ,
al hough he men ioned s udies p o ided sound a gumen s abou he candida e enzymes
esponsible o he obse ed bio ans o ma ion eac ions, hei iden i y could no be
i e u ably p o ed. Mo eo e , ano he limi a ion o his p ocedu e is ha i is no possible
o ex ac all he enzymes no pe o m long- e m expe imen s since he enzymes can
apidly lose hei ac i i y, which could comp omise he compa ison wi h esul s in i o.
Fu he mo e, in i o assays a e limi ed in p ope ly ep esen ing complex biological
p ocesses, and i canno be ully assu ed ha eac ions will occu in he same way and
ex en as unde eal bio eac o condi ions, whe e biomass and subs a e he e ogenei y is
huge and compe i ion and inhibi ion mechanisms migh happen among enzymes and
mic oo ganisms. None heless, hei esul s a e undoub edly aluable o sol e he puzzle
o OMPs bio ans o ma ion.
Chap e 1
34
Fig. 1.3. Main s ages needed o pe o m in i o assays wi h sole pu i ied enzymes o a
cell- ee lysa e.
1.4.2. Use o inhibi o s in mic obial and enzyma ic cul u es
The use o enzyma ic inhibi o s is a as and simple app oach o de e mine he ela i e
con ibu ion o speci ic enzyma ic ac i i ies p esen in a mic obial popula ion. The
me hodology consis s in linking a dec ease in OMPs bio ans o ma ion o he ac i i y o
he inhibi ed enzyme, which o e s an in e es ing s a ing poin o un a el OMPs
bio ans o ma ion pa hways. Resul s can imply ha he inhibi ed enzymes and
mic oo ganisms a e esponsible o he bio ans o ma ion (di ec in ol emen ) o ha
hei inhibi ion leads o he subsequen supp ession o he mic obial and enzyma ic
ac i i ies ca ying ou he p ocess (indi ec in ol emen ) (Men e al., 2017). This
app oach is pa icula ly use ul when he inhibi o is e y enzyme-speci ic, making i s
selec ion a key aspec ha equi es knowledge on he inhibi ed enzymes and he
associa ed bio ans o ma ion eac ions. In ac , a common p oblem occu s when he
enzyma ic speci ici y o he inhibi o is b oade han expec ed, possibly because i had
no been es ed be o e wi h as many enzymes as hose p esen in was ewa e mixed
mic obial cul u es (Gonzalez-Gil e al., 2019b; Helbling e al., 2012). Thus, pe o ming
mul iple expe imen s wi h an ex ended ange o inhibi o s ha heo e ically hinde he
same eac ion could help o inc ease con idence in he esul s (Men e al., 2017).
The ole o AOB in OMPs bio ans o ma ion has ypically been de e mined h ough
inhibi ion s udies wi h allyl hiou ea (ATU) (Fe nandez-Fon aina e al., 2016; Ma go e
al., 2016; Wu e al., 2020). Howe e , Men e al. (2017), who used wo ammonia oxida ion
Biomass
Wash
(bu e )
,
concen a e,
homogenize
Cell lysis
(Ul asonica ion o
bead bea ing)
Remo e cellula
emains
(Cen i uga ion,
il a ion)
Cell- ee
lysa e
In i o
assays
wi h
OMPs
Addi ion o
co ac o s,
inhibi o s
Addi ion o
su ac an s, ca ion
exchange esins
S udy o he
biological p ocess
(Species, unc ions,
me abolic pa hways)
Selec key
enzymes
Measu e he
enzyma ic
ac i i y in he
bio eac o
Ob ain pu i ied
enzyme
(comme cially,
labo a o y)
Find op imal
assay
condi ions
In i o
assays
wi h
OMPs
Sole pu i ied enzymes
Cock ail o ex ac ed enzymes
Measu e
p o ein con en
and enzyma ic
ac i i ies
In oduc ion and con ex
35
inhibi o s, ATU and oc yne (OCT), concluded ha ATU could o e es ima e he ole o
AOB by no being i s inhibi o y e ec as speci ic as p e iously hough . They obse ed
ha 17 compounds showed signi ican ly educed bio ans o ma ion wi h ATU compa ed
o OCT, pa icula ly hose con aining hioe he and phenylu ea g oups. Since hey
de e mined ha ATU ba ely a ec ed he e o ophic espi a ion and he essen ial
he e o ophic enzymes implica ed in ene gy conse a ion and cen al me abolism, hey
sugges ha ATU may ha e inhibi ed o he non-essen ial he e o ophic enzymes possibly
in ol ed in OMPs bio ans o ma ion.
Inhibi ion s udies ha e also been pe o med o elucida e he ole o o he enzymes
and mic oo ganisms. K ah e al. (2016) ca ied ou expe imen s wi h ex ac ed na i e
enzymes om ac i a ed sludge and, using pep idase inhibi o s, de e mined he
in ol emen o pep idases in he hyd olysis o amide bonds p esen in OMPs. They used
h ee inhibi o s a ge ing he ac ion o endopep idases (E-64 o cys eine pep idases (EC
3.4.22.-); 4-benzenesul onyl luo ide hyd ochlo ide (AEBSF) o se ine pep idases (EC
3.4.21.-) and peps a in A o aspa ic pep idases (EC 3.4.23.-)) and documen ed hei
in ol emen in he bio ans o ma ion o a enolol and beza ib a e; al hough hey poin ou
he need o pe o ming u he s udies o con i ma ion since he inhibi o s could ha e
a ec ed he ac i i y o o he non-endopep idase enzymes. Gonzalez-Gil e al. (2019b)
pe o med inhibi ion assays in i o wi h ex ac ed na i e enzymes om anae obic sludge
using E-64, AEBSF and peps a in A as pep idase inhibi o s and cas anospe mine and 2-
b omoe hanesul ona e (BES) as glycosidase and me hanogenic inhibi o s, espec i ely.
They obse ed ha he bio ans o ma ion o cla i h omycin, climbazole, ci alop am and
e y h omycin was impai ed unde he ac ion o he pep idase inhibi o s and ha a enolol
bio ans o ma ion was ully inhibi ed unde he ac ion o AEBSF, sugges ing he
pa icipa ion o pep idase enzymes in hei emo al. Howe e , hey also epo ed ha he
bio ans o ma ion o ace yl-sul ame hoxazole was conside ably inhibi ed unde he
ac ion o BES e en hough me hyl-CoM educ ase is unlikely in ol ed in he p ocess,
indica ing ha he ac ion o he inhibi o is mo e unspeci ic han expec ed. Mo eo e ,
educed emo al o e y h omycin and cla i h omycin in assays wi h cas anospe mine was
no obse ed despi e being good candida es o glycosylase ac ion due o hei cladinose
moie y, sugges ing ha o he glycosylases no a ec ed by he inhibi o could pa icipa e
in he bio ans o ma ion (Gonzalez-Gil e al., 2019b).
Chap e 1
36
1.4.3. T ans o ma ion p oduc analysis
As p e iously men ioned, bio ans o ma ion o mos OMPs in WWTPs does no lead
o comple e mine aliza ion, gi ing ise o he o ma ion o TPs. Since OMPs
bio ans o ma ion is mainly in luenced by he a ini y o hei chemical s uc u e wi h he
unspeci ic enzymes exp essed du ing p ima y subs a e biodeg ada ion (Han T an e al.,
2017), TPs iden i ica ion and p edic ion would be possible i he enzyma ic cycles we e
deeply unde s ood and he s uc u es o g ow h subs a es and OMPs could be co ela ed.
In like manne , TPs s uc u e analysis and elucida ion can be an excellen ool o ob ain
in o ma ion on OMPs biodeg adabili y and link enzyma ic ac i i ies aking pa du ing
bio ans o ma ion (Table 1.1). Howe e , un il ecen ly, sca ce in o ma ion was a ailable
due o he challenging ask o iden i ying and quan i ying unknown TPs p esen a
ex emely low concen a ions in complex ma ices (L. Zhang e al., 2013). Fo una ely,
he de elopmen and op imiza ion o e sa ile analy ical ools ha e helped o sea ch and
iden i y many suspec and non- a ge compounds wi h easonable accu acy and ce ain y
(Fenne e al., 2021; Gulde e al., 2016).
De e mining key enzymes h ough TPs analysis p o ides se e al ad an ages, such
as: he expe imen al design is gene ally simple; lab esul s can ealis ically be
ex apola ed o WWTPs (Ke n e al., 2010); he isks o inding and acqui ing comme cial
enzymes o in i o assays o o su e ing enzyma ic ac i i y loss when wo king wi h
enzyma ic lysa es a e a oided (sec ion 1.4.1.); and he subs an ial cos s o o he
echniques a e eluded (sec ion 1.4.4.). Fo such eason, o da e, mos candida e enzymes
in ol ed in OMPs bio ans o ma ion ha e been sugges ed applying his me hodology
(Ache mann e al., 2018b; Gulde e al., 2016; Kennes-Veiga e al., 2021b; Yu e al., 2018).
Howe e , TPs elucida ion canno lead o a di ec and comple e con i ma ion o he
esponsible enzyma ic ac i i ies and ce ain cons ain s migh a ec TPs de ec ion and
iden i ica ion h ough mass spec ome y app oaches, such as when: hey a e p esen a
concen a ions below he de ec ion limi ; hey possess molecula masses ou side he mass
ange o he ull scan; hey a e highly uns able and eadily ans o mable; hei s uc u es
a e oo simple and lead o analy ical e o s; hey ha e compound-speci ic p ope ies
limi ing he analy ical ioniza ion e iciency; hey ha e s uc u al isome s; in e e ing ions
wi h he same mass as he TPs a e p esen ; and he e is a lack o e e ence s anda ds ha
allow achie ing highe con idence le els in he p oposed TPs s uc u es (Helbling e al.,
2010b; Ke n e al., 2010; Schymanski e al., 2014). Thus, i is ad isable o combine esul s
In oduc ion and con ex
37
om se e al app oaches, such as in i o assays, enzyma ic inhibi ion and TPs analysis,
which allows de e mining key enzymes wi h much highe con idence, as pe o med o
iden i y he con ibu ion o se ine p o eases (EC 3.4.21.-) du ing he bio ans o ma ion o
a enolol in anae obic condi ions (Gonzalez-Gil e al., 2019b).
Filling he knowledge gap o TPs o ma ion and linking hem o esponsible enzymes
jus wi h labo a o y s udies is a conside able challenge since such assays can be expensi e
and ime-consuming, bio ans o ma ion is in luenced by mul iple p ocess and
en i onmen al ac o s and mic oo ganisms may de elop new ca abolic pa hways o e
ime (Kol enbach e al., 2014; Men e al., 2017; Yuxin Wang e al., 2020). The e o e, he
use o modeling ools, such as Pa hP ed (Mo iya e al., 2010), CRAFT (CRAFT, 2009),
OECD Toolbox (OECD, 2020), en iPa h (Wicke e al., 2016) and he EAWAG-PPS
(EAWAG-BBD/PPS), which a e based on bio ans o ma ion ules ex ac ed om
mic obial me abolic pa hways and enzyma ic eac ions epo ed in he li e a u e, may
p o ide an app op ia e and cos -e ec i e app oach o help p edic ans o ma ion
eac ions a he di e en molecula unc ional g oups (Helbling e al., 2010b). They can
con ibu e o building pa hway knowledge, de e mine he biodeg adabili y o OMPs and
TPs, and pe o m isk assessmen s. Besides, hey a e ex emely use ul when c ea ing lis s
o expec ed TPs in suspec -sc eening app oaches (Ache mann e al., 2018b; Gulde e al.,
2016; Kennes-Veiga e al., 2021b). Howe e , hei main cons ain is ha hey a e
excep ionally sensi i e bu poo ly selec i e, leading o he p edic ion o oo many TPs
and causing nume ous alse posi i es, likely due o he limi ed da a a ailable o mic obial
p ocesses and o no conside ing he e ec s caused by moie ies su ounding he a ge
unc ional g oups in he bio ans o ma ion ules. Ano he limi a ion is ha mos ules and
mic obial pa hways a e based on li e a u e in o ma ion ob ained om s udies pe o med
wi h pu e o en iched cul u es, unspeci ic en i onmen al condi ions o whe e xenobio ics
a e used as p ima y subs a es, esul ing in bio ans o ma ion eac ions unlikely o happen
du ing he come abolic bio ans o ma ion o ace-le el OMPs by mixed cul u es (Gulde
e al., 2016). Thus, combining modeling ools and lab expe imen s unde de ined and
en i onmen ally ealis ic condi ions is necessa y o imp o e he p edic ion o TPs and
lea n abou new bio ans o ma ion pa hways.
1.4.4. Omics app oach
The ecen e olu ion and imp o emen o sequencing echniques, he inc eased
knowledge on sequence in o ma ion and he de elopmen o p o ein and genome
Chap e 1
38
da abases, such as Eawag-BBD/PPS and en iPa h (EAWAG-BBD/PPS; Wicke e al.,
2016), ha e allowed using me a-omics associa ion s udies o elucida e OMPs
bio ans o ma ion pa hways and se up hypo heses o po en ially in ol ed enzyma ic
ac i i ies (Table 1.1) (K ah e al., 2016). Wi h ha pu pose, me agenomics, epo ing
which mic oo ganisms a e p esen in a sample and i s me abolic po en ial;
me a ansc ip omics, o e ing p ecise in o ma ion on he mic obial unc ions happening
a a gi en ime; and p o eomics analyses, showing he ac i e enzyme pool p esen in he
mic obiome, ha e been inc easingly pe o med. Me agenomics echniques ha e been
widely applied in expe imen s wi h OMPs h ough mode n high- h oughpu DNA
sequencing echnologies, while he use o me a ansc ip omics and me ap o eomics is
mo e ecen and he e is deba e abou hei sui abili y (Fenne e al., 2021).
Me a ansc ip omics has a highe sensi i i y o de ec low-abundance gene ansc ip s
han me ap o eomics o de ec exp essed p o eins, and ansc ip s ha e been shown o
co ela e p ope ly wi h he p o ein abundance le el, bu i gene ally implies a highe cos .
Addi ionally, me ap o eomics may p o ide a highe po en ial o ob ain mechanis ic
insigh s hanks o desc ibing he enzyme pool mo e di ec ly (Ache mann e al., 2020;
Fenne e al., 2021), bu p o ein iden i ica ion equi es me agenomic da a om he
samples, along wi h de ailed in e p e a ion and anno a ion (Fenne e al., 2021).
The in oduc ion and accumula ion o OMPs in bio eac o s a ec mic obial
communi y s uc u e and leads o changes in he exp ession le els o biodeg ada ion
genes and gene p oduc s, as obse ed by Ha b e al. (2016) using high- h oughpu 16S
RNA gene sequencing, me a ansc ip omics and gene da abases. Thus, -omics
associa ion s udies ha e been b oadly used o ind bioma ke s ha can help o unde s and
and p edic he capaci ies o mic obial communi ies and he in luence o speci ic WWTPs
pa ame e s (Helbling e al., 2012; Da id R. Johnson e al., 2015a). Fo example, Cydzil-
Kwia kowska e al. (2020) de e mined a linea co ela ion be ween BPA concen a ion
and he exp ession le els o he gen bisdA, which encodes o e edoxin. Simila ly, Zhou
e al. (2015), in an expe imen wi h Sphingobium sp. BiD32 a a ying concen a ions o
BPA, s udied i s pa hway and key enzymes h ough genomic, p o eomic and TPs analysis.
They obse ed he up egula ion o 43 p o eins belonging o he dehyd ogenase,
dioxygenase, hyd a ase, hyd oxylase and cycloisome ase class; con i med ha p-
hyd oxybenzoa e hyd oxylase was in ol ed in he i s bio ans o ma ion s ep and
iden i ied he espec i e TP as a gene ic bioma ke o BPA bio ans o ma ion. Helbling
In oduc ion and con ex
39
e al. (2015), wi h a mul i a ia e model and bac e ial 16S RNA analysis, de e mined ha
speci ic phylogene ic g oups can also se e as bioma ke s o mic obial communi y
ac i i y owa ds OMPs bio ans o ma ion, al hough axonomic bioma ke s may only be
use ul when he e is no unc ional edundancy and bio ans o ma ion is no ex ensi ely
dis ibu ed among axa. In his sense, Vuono e al. (2016), analyzing 16S RNA genes
and RNA gene exp ession, obse ed ha a e axa had highe a ios o RNA o DNA
and a supe io p o ein syn hesis po en ial, which sugges s hei ele an ole in eac o
pe o mance and OMPs bio ans o ma ion, and con i ms ha unc ional and axonomic
ichness can posi i ely in luence OMPs emo al (S adle e al., 2018).
The main ad an age o me a-omics echniques is ha hey in end o cha ac e ize he
comple e se o genes o gene p oduc s p esen in a mic obial communi y a a gi en ime,
a oiding he need o he speci ic and obus knowledge equi ed o he p e iously
desc ibed app oaches (sec ions 1.4.1, 1.4.2 and 1.4.3) (Ache mann e al., 2020; K ah e
al., 2016). Besides, me a-omics associa ion no only in o ms abou key enzyma ic
ac i i ies bu also poin s owa ds speci ic mic oo ganisms esponsible o
bio ans o ma ion and p o ides in o ma ion on he subcellula loca ion and o ganismal
o igin o candida e enzymes (K ah e al., 2016). Such knowledge is highly impo an since
i can allow p edic ing changes in OMPs bio ans o ma ion a es and pa hways (S adle
e al., 2018) gi en ha : (i) bio ans o ma ion s eps may be ca ied ou by di e en
o ganisms (Ache mann e al., 2020; Helbling e al., 2015; Da id R. Johnson e al., 2015a);
(ii) enzymes o di e en mic obial o igins can possess a ying ca aly ic ac i i ies
(Gonzalez-Gil e al., 2019b, 2017; Da id R. Johnson e al., 2015a); (iii) he same TP o
an OMP can be p oduced by di e en mic oo ganisms and enzymes (Zhou e al., 2015);
and (i ) he same gene may associa e wi h di e en enzyma ic ac i i ies depending on he
eac o con igu a ion (Ha b e al., 2016).
The essen ial limi a ion o -omic associa ion mining is he gene a ion o a la ge
numbe o alse posi i es ha lead o non-causal co ela ions, adding g ea complexi y o
he iden i ica ion o genes o gene p oduc s o in e es i hypo heses a e no educed when
he la ge da ase s a e ob ained (Ache mann e al., 2020; Da id R. Johnson e al., 2015b).
Mo eo e , a di ec s a emen on he in ol emen o an enzyme in OMPs
bio ans o ma ion is o en no possible since, o ins ance, he up egula ion o ansc ip s
does no always imply an inc ease in enzyma ic ac i i y and he physiological unc ions
o p omiscuous enzymes canno be consis en ly p edic ed based on he knowledge o he
Chap e 2
46
bio eac o a e also p esen in WWTPs ha deal wi h mo e complex ca bon subs a es.
Mo eo e , o he ace nu ien s we e also added o p omo e he g ow h o ae obic
he e o ophic mic oo ganisms (Table 2.3) and allyl hiou ea solu ion was added in he
eeding wi h a concen a ion o 5 mg L-1 o a oid ni i ica ion.
Table 0.2. Feeding composi ion and concen a ions used o ob ain a chemical oxygen
demand (COD) o 0.2 g L-1. In he expe imen s wi h o he COD concen a ions, he
concen a ions we e calcula ed p opo ionally.
Compounds
mg L-1
Sodium ace a e (CH
3
COONa·3H
2
O)
210
Ace ic Acid (CH
3
COOH)
95
Ammonium chlo ide (NH
4
Cl)
40
Po assium dihyd ogen phospha e (KH
2
PO
4
)
10
Calcium chlo ide (CaCl
2
)
5.5
Magnesium sul a e (MgSO
4
)
5.5
Table 0.3. T ace me als composi ion and concen a ions used in he eeding o he ae obic
he e o ophic eac o .
Compounds
mg L-1
FeCl
3
·6H
2
O
0.15
H
3
BO
3
0.015
CuSO
4
·5H
2
O
0.003
KI
0.003
ZnSO
4
·7H
2
O
0.012
CoCl
2
·6H
2
O
0.015
MnCl
2
·4H
2
O
0.012
2.2. EXPERIMENTAL DESIGN
Di e en ypes o eac o con igu a ions (con inuous, sequen ial ba ch and ba ch
eac o s) we e ope a ed o unde s and he come abolic bio ans o ma ion o a ange o
OMPs unde ae obic he e o ophic condi ions.
Ma e ials, me hods and equipmen
47
2.2.1. Con inuous eac o s
5 L con inuously s i ed lab-scale eac o s connec ed o a 2 L se le we e used in
Chap e s 3 and 4, while a eac o o 2 L connec ed o a 1 L se le was used in Chap e
5 (Fig. 2.1.). The eac o s we e inocula ed wi h sludge om a con en ional ac i a ed
sludge eac o o a WWTP nea San iago de Compos ela (Spain), se ing an in- eac o
biomass concen a ion o app oxima ely 1.0 g ola ile suspended solids (VSS) L-1, and
ope a ed a 25ºC. The WWTP is designed o 184000 popula ion equi alen s, ecei es an
in luen COD anging be ween 0.2 - 0.7 g L-1 and ope a es wi h app oxima e SRT and
hyd aulic e en ion ime (HRT) alues o 10 d and 8 h, espec i ely. In he eac o s, he
HRT was se o 1 d o p o ide su icien ime o he mic oo ganisms o achie e he
maximum bio ans o ma ion hey a e capable o , a he same ime as minimizing possible
changes in he mic obial popula ion caused by an HRT oo long. Besides, he SRT was
main ained a ound 5 d o minimize he p esence o slow-g owing mic oo ganisms and
a o he he e o ophic ac i i y (Ache mann e al., 2018b).
Fig. 2.1. Schema ic ep esen a ion o he con inuous ae obic he e o ophic eac o s.
Ae a ion was p o ided o he eac o s ensu ing oxygen concen a ions be ween 3.5
and 7.5 mg O2 L-1. A e a s a -up pe iod o a ew days, he OMPs we e spiked in he
eeding a he desi ed concen a ions, which we e selec ed acco ding o ypical WWTPs
in luen concen a ions (Table 2.1.).
The ope a ion o he eac o s was mainly moni o ed h ough he s anda d analy ical
me hods (APHA, 2017) desc ibed in sec ion 2.3. Soluble COD, ammonium, ni a e and
ni i e concen a ions, o al suspended solids (TSS), VSS, pH and empe a u e we e
measu ed wo o h ee imes pe week in he eac o in luen ( eeds ock) and e luen .
Measu emen s o OMPs and TPs we e also pe o med when s eady s a e condi ions we e
eached a he di e en expe imen al condi ions applied. Addi ionally, in Chap e 5,
samples we e aken o genomic and me a ansc ip omic analysis.
Chap e 2
48
2.2.2. Ba ch expe imen s
Ba ch assays we e se o obse e i a ia ions in he he e o ophic mic obial ac i i y,
de e mined as he maximum COD speci ic ac i i y, a ec ed OMPs bio ans o ma ion
kine ics and, pa icula ly, he kbiol (Chap e 3). The expe imen s we e pe o med wi h
COD ini ial concen a ions o 0.2, 0.4, 0.6 and 0.8 g L-1 by a ying he sodium ace a e
and ace ic acid concen a ion and main aining he mic o and mac onu ien s used in he
con inuous eac o . The biomass was aken om he con inuous eac o s wo king a he
espec i e o ganic loading a es (OLR) (Chap e 3) o ensu e ha he mic obial
popula ion was adap ed o he expe imen al condi ions o he assays. Fo each ba ch, 18
lasks we e p epa ed, h ee o each ime poin o ha e iplica es (0, 1, 3, 8, 24 and 48 h).
A each o he men ioned imes, 3 lasks we e aken o de e mine he concen a ion o he
OMPs. The VSS concen a ion was se in all cases o app oxima ely 0.80 g VSS L-1 and
he empe a u e and s i ing we e ixed a 25qC and 150 pm (Inno a 4300 Incuba o
Shake –New B unswick Scien i ic). Neu al pH was ensu ed using punc ual addi ions
o NaOH o HCl when necessa y and an oxygen concen a ion abo e 4.5 mg O2L-1 was
main ained du ing expe imen a ion. To moni o he ope a ion, he same analyses as in he
con inuous eac o (soluble COD, ammonium, ni a e and ni i e concen a ions, TSS,
VSS, pH and empe a u e) we e conduc ed o each ime poin , excep o he solids and
ni ogen concen a ions which we e measu ed a imes 0, 24 and 48 h. OMPs analysis
was pe o med (solid and liquid phase) by aking samples om he lasks a each ime
poin .
2.2.3. Sequen ial ba ch eac o s
In Chap e 5, aiming o de e mine he key mic obial and enzyma ic playe s in ol ed
in SMX bio ans o ma ion, a o al o 18 sequen ial ba ch eac o s wi h a wo king olume
o 100 mL we e ope a ed in an incuba o a neu al pH, 25ºC, 150 pm and wi h oxygen
concen a ions anging be ween 3-5 mg O2L-1 o 25 days. Six di e en SMX
concen a ions we e es ed in iplica es: 0 (con ol), 50, 250, 500, 1000 and 2000 μg L-1.
The eac o s we e inocula ed wi h ac i a ed sludge om he same WWTP as he
con inuous eac o s and he ope a ion was pe o med unde s e ile condi ions.
The con en o he sequen ial ba ch eac o s was cen i uged daily a 6000 pm and
10ºC o 10 min o sepa a e he biomass om he supe na an . Then, he exhaus ed
supe na an was emo ed and new eed was added oge he wi h he spike o SMX
Ma e ials, me hods and equipmen
49
co esponding o each eac o . Finally, he lasks we e placed again in he incuba o o
esume ope a ion. All he p ocess was pe o med using a ume hood o ensu e s e ile
condi ions.
Ammonium, ni a e and ni i e concen a ions, TSS and VSS we e de e mined once
pe week, while he pH and ace a e concen a ion we e de e mined daily. Ace a e was
de e mined h ough gas ch oma og aphy using a DB-Wax-Agilen Technologies column
(30mx0.250mmx0.25μm) while he o he pa ame e s we e de e mined acco ding o he
s anda d analy ical me hods (APHA, 2017) desc ibed in sec ion 2.3. Besides, samples
om he eeding and he exhaus ed supe na an o he eac o s we e aken on days 2, 17
and 25 o SMX and TPs analysis. Finally, samples o p o eomic (days 1 and 25) and
16S RNA gene sequencing (day 25) analyses we e also aken.
2.3. ANALYTICAL METHODS
Se e al analy ical me hods we e applied o he expe imen al samples, gene ally
acco ding o S anda d Me hods (APHA, 2017).
2.3.1. Soluble chemical oxygen demand
The chemical oxygen demand (COD) is used o de e mine he concen a ion o he
o ganic ma e p esen in a liquid sample and is de ined as he amoun o oxygen equi ed
o oxidize he o ganic con en . The COD was de e mined ollowing a closed e lux
i ime ic me hod based on he S anda d Me hods 5220C (APHA, 2017), bu wi h mino
modi ica ions. O e all, he p ocedu e uses a s ong chemical oxidan (po assium
dich oma e) in an acid medium, along wi h a ca alys (sil e sulpha e) ha imp o es he
oxida ion o some o ganic compounds. A e diges ion, he un educed po assium
dich oma e is i a ed wi h e ous ammonium sulpha e o de e mine i s consump ion.
Then, using his alue, he amoun o oxidized o ganic ma e p esen in he sample is
calcula ed in e ms o oxygen equi alen s. The eac ion is shown below:
ଶଶି ାଷାଶଶ(1)
The equi ed eagen s, which allow o a de ec ion ange be ween 0.09 and 0.9 g
COD L-1, a e lis ed below:
xS anda d po assium dich oma e diges ion solu ion: 10.216 g o K2C 2O7
and 33 g o HgSO4a e dissol ed in 0.5 L o dis illed wa e p io o he addi ion o
Chap e 2
50
167 mL o concen a ed H2SO4. Then, he solu ion is cooled down o oom
empe a u e and he solu ion is dilu ed o each a o al olume o 1 L.
xSulphu ic acid eagen (ca aly ic solu ion): 10.7 g Ag2SO4a e added o 1
L o concen a ed H2SO4.
xFe oin indica o solu ion: 1.485 g o C18H18N2·H2O and 0.695 g o
SO4Fe·7H2O a e dissol ed in 0.1 L o dis illed wa e .
xPo assium dich oma e solu ion 0.05N: 1.226 g o K2C 2O7a e dissol ed
in 0.5 L o dis illed wa e .
xFe ous ammonium sulpha e i an (FAS) 0.035N: 13.72 g o
Fe(NH4)2(SO4)2·6H2O a e dissol ed in dis illed wa e be o e adding 20 mL o
concen a ed H2SO4and dissol ing o a o al olume o 1 L.
Fi s ly, samples a e il e ed h ough a po e size il e o 0.45 μm (MF-Millipo e,
Millipo e) o de e mine he soluble COD. Then, 2.5 mL o each sample a e placed in 10
mL diges ion glass essels, o which 1.5 mL o he diges ion solu ion and 3.5 mL o he
sulphu ic acid eagen a e added slowly and ca e ully o a oid mixing o he eagen s a
his s age. Simila ly, blanks a e p epa ed using 2.5 mL o dis illed wa e . All essels a e
sealed wi h Te lon, igh ly capped and mixed be o e placing hem in he block diges e
(ECO 16, VELP Scien i ic) p e iously se a 150 ºC o wo hou s. A e diges ion, he
ubes a e cooled o oom empe a u e and hen each sample is ans e ed o a 50 mL
E lenmeye lask which is magne ically s i ed. Then, 1-2 d ops o he e oin indica o
a e added and he solu ion is i a ed using a mic obu e wi h FAS un il a change in colo
om ligh blue o ed/o ange is obse ed.
To de e mine he no mali y o he FAS solu ion, 5 mL o he po assium dich oma e
s anda d solu ion a e mixed wi h 5 mL o dis illed wa e and 3.5 mL o he ca aly ic
solu ion. Then, a e cooling down he sample o oom empe a u e, i a ion is pe o med
as p e iously explained.
Finally, he COD concen a ion is calcula ed aking in o accoun he wo equa ions
below:
ୗ ൌǤଶହ
ూఽ (2)
ቀ
ቁൌሺିሻూఽ଼
(3)
Ma e ials, me hods and equipmen
51
Whe e NFAS is he no mali y o he FAS solu ion (mol L-1); VFAS is he olume o he
FAS solu ion consumed in he i a ion (mL); A and B a e he olumes o he FAS solu ion
consumed by he blank and he sample, espec i ely; V is he sample olume used be o e
dilu ion; DF is he dilu ion ac o applied o he sample and 8 s ands o he con e sion
ac o be ween COD and FAS (g COD mol-1 FAS).
2.3.2. Ni ogen compounds
All ni ogen analyses we e pe o med a e il e ing he samples (0.45 μm, MF-
Millipo e, Millipo e) o emo e solid pa icles, acco ding o he S anda d Me hods
(APHA, 2017).
2.3.2.1. Ni i e
Ni i e concen a ion was de e mined spec opho ome ically using a me hod based
on he p oduc ion o a iole azo dye a a pH anging be ween 2 and 2.5. I consis s o a
coupling be ween diazo ized sulphanilamide and N-(1-naph hyl)e hylenediamine
dihyd ochlo ide (NED dihyd ochlo ide), as desc ibed in he S anda d Me hods (APHA,
2017) unde he 4500-NO2-B p ocedu e (Colo ime ic me hod). The equi ed eagen s
a e:
xSulphanilamide solu ion: 10 g o sulphanilamide (C6H8N2O2S) a e
dissol ed in 0.1 L o concen a ed HCl and 0.6 L o dis illed wa e . When he
solu ion is cool, addi ional dis illed wa e is added un il eaching a o al olume
o 1 L.
xNED solu ion: 0.5 g o NED (C10H7NH(CH2)2NH2·2HCl) a e dissol ed in
0.5 L o dis illed wa e .
The de e mina ion p ocedu e consis s o he addi ion o 0.1 mL o each eagen o 5
mL o sample. Then, a leas 20 minu es o eac ion ime a e equi ed be o e measu ing
he colo ed sample a a wa eleng h o 543 nm in he spec opho ome e (Shimadzu UV-
1800).
The quan i ica ion is done wi h a calib a ion cu e, which is p epa ed by making
dilu ions in he ange o 0-0.25 mg NO2--N L-1 om a s ock solu ion o 10 mg NO2-L-1.
Then, he abso bance o each sample is measu ed spec opho ome ically and ela ed o
he ni i e concen a ion (NO2--N L-1) (Fig. 2.2.).
Chap e 2
52
Fig. 2.2. Calib a ion cu e o he de e mina ion o he concen a ion o ni i e.
2.3.2.2. Ni a e
Ni a e concen a ion was de e mined h ough measu emen s o UV abso p ion a he
wa eleng hs o 220 and 275 nm, which allows a oiding e o s caused by he abso bance
o dissol ed o ganic ma e . The p ocedu e co esponds o he 4500-NO3-B me hod lis ed
in he S anda d Me hods (APHA, 2017). The equi ed eagen s a e he ollowing:
xHCl 1 N: 8.3 mL o HCl (37% w/ , 1.19 g L-1) dissol ed in 0.1 L o
dis illed wa e .
xSul amic acid: Comme cial eagen (H3NSO3).
The de e mina ion p ocedu e consis s o adding 100 μL o HCl 1 N and a pinch o
sul amic acid, which allows a oiding in e e ences in he measu emen caused by ni i e,
o 5 mL o sample. Then, he abso bance is measu ed a 220 and 275 nm using he
spec opho ome e (Shimadzu UV-1800) and he ni a e concen a ion (NO3--N L-1) is
calcula ed using he calib a ion cu e (Fig. 2.3.). The abso bance ela ed o ni a e is
ob ained acco ding o Eq. 4.
ሺଷିെሻൌଶଶ െʹଶହ (4)
αͲǤʹͺ͵ͶͶǦ ͲǤͲͲͳͻͲ
;αͲǤͻͻͻ
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
1.0
mg NO
2
--N/L
Abso bance
Ma e ials, me hods and equipmen
53
Fig. 2.3. Calib a ion cu e o he de e mina ion o he concen a ion o ni a e.
The calib a ion cu e is p epa ed using a 10 mg NO3- L-1 s ock solu ion, which is
dilu ed o ob ain samples anging be ween 0-3 mg NO3--N L-1. Then, he abso bance is
measu ed spec opho ome ically and ela ed o he ni a e concen a ion.
2.3.2.3. Ammonium
A spec opho ome ic me hodology was selec ed o de e mine he concen a ion o
ni ogen in he ammonium o m. The analysis is based on he p oduc ion o indophenol
blue h ough he eac ion o ammonia wi h salicyla e and hypochlo i e, unde he p esence
o sodium ni op usside (Bowe and Holm-Hansen, 1980). Thus, he equi ed eagen s
a e he ollowing:
x Reagen A: 0.28 g o sodium ni op usside (Na2(Fe(CN)5NO)) and 440 g o
sodium salicyla e (C7H5NaO3) dissol ed in 1 L o dis illed wa e .
x Reagen B: 18.5 g o NaOH and 120 g o sodium ci a e (Na3C6H5O7) dissol ed
in 1 L o dis illed wa e .
x Reagen C: 5% (w/w) comme cial solu ion o sodium hypochlo i e (NaClO).
x Reagen D: Solu ion consis ing o a mix u e o eagen B and Reagen C in a 7:1
a io. This solu ion emains s able o 1 h a e p epa a ion.
The de e mina ion p ocedu e consis s o adding 600 μL o eagen A and 1 mL o
eagen D o 5 mL o he p e iously il e ed sample. Then, he mix u e is le o eac o
2 h in an en i onmen p o ec ed om ligh . The measu emen o he colo ed sample was
α͵ǤͻͺͲͻͷΪͲǤͲͲͺͻ
;αͲǤͻͻͻ
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
0.8
mg NO
3
--N/L
Abs1 - 2 · Abs2
Chap e 2
54
pe o med wi h a spec opho ome e (Shimadzu UV-1800) a a wa eleng h o 640 nm
and he quan i ica ion was done wi h help o a calib a ion cu e (Fig. 2.4.).
The calib a ion cu e is p epa ed using a s ock solu ion o 10 mg NH4+L-1, which was
dilu ed in se e al samples in he ange o 0-0.9 mg NH4+-N L-1. Then, he abso bance o
each sample was measu ed in he spec opho ome e and linked o he p epa ed ammonia
concen a ion.
Fig. 2.4. Calib a ion cu e o he de e mina ion o he concen a ion o ammonium.
2.3.3. Solids concen a ion
TSS and VSS we e de e mined acco ding o he me hods 2540D and 2540E,
espec i ely, de ailed in he S anda d Me hods (APHA, 2017).
Fo he de e mina ion o he TSS, a well-mixed sample o he bio eac o s is aken
(aiming o yield a esidue be ween 2.5 and 200 mg) and il e ed h ough a weigh ed glass
ibe il e (Wha man, GF/C, 4.7 cm o diame e , 1.2 μm o po e size), which needs o be
d ied be o e i s use in he o en (a ound 15 min). The esidue e ained in he il e is d ied
in he o en a 105ºC un il a cons an weigh is eached (app oxima ely 2-3 h). The weigh
inc ease in he il e ep esen s he TSS con en in he sample, as shown in he ollowing
equa ion:
ሺȀሻൌି
(5)
αͲǤͶͶͻʹǦ
ͲǤͲͺͳͺ
;αͲǤͻͻͻͺͺ
0.0
0.2
0.4
0.6
0.8
1.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2
1.4
mg NH4+-N/L
Abso bance
Ma e ials, me hods and equipmen
55
Whe e A ep esen s he weigh o he o en-d ied il e , B he weigh o i s a e and
V he olume o he liquid sample aken om he eac o .
The VSS is de e mined by ans e ing he d ied il e s, which allowed o calcula e
he TSS, o a mu le u nace o calcina ion a 550ºC o 20-30 min, un il a cons an
weigh is eached. The weigh loss du ing igni ion co esponds o he ola ile ac ion
(Eq. 6), p o iding an es ima e o he o ganic ma e concen a ion p esen in he solid
ac ion o he bio eac o s.
ሺȀሻൌିେ
(6)
Whe e A s ands o he weigh o he o en-d ied il e , C o he weigh o he il e
a e calcina ion a 550ºC and V o he olume o he liquid sample aken om he
eac o .
2.3.4. Dissol ed oxygen, empe a u e and pH
The pH was measu ed in he eac o s using an elec ode (C ison Ins umen s GLP22,
USA) equipped wi h an au oma ic compensa o y empe a u e de ice connec ed o a pH-
me e (GLP-22, C ison Ins umen s). The elec ode was calib a ed a oom empe a u e
wi h wo s anda d bu e solu ions o pH 7.00 and 4.00.
The dissol ed oxygen was measu ed using a p obe wi h a memb ane-co e ed
gal anic dissol ed oxygen senso (LDO101) connec ed o a digi al mul ime e de ice
(HQ40D, HACH Lange, USA). This same p obe was equipped wi h a he mopa ha was
used o ake empe a u e measu emen s.
2.3.5. O ganic Mic opollu an s
The de e mina ion o OMPs concen a ion in he liquid phase o he bio eac o s
begins wi h a cen i uga ion s ep a 3500 pm o 10 min, while he eeding samples,
which lack a solid ac ion, do no need his s ep. Then, he supe na an s and he eeding
samples a e p e il e ed (AP4004705, Millipo e) and il e ed again a 0.45 μm
(HAWP04700, Millipo e). Las ly, solid-phase ex ac ion (SPE) is pe o med wi h 200
mL samples and 60 mg Oasis HLB ca idges (Wa e s, Mil o d, MA, USA), as desc ibed
by Fe nandez-Fon aina e al. (2013). The quan i ica ion o an ibio ics (ERY, ROX, SMX,
TMP), neu od ugs (FLX, CBZ, DZP) and ho mones (E1, E2, EE2) was pe o med using
an Agilen G1312A liquid ch oma og aphy ins umen wi h a bina y pump and au oma ic
Chap e 2
62
ୢେ
ୢ୲ ൌሺ୲ሻ
െ౫౪ሺ୲ሻ
െቀୢେ
ୢ୲ ቁ୴୭୪ୟ୲୧୪୧ୟ୲୧୭୬ െቀୢେ
ୢ୲ ቁୱ୭୰୮୲୧୭୬ ቀୢେ
ୢ୲ ቁୢୣୱ୭୰୮୲୧୭୬ െ
ቀୢେ
ୢ୲ ቁୠ୧୭୲୰ୟ୬ୱ୭୰୫ୟ୲୧୭୬ (12)
ୢେ
ୢ୲ ൌሺ୲ሻ
െ౫౪ሺ୲ሻ
ቀୢେ
ୢ୲ ቁୱ୭୰୮୲୧୭୬ െቀୢେ
ୢ୲ ቁୢୣୱ୭୰୮୲୧୭୬ (13)
Whe e CW(μg L-1) is he dissol ed OMPs concen a ion, Cs(μg L-1) is he OMPs
concen a ion in he solid phase, Fin (μg d-1) is he inle OMPs low a e, Fou (μg d-1) is
he ou le OMPs low a e and V (L) is he bio eac o olume.
Vola iliza ion is based on he ans e due o an equilib ium p ocess be ween he
liquid and gas phase and depends mos ly on he physicochemical p ope ies o he OMPs
and he ope a ing condi ions o he p ocess. In an ac i a ed sludge p ocess, ola iliza ion
can occu due o s ipping, which is con olled by he ai low, he OMPs concen a ion
and he Hen y’s cons an , and due o su ace ola iliza ion, which usually is no aken
in o accoun as a esul o i s lowe ele ance (L d I C Consul an s, 2001). Vola iliza ion
can be neglec ed when a compound has a Hen y cons an below 10 Pa m3mol-1 and i s
Hc/Kow (Hen y’s cons an di ided by he oc anol-wa e pa i ion coe icien ) is lowe han
10-4 (L d I C Consul an s, 2001). This is he case o he OMPs s udied in his hesis (Table
2.1.).
So p ion is based on he ans e due o an equilib ium p ocess be ween he liquid
and solid phase, including dissol ed and colloidal o ganic ma e , whe e OMPs
simul aneously unde go so p ion and deso p ion phenomena (Ba e e al., 2010).
Equilib ium is assumed o be eached gene ally in a ma e o hou s and depends on he
OMPs physicochemical p ope ies (including hyd ophobici y, ca ion exchange, ca ion
b idging, su ace complexa ion and hyd ogen-bonding (Plósz e al., 2010)), sludge
quali y and ope a ing condi ions (Pomiès e al., 2013). Two mechanisms a e in ol ed in
he so p ion p ocess: (i) abso p ion, occu ing due o hyd ophobic in e ac ions be ween
he alipha ic and a oma ic g oups o he OMPs and he lipophilic cell memb ane and a
ac ions o he mic oo ganisms and (ii) adso p ion, happening due o elec os a ic
in e ac ions o he posi i ely cha ged g oups o he OMPs and he nega i ely cha ged
su aces o he mic obial popula ion (Sipma e al., 2010). Since OMPs a e p esen a low
Ma e ials, me hods and equipmen
63
concen a ions in WWTPs, a linea model is no mally assumed o desc ibe his
phenomenon (Pomiès e al., 2013), as shown below (Eq. 14 and 15):
ቀୢେ
ୢ୲ ቁୱ୭୰୮୲୧୭୬ൌୱ୭୰ୗୗ (14)
ቀୢେ
ୢ୲ ቁୢୣୱ୭୰୮୲୧୭୬ൌୢୣୱ୭୰ୗ(15)
Whe e kso is he so p ion kine ic cons an (L g-1 d-1), kdeso is he deso p ion kine ic
cons an (d-1) and XTSS is he o al suspended solids concen a ion (g L-1).
The pa i ion coe icien (Kd), shown in Eq. 16, is he a io be ween he kine ic
cons an s a equilib ium condi ions:
ୢൌ୩౩౨
୩ౚ౩౨ ൌେ
େଡ଼ (16)
Bio ans o ma ion consis s o he biological emo al o he compounds, which is
ypically calcula ed o he dissol ed OMPs (Pomiès e al., 2013). Pseudo- i s o de
kine ics a e ypically used o model he OMPs bio ans o ma ion a e ( biol), ep esen ed
in Eq. 17 (Schwa zenbach e al., 2003):
ቀୢେ
ୢ୲ ቁୠ୧୭୲୰ୟ୬ୱ୭୰୫ୟ୲୧୭୬ ൌୠ୧୭୪ൌୠ୧୭୪ୗୗ (17)
The biodeg ada ion kine ic cons an s we e calcula ed pe o ming mass balances o
he ba ch expe imen s (Chap e 3), as shown in Eq. 18:
ୠ୧୭୪ሺିଵିଵሻൌେబିେ౪ሺଵା୩ౚଡ଼ሻ
େ౪ଡ଼୲ (18)
Whe e Cw0 (μg L-1) is he dissol ed OMPs concen a ion a ime 0 and Cw (μg L-1)
is he dissol ed OMPs concen a ion a ime , whe e he slope is maximum.
The bio ans o ma ion yield (Eq. 19) and he speci ic bio ans o ma ion a e (Eq. 20)
we e calcula ed o desc ibe he beha io o he OMPs. The yield de e mines he
bio ans o ma ion e iciency o he compounds and he bio ans o ma ion a e p o ides
in o ma ion ega ding he come abolic beha io o he OMPs and he pe o mance o he
eac o .
Chap e 2
64
ሺΨሻൌି౭౫౪ି౩౫౪
ͳͲͲ (19)
ሺρୗୗିଵିଵሻൌି౭౫౪ି౩౫౪
ଡ଼ ͳͲͲ (20)
Whe e Fwou (μg d-1) is he ou le dissol ed OMPs low a e and Fsou (μg d-1)is he
ou le OMPs low a e in he solid phase.
The ou le OMPs low a e in he solid phase (Fsou ) is calcula ed as shown in Eq. 21:
ୱ୭୳୲ ൌୗԢୗୗିୣ୪୳ୣ୬୲ ୣ୪୳ୣ୬୲ୗԢୗୗି୮୳୰ୣ୮୳୰ୣ (21)
Whe e CS’ (μg g-1) is he OMPs concen a ion in he solid phase, Fe luen (L d-1) is he low
o he e luen and Fpu ge (L d-1) is he low o he pu ge.
In Chap e 3, o de e mine i he yields and speci ic bio ans o ma ion a es o he
OMPs we e s a is ically di e en a he es ed p ima y subs a e speci ic biodeg ada ion
a es, R so wa e 3.6.2. was used. The s a is ical es s we e pe o med a a 5% signi icance
le el (p < 0.05).
CHAPTER 3
CHAPTER 3. The o ganic loading a e a ec s o ganic mic opollu an s’
come abolic bio ans o ma ion kine ics unde he e o ophic condi ions
in ac i a ed sludge
SUMMARY
O ganic mic opollu an s (OMPs) a e bio ans o med come abolically in ac i a ed
sludge sys ems. Howe e , he indi idual ole ha he e o ophs play is s ill no clea and
he e is s ill a gap ega ding he in luence o he he e o ophic ac i i y on he come abolic
bio ans o ma ion kine ics and yield o he OMPs. To answe hese ques ions,
expe imen s wi h inc easing p ima y subs a e concen a ions we e pe o med unde
ae obic he e o ophic condi ions in a con inuous s i ed ank eac o ope a ed a se e al
o ganic loading a es (OLR) wi h ixed hyd aulic e en ion ime. Mo eo e , he indi idual
kine ic pa ame e s we e de e mined in ba ch assays wi h di e en ini ial subs a e
concen a ions using he sludges om he con inuous eac o . A se o 15 OMPs
displaying a a ie y o physicochemical p ope ies we e spiked o he eeding in he ng
L-1 -μg L-1 ange. Resul s e eal ha he biodeg ada ion o he p ima y ca bon sou ce
and he bio ans o ma ion o he OMPs occu simul aneously, in e idence o come abolic
beha iou . Mo eo e , he OMPs bio ans o ma ion kine ic cons an (kbiol) shows a linea
dependence wi h he OLR o he p ima y subs a e o mos compounds, sugges ing ha
he he e o ophic ac i i y a ec s he OMPs bio ans o ma ion kine ics. Howe e , unde
ypical ac i a ed sludge sys ems ope a ing condi ions (hyd aulic e en ion imes abo e 8
h), he bio ans o ma ion yield would no be signi ican ly a ec ed.
This chap e , unde he consen o he publishe (Else ie ), was ed a ed a e :
Kennes-Veiga, Gonzalez-Gil, L., Ca balla, M., Lema, J. M. (2021). The o ganic loading
a e a ec s o ganic mic opollu an s’ come abolic bio ans o ma ion kine ics unde
he e o ophic condi ions in ac i a ed sludge. Wa e Resea ch, 189, 116587. ISSN: 0043-
1354.
Fu he in o ma ion can be ound in he “Lis o publica ions” (page 145).
Chap e 3
66
3.1. INTRODUCTION
To cha ac e ize OMPs bio ans o ma ion, a pseudo- i s o de kine ic model is
commonly assumed and he esul ing bio ans o ma ion kine ic cons an (kbiol) la gely
depends on he physicochemical p ope ies o each pa icula pollu an (Lema and Sua ez,
2017). In ac , Falås e al. (2016) concluded ha kbiol a ies mo e depending on he na u e
o he OMPs han on he expe imen al condi ions. Howe e , kbiol, as a kine ic cons an , is
also a ec ed by he eac o ope a ing condi ions (such as empe a u e, pH and oxida ion
educ ion po en ial), he p esence and a ailabili y o co-subs a es and he biochemical
e sa ili y o he sludge, as se e al s udies demons a e (Ba celó, 2012; Pe o ic e al.,
2013). Fo ins ance, Al a ino e al. (2016) epo ed kbiol alues o 0.09 and 0.05 L g VSS-
1d-1 o SMX unde ae obic he e o ophic and au o ophic deni i ying condi ions,
espec i ely; Gulde e al. (2014) epo ed kbiol inc emen s o one o de o magni ude o
a enolol a pH 8 compa ed o pH 6 unde ac i a ed sludge condi ions and Li e al. (2005)
epo ed ha E2 inc eased i s kbiol alue om 1.8 o 3.3 L g VSS-1 h-1 when inc easing he
empe a u e om 20 o 35ºC in ba ch expe imen s wi h ac i a ed sludge. None heless,
con e sely, he e a e so a no conclusi e s udies ega ding he in luence o he OLR and
he he e o ophic ac i i y on kbiol.
The ole o he OLR in he emo al o he OMPs is, in ac , a opic o cu en and
gene al in e es in biological sys ems, bu esul s s ill do no show a clea end. Fo
ins ance, Ko a e al. (2020) showed ha inc easing he OLR imp o ed he emo al o 3
ou o 5 OMPs spiked in a me hanogenic-ae obic mo ing bed bio ilm eac o and Moya-
Llamas e al. (2018) ound be e biodeg ada ions o 6 OMPs a highe OLRs in a UASB
eac o coupled o a MBR. On he con a y, in a mo ing bio ilm bed eac o , Ab ahi e al.
(2018) epo ed o some OMPs a maximum emo al a he highes OLR es ed and o
o he s a he lowes OLR and Ca nei o e al. (2020) epo ed a nega i e impac o
inc easing OLRs in he biodeg ada ion o ci alop am and SMX in anae obic ix bed
bio ilm eac o s. Finally, di e en ly om hese s udies, Gonzalez-Gil e al. (2018) ound
no co ela ion be ween a ia ions in he OLR and he bio ans o ma ion o mos OMPs
in me hanogenic diges e s.
The objec i e o he p esen chap e is o ex end he knowledge behind OMPs
bio ans o ma ion p ocesses unde exclusi ely ae obic he e o ophic condi ions. Mo e
speci ically, esea ch is ocussed on assessing he OMPs come abolic bio ans o ma ion,
The OLR a ec s OMPs come abolic bio ans o ma ion kine ics
67
aiming o de e mine he ela ionship be ween he in ensi y o he he e o ophic ac i i y
and he OMPs bio ans o ma ion a e, wi h a pa icula ocus on e alua ing he e ec on
kbiol. The implica ions o unde s anding such linkage could be o g ea impo ance o
design biological sys ems able o maximize OMPs emo al. To each hese goals, a se ies
o expe imen s in a con inuous s i ed ank eac o ope a ed a di e en OLRs we e
pe o med, as well as ba ch expe imen s se wi h di e en ini ial COD concen a ions.
3.2. MATERIALS AND METHODS
An ae a ed 5 L con inuous s i ed ank eac o connec ed o a 2 L se le (sec ion
2.2.1.) se a an HRT o 1 d and a SRT a ound 5 d was inocula ed wi h ac i a ed sludge
a a concen a ion o 1 g L-1. I was ope a ed a ou di e en OLRs (0.2, 0.4, 0.6 and 0.8
g COD L-1 d-1, ha esul ed in 0.4, 0.6, 1.1 and 2.7 g COD g VSS-1 d-1), selec ed ying o
co e ypical condi ions o ac i a ed sludge WWTPs (Lema and Sua ez, 2017; Me cal
& Eddy, 2014). Each expe imen al s age was main ained o one mon h and he OLR was
changed by a ying he o ganic ma e concen a ion in he eeding while keeping
cons an he HRT. The eeding was based on ace a e as o ganic ca bon sou ce (sec ion
2.1.2.) and con ained ERY, ROX, TMP, SMX, IBP, NPX, CBZ, DZP and TCS a
concen a ions o 10 μg L-1; E1, E2 and EE2 a 1 μg L-1 and ADBI, HHCB and AHTN a
40 μg L-1 (sec ion 2.1.1). Once s eady s a e was eached a each OLR, inle and ou le
OMPs concen a ions we e measu ed (solid and liquid phase) by aking h ee samples
om he eeding and he eac o essel in h ee consecu i e days.
Besides, analysis o he in luen and e luen o he eac o was pe o med o
de e mine he ypical ope a ional pa ame e s acco ding o he s anda d analy ical me hods
desc ibed in Chap e 2. The soluble COD, ammonium, ni a e and ni i e concen a ions,
SSV, pH and empe a u e we e measu ed wo o h ee imes pe week.
Addi ionally, 4 ba ch assays using he same g oup o OMPs we e se o analyze hei
bio ans o ma ion kine ics and kbiol. The expe imen s we e ae a ed and pe o med a
neu al pH, 25ºC, 150 pm and COD ini ial concen a ions o 0.2, 0.4, 0.6 and 0.8 g L-1
by a ying he o ganic ca bon concen a ion while main aining he same mic o and
mac onu ien s used in he he e o ophic eac o . The inoculum was aken om he
con inuous eac o s wo king a he espec i e OLRs, aiming a an ini ial concen a ion o
0.80 g VSS L-1. Fo each ba ch assay, 18 lasks we e p epa ed, h ee o each ime poin
o ha e iplica es (0, 1, 3, 8, 24 and 48 h). A he men ioned imes, 3 lasks we e aken
Chap e 3
68
o de e mine he concen a ion o he OMPs in he solid and liquid phases. The same
con en ional ope a ional pa ame e s as in he con inuous eac o we e de e mined a each
ime poin , excep o he solids and ni ogen concen a ions which we e measu ed a
imes 0, 24 and 48 h.
3.3. RESULTS AND DISCUSSION
3.3.1. Bio ans o ma ion yield
Fig. 3.1 shows he bio ans o ma ion yield, i.e. he bio ans o ma ion pe cen age, o
he selec ed OMPs a he 4 OLRs in he ae obic he e o ophic eac o . The
bio ans o ma ion ex en a ied conside ably among OMPs, being hei beha iou
cha ac e ized as (i) low bio ans o ma ion (below 20%) o CBZ and DZP, (ii) medium-
low bio ans o ma ion (20-50%) o TMP, (iii) medium-high bio ans o ma ion (50-80%)
o ERY and (i ) high bio ans o ma ion (o e 80%) o ROX, SMX, IBP, NPX, TCS,
ADBI, HHCB, AHTN, E1, E2 and EE2. These esul s a e consis en wi h li e a u e
in o ma ion (Al a ino e al., 2018b; Fe nandez-Fon aina e al., 2016).
Fig. 3.1. OMPs bio ans o ma ion yield achie ed in he ae obic he e o ophic eac o
ope a ed wi h ixed HRT (1 day) a 4 di e en speci ic OLRs o he p ima y subs a e.
0
10
20
30
40
50
60
70
80
90
100
ERY
ROX
SMX
TMP
IBP
NPX
TCS
ADBI
HHCB
AHTN
E1
E2
EE2
CBZ
DZP
Bio ans o ma ion (%)
0.4 g COD/g VSS·d 0.6 g COD/g VSS·d 1.1 g COD/g VSS·d
2.7 g COD/g VSS·d
The OLR a ec s OMPs come abolic bio ans o ma ion kine ics
69
I can be obse ed ha he bio ans o ma ion yield o mos OMPs emained cons an
h oughou he di e en OLRs (bio ans o ma ion di e ences below 10 pe cen age poin s
and no s a is ically signi ican ; p > 0.05 ). Only one alue o ERY (expe imen wi h 1.1
g COD g VSS-1 d-1) and ano he o IBP (expe imen wi h 2.7 g COD g VSS-1 d-1),
signi ican ly (p < 0.05) de ia ed (be ween 15-20 pe cen age poin s) om he alues o
he o he h ee condi ions. These e en s o ERY and IBP canno be a ibu ed o a speci ic
end o beha iou . The e o e, o e all, he esul s o Fig. 3.1 indica e ha inc easing he
ae obic he e o ophic come abolism did no a ec he OMPs bio ans o ma ion yield
unde he condi ions es ed. Se e al hypo heses could explain his beha iou . The i s
one is ha he enzymes in ol ed in he OMPs bio ans o ma ion migh no be
inc easingly s imula ed a highe OLRs. Acco ding o S adman (1970), in ca abolic
pa hways, enzymes can be classi ied as “cons i u i e” enzymes, whose concen a ion is
independen o he p esence o hei subs a es, and “inducible” enzymes, ha a e
p oduced when hei immedia e subs a es o sui able de i a i es a e p esen . I he
enzymes in ol ed in he bio ans o ma ion o he OMPs belong o he i s class and we e
no inc easingly s imula ed, he yield could ha e emained s able e en a highe OLRs. A
second hypo hesis is ha he maximum come abolic a e owa ds OMPs is al eady
eached a he lowes OLR and, he e o e, inc easing he deg ada ion a e o he p ima y
subs a e (ace a e) does no ha e an e ec on he OMPs. In his ega d, i is commonly
assumed ha he oxida ion a e o a non-g ow h subs a e (OMPs) should always be
linked o he oxida ion a e o a g ow h subs a e (ace a e) p opo ionally; howe e , some
s udies ha e shown ha his is no necessa ily always he case (C iddle, 1993; M. H. Kim
e al., 2020). Thi dly, he OMPs may ha e al eady achie ed hei bio ans o ma ion limi s
due o he modynamic cons ain s. This e en could be caused by e e sibili y o he
biological eac ions, leading o a chemical equilib ium be ween he pa en compound and
he TPs, as p e iously sugges ed (Gonzalez-Gil e al., 2019a). Las ly, he p ima y
me abolism may inc ease he speci ic bio ans o ma ion a e, bu he HRT may ha e been
high enough o hide his e ec , showing he same bio ans o ma ion yield in all cases. In
common biological ea men s, as in ac i a ed sludge, he op imal HRT o OMPs
emo al is 24 h o longe (Boonno a e al., 2019). Ne e heless, i has been p o en ha
lowe imes may be su icien o achie e he maximum bio ans o ma ion ex en o
se e al compounds (Boonno a e al., 2019; Ejhed e al., 2018). O e all, he esul s shown
in Fig. 3.1 a e insu icien o de e mine which o hese ou hypo heses is mo e likely,
being necessa y o e alua e he beha io o he bio ans o ma ion a e.
Chap e 3
70
3.3.2. Speci ic bio ans o ma ion a e
In Fig. 3.2, i can be obse ed he di ec ela ionship be ween he OMPs
bio ans o ma ion a e and he speci ic sludge ac i i y: he highe he speci ic
biodeg ada ion a e o he g ow h subs a e, he highe he OMPs speci ic
bio ans o ma ion a e. The dependence o he bio ans o ma ion a e o he seconda y
subs a es (OMPs) wi h he biodeg ada ion o he p ima y subs a e (ace a e) is clea p oo
o come abolism (Lema and Sua ez, 2017). When he concen a ion o he p ima y
subs a e is inc eased (i.e., a highe OLR is applied), a highe mic obial ac i i y o he
biomass is achie ed. Hence, i an inc emen o he OMPs bio ans o ma ion a e also
occu s, i indica es ha he OMPs a e been come abolized by he same enzymes in ol ed
in he me abolism o he g ow h subs a e. In Fig. 3.2, he di e ences in he speci ic
bio ans o ma ion a es we e s a is ically signi ican (p < 0.05) o all expe imen al
condi ions and all OMPs es ed.
A)
0.0
0.5
1.0
1.5
2.0
E1 E2 EE2
Bio ans o ma ion a e
(
P
g OMP bio ans o med /g VSS·d)
0.4 g COD/g VSS·d 0.6 g COD/g VSS·d
1.1 g COD/g VSS·d 2.7 g COD/g VSS·d
The OLR a ec s OMPs come abolic bio ans o ma ion kine ics
71
B)
C)
Fig. 3.2. Speci ic OMPs bio ans o ma ion a e (Pg OMP g VSS-1 d-1) a 4 speci ic
biodeg ada ion a es o he p ima y subs a e (g COD g VSS-1 d-1). A) OMPs ed a 1 Pg
L-1 d-1. B) OMPs ed a 40 Pg L-1 d-1. C) OMPs ed a 10 Pg L-1 d-1.
0
10
20
30
40
50
60
ADBI HHCB AHTN
Bio ans o ma ion a e
(
P
g OMP bio ans o med /g VSS·d)
0.4 g COD/g VSS·d 0.6 g COD/g VSS·d
1.1 g COD/g VSS·d 2.7 g COD/g VSS·d
0
5
10
15
20
25
30
ERY ROX SMX TMP IBP NPX TCS
Bio ans o ma ion a e
(
P
g OMP bio ans o med /g VSS·d)
0.4 g COD/g VSS·d 0.6 g COD/g VSS·d
1.1 g COD/g VSS·d 2.7 g COD/g VSS·d
Chap e 3
linea ly. The excep ions o HHCB and ADBI could indica e ha hey would ha e equi ed
lowe o highe mic obial ac i i ies, p o ing ha kbiol is in luenced bo h by he mic obial
ac i i y and he na u e o he compound.
To be e unde s and he in luence o he mic obial ac i i y on he bio ans o ma ion
o he OMPs, Table 3.1 shows an es ima ion o he bio ans o ma ion a es ha could be
achie ed o ROX, NPX, SMX and HHCB in a eal ac i a ed sludge sys em designed o
only emo e o ganic ma e , as well as he HRT ha hese OMPs would equi e o
comple e bio ans o ma ion acco ding o he expe imen al kbiol alues (Fig. 3.4). Fo
ins ance, in he case o NPX, assuming an inle concen a ion o 10 Pg L-1, a kbiol
equi alen o 0.7 L g VSS-1 d-1 would p o ide a bio ans o ma ion a e o 14 Pg NPX L-1
d-1 and an HRT o 17 h would be equi ed. On he o he hand, a kbiol o 3.8 L g VSS-1 d-
1would lead o a bio ans o ma ion a e o 76 Pg NPX L-1 d-1 and an HRT o 3 h could be
su icien o o al NPX emo al. The esul s indica e ha highe he e o ophic ac i i ies
could conside ably educe HRT equi emen s hanks o imp o ed OMPs speci ic
bio ans o ma ion a es. Mo eo e , excep o DZP and CBZ, which a e ecalci an unde
he e o ophic condi ions, all he OMPs would be almos comple ely emo ed a e 8 h
(Table 3.1). Acco dingly, p omo ing he he e o ophic ac i i y wi h highe OLRs would
no ha e signi ican e ec s on he bio ans o ma ion yield a ypical HRTs o ac i a ed
sludge sys ems, suppo ing he indings obse ed in Fig. 3.1.
78
The OLR a ec s OMPs come abolic bio ans o ma ion kine ics
79
Table 3.1. Bio ans o ma ion a e and HRT equi ed o ull bio ans o ma ion o ROX, NPX, SMX and HHCB in a eal WWTP based
on he kbiol alues ob ained in he ba ch assays. The XVSS alue used is a ypical solids concen a ion in WWTPs
(Me cal & Eddy,
2014), and he C
w
alue is in he ange o ypical in luen OMPs concen a ions in WWTPs (Luo e al., 2014; Pe ie e al., 2014).
OMPs
Speci ic bio . a e
(g COD g VSS
-1
d-1)
kbiol
(L g VSS-1 d-1)
X
VSS
(g L
-
1)
CW
(ߤ
ߤ
OMP L
-
1)
Bio ans o ma ion a e
(
ߤ
g OMP L-1 d-1)
HRT equi ed o 100%
bio ans o ma ion (h)
ROX
0.4
1.6
210
32
7.6
0.6
2.4
49
4.9
1.1
3.4
68
3.5
2.7
4.0
81
3.0
NPX
0.4
0.7
210
14
17.1
0.6
2.8
56
4.3
1.1
3.4
68
3.5
2.7
3.8
76
3.2
SMX
0.4
1.0
210
20
12.0
0.6
2.0
40
6.0
1.1
2.6
52
4.6
2.7
2.9
58
4.1
HHCB
0.4
5.0
210
100
2.4
0.6
3.9
78
3.1
1.1
6.7
134
1.8
2.7
5.2
104
2.3
Chap e 3
3.4. CONCLUSION
In his chap e , expe imen al da a e idenced ha highe OLRs lead o highe OMPs
bio ans o ma ion a es and ha he emo al o he o ganic ma e and he OMPs occu s
simul aneously, p o ing come abolism as he main mechanism behind he
bio ans o ma ion o OMPs unde ae obic he e o ophic condi ions. The enhancemen o
he OMPs bio ans o ma ion a e occu ed due o inc eases in hei espec i e kbiol alues,
showing ha he bio ans o ma ion kine ic cons an is no only dependen on he
compound and he en i onmen al condi ions, bu also on he in ensi y o he me abolic
ac i i y. Besides, he in luence o he he e o ophs in kbiol is p o ed o be compound
dependen . On he o he hand, he come abolic end does no necessa ily in ol e an
imp o emen in he bio ans o ma ion yield o he compounds p o iding ha a su icien
HRT is applied. In ac , in his wo k, i is shown ha he e ec i eness did no imp o e a
highe speci ic deg ada ion a es o he p ima y subs a e. Hence, he a ia ion o he
WWTP o ganic load, he managemen o he o ganic subs a es and he con ol o he
mic obial ac i i y appea as key pa ame e s go e ning OMPs bio ans o ma ion.
80
CHAPTER 4
CHAPTER 4. He e o ophic enzyma ic bio ans o ma ions o o ganic
mic opollu an s in ac i a ed sludge
SUMMARY
While he e o ophic mic oo ganisms cons i u e he majo ac ion o ac i a ed sludge
biomass, he ole o he e o ophs in he bio ans o ma ion o o ganic mic opollu an s
(OMPs) has no been ully elucida ed. Ye , such knowledge is essen ial, pa icula ly when
concei ing no el was ewa e ea men plan s based on a wo-s age p ocess including an
A-s age unde he e o ophic condi ions and a B-s age based on anammox ac i i y.
Bio ans o ma ion o OMPs in ac i a ed sludge is hough o mos ly occu
come abolically hanks o he ac ion o low speci ici y enzymes in ol ed in he
me abolism o he p ima y subs a es. Fo a be e unde s anding o he p ocess, i is
impo an o de e mine such enzyma ic ac i i ies and he unde lying mechanisms
in ol ed in OMPs bio ans o ma ion. This ask has p o en o be di icul due o he lack
o in o ma ion abou he enzyma ic p ocesses and he complexi y o he biological
sys ems p esen in ac i a ed sludge. In his chap e , a con inuous ae obic he e o ophic
eac o spiked wi h 20 OMPs a en i onmen al concen a ions was ope a ed o (i) assess
he po en ial o he e o ophs du ing he come abolic bio ans o ma ion o OMPs, (ii)
iden i y bio ans o ma ion eac ions ca alysed by ae obic he e o ophs and (iii) p edic
possible he e o ophic enzyma ic ac i i ies esponsible o such bio ans o ma ions.
Con adic ing p e ious epo s on he dominan ole o ni i ie s in OMPs emo al du ing
ac i a ed sludge ea men , he he e o ophic popula ion p o ed i s capaci y o
bio ans o m he OMPs o ex en s equi alen o epo ed alues in ni i ying ac i a ed
sludge plan s. Besides, 12 ans o ma ion p oduc s po en ially o med h ough he ac i i y
o se e al enzymes p esen in he e o ophs, including monooxygenases, dioxygenases,
hyd olases and ans e ases, we e iden i ied.
This chap e , unde he consen o he publishe (Else ie ), was ed a ed a e :
Kennes-Veiga, D. M., Vogle , B., Fenne , K., Ca balla, M., Lema, J. M. (2021).
He e o ophic enzyma ic bio ans o ma ion o o ganic mic opollu an s in ac i a ed
sludge. Science o he To al En i onmen , 780, 146564. ISSN: 0048-9697.
Fu he in o ma ion can be ound in he “Lis o publica ions” (page 145).
Chap e 4
82
4.1. INTRODUCTION
Since OMPs a e equen ly no ully mine alized du ing come abolic
bio ans o ma ion, he assessmen o he TPs is also impo an because hey pose he
po en ial o be as o e en mo e oxic han hei pa en compounds o he ecosys em
(Be kne and Thie bach, 2014; Celiz e al., 2009; Gulde e al., 2016). Besides, TPs
iden i ica ion can be a e y help ul ool o de e mine he eac ions occu ing in a speci ic
en i onmen and p o ide hin s abou he enzyma ic ac i i ies ca alysing OMPs
bio ans o ma ion. Fo ins ance, ollowing his app oach, Gulde e al. (2016)
demons a ed ha N-acyl ans e ases could be in ol ed in he bio ans o ma ion o
se e al amine-con aining OMPs in ac i a ed sludge.
In his chap e , we aim o add ess he con ibu ion o he e o ophs o he
bio ans o ma ion o OMPs in ac i a ed sludge p ocesses and, pa icula ly, hei capaci y
o bio ans o m a ange o OMPs. Besides, we a emp o de ec se e al TPs as a ool o
iden i y key OMP bio ans o ma ion eac ions ca alysed by ae obic he e o ophs and
deciphe possible enzyma ic ac i i ies ca ying ou such bio ans o ma ions. To his end,
we e alua ed he bio ans o ma ion o a se o OMPs wi h di e en physicochemical
p ope ies in an ae obic he e o ophic eac o and used liquid ch oma og aphy coupled o
high- esolu ion mass spec ome y o iden i y TPs.
4.2. MATERIALS AND METHODS
A 5 L con inuously s i ed lab-scale eac o connec ed o a 2 L se le (sec ion 2.2.1.)
was ope a ed a 25ºC wi h an OLR o 0.6 g COD L-1 d-1, HRT o 1 d and SRT o
app oxima ely 5 d o de e mine he key enzyma ic ac i i ies in ol ed in he
bio ans o ma ion o se e al OMPs. The eac o was inocula ed wi h ac i a ed sludge a
a concen a ion o 1 g L-1 and he expe imen al s age was main ained o one mon h. The
eeding included ATU o a oid he de elopmen o ni i ying ac i i y and was based on
ace a e as o ganic ca bon sou ce (sec ion 2.1.2.). Besides, 20 OMPs (sec ion 2.1.1.) we e
spiked a concen a ions o 10 μg L-1 o he pha maceu icals, 1 μg L-1 o he ho mones
and 40 μg L-1 o he ag ances.
Once s eady s a e was eached, inle and ou le OMPs and TPs concen a ions we e
measu ed (solid and liquid phase) by aking iplica e samples om he eeding and he
eac o essel in h ee consecu i e days. Besides, analysis o he in luen and e luen o
He e o ophic enzyma ic bio ans o ma ions o OMPs
83
he eac o was pe o med o de e mine he ypical ope a ional pa ame e s acco ding o
he s anda d analy ical me hods desc ibed in Chap e 2. The soluble COD, ammonium,
ni a e and ni i e concen a ions, TSS, SSV, pH and empe a u e we e measu ed wo o
h ee imes pe week.
4.3. RESULTS AND DISCUSSION
4.3.1. Reac o pe o mance
The eac o was ope a ed o 28 days and s eady-s a e condi ions we e achie ed a e
one week. Du ing expe imen a ion, neu al pH was main ained o maximize he ac i i y
o he mic oo ganisms as well as o a oid any e ec s o a ying pH le els on OMPs
emo al. Al hough equen ly ecei ing li le a en ion, pH is a key pa ame e a ec ing
OMPs bio ans o ma ion. I s inc ease by jus one pH uni can conside ably p omo e he
emo al o OMPs wi h ca ionic-neu al speci ica ion and hinde ha o compounds wi h
neu al-anionic specia ion, as shown by Gulde e al. (2014).
Biomass concen a ion emained qui e s able a 1.4 g L-1 by con olling he pu ge
(Fig. 4.1), pe o med almos daily om he se le , and he con ol o he eci cula ion,
se a a a io o 100% o he in luen low (Eq. 4). The sou ce o o ganic ma e was
ace a e, which was ully consumed (95-100%), leading o e luen COD concen a ions
consis en ly below 0.03 g L-1 (Fig. 4.1). Besides, ope a ion occu ed in he absence o
ni i ica ion (Fig. 4.1), indica ing ha all he ni ogen consumed was used only o
mic obial g ow h. Among he non-consumed ni ogen, some soluble non-deg adable
o ganic ni ogen om endogenous espi a ion may ha e been p esen .
The ne was e ac i a ed sludge p oduced was es ima ed a 0.15 g VSS L-1, including
he ac i e he e o ophic biomass o med daily and he cell deb is, which accoun s o 10-
20% o he decayed biomass and canno be deg aded due o hei ex emely low
hyd olysis kine ics (Liu and Wang, 2015). Mo eo e , he e was also an endogenous decay
a e o biomass in he eac o , p opo ional o he ac i e biomass concen a ion (XVSS),
ha accoun ed o 0.14 g VSS L-1 d-1. The endogenous decay ep esen s he cell biomass
loss and includes an in e nal decay (cell le el), in ol ing he oxida ion o s o ed
subs a es o p oduce ene gy o cell main enance, and an ex e nal decay (communi y
le el), such as cell dea h o p eda ion by highe o ganisms (Liu and Wang, 2015). Ou
eac o p esen ed mos ly ae obic he e o ophic bac e ial s ains, as well as highe li e-
Chap e 4
o ms, such as o i e s and p o ozoans, based on mic oscopic obse a ion. The high li e
o ms ha e also been shown o pa icipa e in he emo al o OMPs, as epo ed by Gulde
e al. (2018) o p o ozoa, which seem o be in ol ed in he ion apping o amine-
con aining compounds and he hyd olysis o selec es e s and phenylu ea compounds.
84
He e o ophic enzyma ic bio ans o ma ions o OMPs
85
A)
B)
0.0
0.1
0.2
0.3
0.4
0.5
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
0 5 10 15 20 25 30
Pu ge (L)
Vola ile suspended solids (g/L)
Time (d)
SSV (g/L) Pu ge (L)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
0
20
40
60
80
100
0 5 10 15 20 25 30
COD concen a ion (g/L)
COD emo al (%)
Time (d)
COD emo al (%) In luen COD (g/L)
E luen COD (g/L)
Chap e 4
86
C)
Fig. 4.1. Ope a ional pa ame e s ob ained in he con inuous ae obic he e o ophic eac o .
A) VSS concen a ion and pu ge pe o med. B) COD emo al along wi h COD in luen
and e luen concen a ions. C) In luen N-NH4+and e luen N-NH4+, N-NO3-and N-
NO2-concen a ions.
4.3.2. He e o ophic OMPs bio ans o ma ion in ac i a ed sludge sys ems
Fig. 4.2 shows he a e o he selec ed OMPs in he con inuous he e o ophic eac o .
So p ion appea s o be minimal o mos compounds (<10%), excep o FLX, o which
i accoun s o 15-20% o he o al mass balance. Resul s show ha 17 ou o he 20 OMPs
we e highly emo ed (abo e 80%) due o he e o ophic ac i i y and only 3 compounds
we e sligh ly (TMP) o no emo ed a all (CBZ, DZP). These esul s ag ee wi h he
bio ans o ma ion obse ed in ac i a ed sludge uni s (Al a ino e al., 2014; Luo e al.,
2014; Pe ie e al., 2014), excep o DCF, o which we ob ain highe alues han unde
ypical ac i a ed sludge condi ions (Al a ino e al., 2014; Fe nandez-Fon aina e al.,
2016). None heless, in o he biological ea men s, high DCF e iciencies ha e also been
achie ed, as in a hyb id bio ilm-ac i a ed sludge p ocess (Jewell e al., 2016b) and in a
ni i ying mo ing bed bio ilm eac o (To esi e al., 2016b). In ou expe imen s, DCF
0
10
20
30
40
0 5 10 15 20 25 30
Concen a ion (mg/L)
Time (d)
In luen N-NH4+ (mg/L) E luen N-NH4+ (mg/L)
E luen N-NO2- (mg/L) E luen N-NO3- (mg/L)
He e o ophic enzyma ic bio ans o ma ions o OMPs
87
was bio ans o med a he ex ensi ely, i.e., a 80% (Fig. 4.2), which could indica e he
p esence o ce ain he e o ophic gene a capable o bio ans o ming i mo e e icien ly.
In his ega d, Nguyen e al. (2019) showed ha unde exposu e o a p ima y ca bon
subs a e and DCF, ce ain gene a o ac i a ed sludge bac e ia can signi ican ly inc ease
hei abundance, sugges ing ha hey migh gain a compe i i e ad an age om i s
come abolic emo al.
Fig. 4.2. Fa e o he OMPs in he con inuous ae obic he e o ophic eac o ope a ed wi h
ixed HRT (1 d) and OLR (0.6 g COD L-1 d-1).
To unde s and he a iabili y in OMPs bio ans o ma ion epo ed in he li e a u e,
Fig. 4.3 compa es he bio ans o ma ion pe o mance o ou ae obic he e o ophic eac o
wi h ha epo ed unde ni i ying condi ions exclusi ely (sys ems no ed wi h o ganic
ca bon o ha ha e inhibi ed he he e o ophic ac i i y) and in ac i a ed sludge sys ems
p esen ing bo h he e o ophic and ni i ying ac i i ies. Fi s ly, i can be obse ed ha
he e o ophs each bio ans o ma ions simila o he alues o he ac i a ed sludge plan s,
indica ing ha hey can g ea ly con ibu e o he emo al o he OMPs, and ha , o
ce ain compounds, he e may no be he need o se up WWTPs wi h ni i ying ac i i ies.
The e is a limi ed numbe o s udies e alua ing he bio ans o ma ion o he selec ed
OMPs wi h exclusi ely ni i ying popula ions. In such wo ks, hey ha e also epo ed
0
10
20
30
40
50
60
70
80
90
100
OMPs a e (%)
Bio ans o ma ion So p ion E luen
Chap e 4
94
Table 4.1. Summa y o he sugges ed TPs s uc u es ound in he ae obic he e o ophic
eac o , including he con idence le el, a om changes ela i e o he pa en OMPs and
bio ans o ma ion eac ions and enzymes likely in ol ed in he p ocess. The TP
s uc u es placed in b acke s a e only sugges ions o possible s uc u es. The unc ional
g oups bonded wi h do ed lines could be placed somewhe e else in he molecule.
OMPs
TP
Le el
A om
chang
e
Reac ion
Candida e
enzymes
ADBI
3
+ O
Hyd oxyla ion
EC 1.14.-
EC
1.13.-
ADBI
3
+ 2O
-
2H
Hyd oxyla ion
Addi ional
oxida ion
EC 1.14.-
EC 1.13.-
EC 1.1.
-
HHCB
4
+ 2O
Hyd oxyla ion
EC 1.14.-
EC 1.13.-
E1
4
+ 2O
Hyd oxyla ion
EC 1.14.-
EC 1.13.-
E2
3
+ O
Hyd oxyla ion
EC 1.14.-
EC 1.13.-
He e o ophic enzyma ic bio ans o ma ions o OMPs
95
DCF
3
+ O
Hyd oxyla ion
EC 1.14.-
EC 1.13.-
NP
3
+ 3O
-
2H
Hyd oxyla ion
Addi ional
oxida ion
EC 1.14.-
EC 1.13.-
EC 1.1.
-
TMP
3
+ O
-
N, H
Hyd olysis
EC 3.5.99.-
DZP
3
-
C,
2H
Deme hyla ion
EC 1.14.-
FLX
3
+ 2O
- C, N,
5H
Deme hyla ion
Deamina ion
Oxida ion
EC 1.14.-
EC 1.4.
-
EC 1.2.1.-
FLX
2
+ 4C,
3O, 4H
Conjuga ion
EC 2.3.
-
SMX
3
+ 7C,
4N,
2O, 4H
Conjuga ion
EC 2.5.
-
EC 3.5.4.11
Chap e 4
96
4.4. CONCLUSION
This chap e highligh s he ele an ole o ae obic he e o ophs in he come abolic
bio ans o ma ion o OMPs, speaking o hei capaci y o con ibu e o he o e all
emo al o OMPs in ac i a ed sludge plan s and ques ioning he equi emen o
main aining a ni i ying ac i i y. In ac , his s udy shows ha he e o ophs can achie e
simila ex en s o bio ans o ma ion o he selec ed OMPs as hose epo ed in se e al
li e a u e s udies wi h ac i a ed sludge (wi h ni i ying, deni i ying and he e o ophic
ac i i ies) and pu ely ni i ying sys ems. Based on he TPs p oduced, i was possible o
iden i y he main eac ions in ol ed in OMPs bio ans o ma ion unde ae obic
he e o ophic condi ions, which a e oxida ion (hyd oxyla ion, dehyd ogena ion,
deamina ion and deme hyla ion), hyd olysis and conjuga ion ou es. An o e all analysis
o all esul s allows selec ing mono- and dioxygenases, dehyd ogenases, hyd olases and
ans e ases as some o he main enzyma ic ac i i ies likely esponsible o OMPs
bio ans o ma ion unde he e o ophic condi ions. Thus, his chap e highligh s he
ele an con ibu ion o he e o ophs o OMPs emo al, which a e gaining impo ance in
he concep ion o new WWTPs, and deepens he knowledge on hei bio ans o ma ion
mechanisms.
CHAPTER 5
CHAPTER 5. Sul ame hoxazole igge s speci ic enzyma ic
ac i i ies unde ae obic he e o ophic condi ions: A
me ap o eomic app oach
SUMMARY
Was ewa e ea men plan s (WWTPs) a e a po en ial ho spo o he de elopmen
o an ibio ic esis ance genes, c ea ing he need o s udy he a e o an ibio ics and he
e olu ion o he mic obio a when exposed o hese compounds. This chap e aims a
de e mining whe he he p esence o sul ame hoxazole (SMX), e en a low
concen a ions, modi ies he mic obial s uc u e and enzyma ic exp ession o a
he e o ophic ac i a ed sludge sys em. To ha end, a combined app oach including
me ap o eomic, genomic and ans o ma ion p oduc analyses was ollowed. Resul s
e ealed he o ma ion o he me aboli es N4-ace yl-SMX and 2,4(1H,3H)-
p e idinedione-SMX, poin ing ou he p esence o se e al bio ans o ma ion pa hways.
Besides, when exposed o inc easing SMX concen a ions, i e bac e ial amilies
belonging o he phylum P o eobac e ia ollowed cha ac e is ic ends, he genus
Co ynebac e ium inc eased i s abundance and i e enzymes in ol ed in i s cen al
me abolism showed a di e en ial exp ession, sugges ing hei ele ance o mi iga e SMX
isks. O e all, his chap e con i ms he po en ial o me ap o eomic echniques o un a el
o ganic mic opollu an s’ bio ans o ma ion mechanisms in WWTPs and elucida e he
bac e ial and enzyma ic key playe s.
This chap e was ed a ed a e he ollowing unde - e iew publica ion:
Kennes-Veiga, D. M., T ueba-San iso, A., Galla do-Ga ay, V., Lema, J. M., Ca balla, M.
Sul ame hoxazole igge s speci ic enzyma ic ac i i ies unde ae obic he e o ophic
condi ions: A me ap o eomic app oach. En i onmen al Science and Technology. ISSN:
1520-5851. Unde e iew.
Fu he in o ma ion can be ound in he “Lis o publica ions” (page 145).
Chap e 5
98
5.1. INTRODUCTION
The p esence o an ibio ics in WWTPs poses a se ious en i onmen al and heal h isk
due o he de elopmen o an ibio ic- esis an genes and bac e ia (Pazda e al., 2019;
Wang e al., 2021; Wo ld Heal h O ganiza ion, 2021). Sul onamides a e a g oup o
an ibio ics pa icula ly impo an due o hei in ensi e u iliza ion wo ldwide and, among
hem, SMX is he mos b oadly consumed one (Ache mann e al., 2018a; Ca alho and
San os, 2016). In 2020, due o he ongoing g owing conce n, SMX was included in he
“Su ace Wa e Wa ch Lis ” published by he Wa e F amewo k Di ec i e o he
Eu opean Union o moni o and ga he da a abou i s po en ial isks o he aqua ic
en i onmen (Eu opean Commission, 2020).
The knowledge o he enzymes and mic oo ganisms in ol ed in SMX
bio ans o ma ion in ac i a ed sludge sys ems is s ill e y limi ed. Howe e ,
me ap o eomics o e s a sui able app oach o sol e hese ques ions since i p o ides a
global iew o he p o eins exp essed by a mic obial communi y a a speci ic momen
(Rod íguez-Vale a, 2004), allowing o s udy biological p ocesses in hei na i e
en i onmen while a oiding he ime-consuming labo o isola ing mic oo ganisms
(Lace da and Rea don, 2009; Yuqiu Wang e al., 2020).
The goal o his chap e was o ob ain a be e insigh in o he unde lying mechanisms
in ol ed in he ae obic he e o ophic bio ans o ma ion o SMX. Speci ically, i is
explo ed how SMX concen a ions a ec he bio ans o ma ion capaci y, axonomic
composi ion, and enzyma ic exp ession o a he e o ophic ac i a ed sludge mic obial
communi y. Fo ha pu pose, a combina ion o me ap o eomic, genomic and TPs
analyses was applied.
5.2. MATERIALS AND METHODS
A 2 L con inuously s i ed lab-scale eac o , connec ed o a 1 L se le (sec ion 2.2.1.),
was ope a ed in h ee di e en s ages. Fi s ly, he bio eac o was ed wi hou SMX o
wo mon hs (con ol s age), hen wi h 50 μg L-1 o SMX o 45 days (s age I) and inally
wi h 1000 μg L-1 o SMX o ano he 45 days (s age II). The eeding included ATU o
a oid he de elopmen o ni i ying ac i i y, was based on ace a e as o ganic ca bon
sou ce (sec ion 2.1.2.) and he eac o was ope a ed wi h an OLR o 0.5 g COD L-1 d-1,
an HRT o 1 d and a SRT o app oxima ely 5 d. Once s eady s a e was eached a each
SMX igge s speci ic enzyma ic ac i i ies
99
s age, inle and ou le SMX concen a ions (sec ion 2.3.5.) we e measu ed by aking h ee
samples om he eeding and he eac o essel in h ee consecu i e days. Besides, a he
end o each s age, iplica e samples o TPs and 16S RNA gene sequencing analyses
we e aken om he eac o (sec ions 2.3.6. and 2.3.7.). Fu he mo e, analyses o he
in luen and e luen o he eac o we e pe o med o de e mine he ypical ope a ional
pa ame e s acco ding o he s anda d analy ical me hods desc ibed in Chap e 2. The
soluble COD, ammonium, ni a e and ni i e concen a ions, SSV, pH and empe a u e
we e measu ed wo o h ee imes pe week.
Addi ionally, a o al o 18 sequen ial ba ch eac o s (sec ion 2.2.2.) di ided in o six
g oups o iplica es we e spiked wi h SMX o ob ain he ollowing concen a ions: 0
(con ol), 50, 250, 500, 1000 and 2000 μg L-1. The eac o s we e inocula ed wi h ac i a ed
sludge a a concen a ion o 1 g L-1, ope a ed a 25ºC and he eeding was also based on
ace a e as o ganic ca bon sou ce (sec ion 2.1.2.). The expe imen las ed 25 d and was
pe o med unde s e ile condi ions. The con en o he eac o s was cen i uged daily o
sepa a e he biomass om he supe na an , which was emo ed be o e adding new eed
oge he wi h he SMX spike. The ope a ion was moni o ed by measu ing he ypical
pa ame e s acco ding o he s anda d analy ical me hods desc ibed in Chap e 2. TSS,
VSS and ammonium, ni a e, ni i e and oxygen concen a ions we e de e mined weekly,
while he ace a e concen a ion and pH, daily. Besides, SMX and TPs concen a ions we e
de e mined on days 2, 17 and 25. Finally, samples o p o eomic (days 1 and 25, sec ion
2.3.8.) and 16S RNA gene sequencing (day 25) analyses we e aken o de e mine he key
enzyma ic and mic obial ac i i ies in ol ed in he bio ans o ma ion o SMX.
5.3. RESULTS AND DISCUSSION
5.3.1. SMX bio ans o ma ion unde ae obic he e o ophic condi ions
In he con inuous bio eac o , he p esence o SMX did no a ec he consump ion o
he p ima y ca bon sou ce (ace a e), possibly due o one o he ollowing easons: i) he
highes SMX concen a ions e alua ed a e s ill below inhibi o y le els and ii) he ole o
speci ic bac e ia in ol ed in he bio ans o ma ion o SMX mi iga ed he po en ial
nega i e e ec s o he an ibio ic o e o he mic oo ganisms. The bio ans o ma ion
e iciency o SMX was independen o i s concen a ion in he eeding, being jus sligh ly
lowe in s age I (70 %) han in s age II (80 %). As a consequence, he speci ic
Chap e 5
100
bio ans o ma ion a e inc eased conside ably be ween he wo s ages, a ying om 0.8
o 10.6 μg OMP g-1 VSS h-1.
Simila ly, in he sequen ial ba ch eac o s, e en he highes SMX concen a ions did
no a ec he biodeg ada ion o ace a e, whose emo al emained cons an a 140 mg L-1
h-1. The a e age SMX bio ans o ma ion anged be ween 62 and 78 % depending on he
spiked concen a ion (Table 5.1) and showed clea cha ac e is ic ends. Fi s ly,
ega dless o he SMX in luen concen a ions, he bio ans o ma ion yield inc eased
om day 2 o day 17 and dec eased on day 25 o alues e en lowe han hose obse ed
on day 2. Secondly, on days 2 and 25, lowe ini ial SMX concen a ions lead o highe
bio ans o ma ion yields, while his end was no obse ed on day 17. The imp o ed
bio ans o ma ion on day 17 compa ed o day 2, as well as he educed bio ans o ma ion
yield o SMX on day 2 a highe ini ial concen a ions can be a ibu ed o he acclima ion
phase o he mic oo ganisms capable o bio ans o ming SMX. Thei highe abundance
on day 17 migh ha e allowed eaching a cons an bio ans o ma ion ex en in he 80-90
% ange in all bio eac o s. This ag ees wi h he indings o Li e al. (2016), who a e an
ex ended lag phase p opo ional o he doses amended, obse ed bio ans o ma ion o
mul iple an ibio ics by bac e ia om di e en gene a. None heless, he dec eased
bio ans o ma ion on day 25, in ensi ied a highe SMX concen a ions, migh be
associa ed wi h he accumula ion o TPs ha limi he bio ans o ma ion o he pa en
compound due o e e sibili y e en s (Gonzalez-Gil e al., 2019a), he modynamic
limi a ions (Gonzalez-Gil e al., 2018a) o by exe ing oxici y o e a ce ain
concen a ion (Cao e al., 2019; Pé ez e al., 2005), hus ou weighing he inc eased
p esence o SMX deg ade s.
These esul s highligh he capaci y o he ae obic he e o ophic popula ion o
ex ensi ely bio ans o m SMX, ollowing he esul s o p e ious wo ks (Fe nandez-
Fon aina e al., 2016; Kennes-Veiga e al., 2020; Majewsky e al., 2011). In ac ,
he e o ophs could be mo e a o able o bio ans o m SMX han ni i ying
mic oo ganisms (Fe nandez-Fon aina e al., 2016; T an e al., 2013), sugges ing ha
s imula ing he he e o ophic ac i i y in ac i a ed sludge sys ems may posi i ely
in luence i s bio ans o ma ion.
SMX igge s speci ic enzyma ic ac i i ies
101
Table 5.1. SMX bio ans o ma ion yield in he sequen ial ba ch eac o s h oughou he
expe imen .
In luen SMX
concen a ion
(μg/L)
Bio ans o ma ion (%)
Day 2
Day 17
Day 25
A e age
50
80
86
68
78 8
250
77
87
63
76 10
500
72
79
60
70 8
1000
70
79
36
62 19
2000
63
86
43
64 18
5.3.2. TPs iden i ica ion unde ae obic he e o ophic condi ions
Two SMX TPs we e de ec ed in he con inuous bio eac o : 2,4(1H,3H)-
p e idinedione-SMX (P O-SMX) and N4-ace yl-SMX. The la e was only obse ed
du ing s age II, which can be a ibu ed o wo easons: (i) N4-ace yl-SMX may ha e been
also p esen du ing s age I, bu a a concen a ion below he limi o iden i ica ion, o (ii)
addi ional bio ans o ma ion ou es may ha e appea ed a highe SMX doses due o
a ia ions in he concen a ions o he me aboli es ha a ec ed he me abolic lux and
pa hway selec ion (Wegne e al., 2015). Ac ually, OMPs in luen concen a ion is
ecognized as a key pa ame e a ec ing bio ans o ma ion yields, a es and pa hways
(Onesios-Ba y e al., 2014; Rios-Miguel e al., 2021; Yuxin Wang e al., 2020). Fo
ins ance, Jewell e al. (2016) obse ed a ia ions in he bio ans o ma ion pa hway and
TPs o ma ion o TMP depending on he ini ial spiked concen a ion.
Di e en ly, in he sequen ial ba ch eac o s, only P O-SMX was de ec ed. I was
obse ed in all eac o s spiked wi h SMX a all sampling poin s, excep o he samples
aken on day 2 om he eac o ed wi h 50 μg L-1 o SMX, which is a ibu ed o i s
expec ed low concen a ion in he sample.
Mul iple SMX TPs o med h ough a wide ange o eac ions such as hyd oxyla ion,
ace yla ion, deamina ion o ni a ion ha e been p e iously epo ed (Majewsky e al.,
2014). P O-SMX has been de ec ed be o e in WWTPs e luen s and lab expe imen s
using ac i a ed sludge (Ache mann e al., 2018a). I s o ma ion occu s h ough he p e in-
conjuga ion pa hway when sul onamides in e ac wi h dihyd op e oa e syn he ase,
hinde ing olic acid syn hesis h ough compe i i e inhibi ion. The pa hway begins wi h
SMX conjuga ion, is ollowed by oxida ion o p e in-SMX and con inues wi h a
Chap e 5
102
hyd olysis s ep o o m P O-SMX hanks o he ac ion o p e in deaminase (EC 3.5.4.11).
P O-SMX can be u he b oken down h ough a ious s eps possibly in ol ing oxida ion
and deca boxyla ion eac ions (Ache mann e al., 2018a). Mos TPs o med in he
sul onamide p e in-conjuga ion pa hway a e modi ied a he pa a amino g oup and s ill
possess he sul anilamide oxicopho e, equi ing a de ailed isk assessmen due o hei
an ibio ic ac i i y, capaci y o p omo e an ibio ic esis ance and abili y o be back-
ans o med o SMX (Majewsky e al., 2015, 2014; Yun e al., 2012).
N4-ace yl-SMX is o med h ough N-acyla ion o SMX, likely ca alyzed by N-
a ylamine ace yl ans e ase (EC 2.3.1.5) (Fische and Majewsky, 2014; X. Li e al., 2016;
Tang e al., 2018). I ep esen s he main SMX human me aboli e and has been o en
de ec ed in WWTPs e luen s (Ache mann e al., 2018a; Majewsky e al., 2014; L. J. Zhou
e al., 2019). Besides, i possesses an ibac e ial ac i i y and exe s oxici y, al hough a
educed le els compa ed o SMX (Majewsky e al., 2014), and i can be apidly
back ans o med o he pa en compound (Ache mann e al., 2018a; Radke e al., 2009),
hinde ing he accu a e assessmen o i s en i onmen al ele ance.
5.3.3. Impac o SMX on mic obial communi y s uc u e
The esul s ob ained in he con inuous bio eac o by DNA me aba coding a he
amily le el show a clea shi in he bac e ial communi y ela ed o he p esence o SMX
and i s ini ial concen a ion in he eeding (Fig. 5.1.). This inding ag ees wi h p e ious
s udies poin ing ou ha he mic obial s uc u e can change unde he selec ion p essu e
o many OMPs (Aguila -Rome o e al., 2020; Vasiliadou e al., 2018; G. Zhou e al.,
2019). Howe e , as p e iously men ioned and di e en om o he s udies (Jiang e al.,
2017), he consump ion o he main ca bon sou ce was no a ec ed by hese changes.
Addi ionally, he a ia ions in he mic obial communi y be ween s ages did no ansla e
in o changes in he SMX emo al e iciency, which was main ained in he 70-80 % ange.
O e all, he highligh is he cha ac e is ic ends ollowed by i e bac e ial amilies
(Rhodobac e aceae,Comamonadaceae,Xan hobac e aceae,De osiaceae and
Hyphomic obiaceae) belonging o he phylum P o eobac e ia. Such phylum has been
desc ibed be o e as one o he mos dominan and p e alen in ae obic ac i a ed sludges
and i has been linked o OMPs emo al (Bains e al., 2019; Balcom e al., 2016; Galla do-
Al ami ano e al., 2019; Vasiliadou e al., 2018).
Commamodaceae was he only bac e ial amily nega i ely a ec ed by he p esence
o SMX a all s ages, a ying i s ela i e abundance om 17.5 % in he con ol s age o
SMX igge s speci ic enzyma ic ac i i ies
103
3.9 and 1.1 % du ing s ages I and II, espec i ely. In e es ingly, Commamodaceae is he
only Be ap o eobac e ia o he 5 amilies, since he o he s belong o he
Alphap o eobac e ia class, which has been shown o h i e in ac i a ed sludge sys ems
in p esence o se e al OMPs (Da ids e al., 2017).
Hyphomic obiaceae was posi i ely a ec ed when SMX was added o he eac o . I
was no de ec ed in he con ol s age bu hen i s abundance inc eased o 0.6 and 4.0 %
wi h SMX du ing s ages I and II, espec i ely. In p e ious s udies, i s abundance was
ela ed o imp o ed OMPs emo al (Coll e al., 2020; Zhang e al., 2020), al hough in his
wo k i was no obse ed a a ia ion in he emo al e iciency o SMX be ween s ages.
De osiaceae showed a as and s able adap a ion o SMX. I was posi i ely a ec ed
by he p esence o SMX a 50 μg L-1, inc easing i s abundance om 0.6 % in he con ol
s age o 10.6 %, which hen sligh ly d opped when SMX was spiked a 1000 μg L-1.
The sum o he ela i e abundances o Rhodobac e aceae and Xan hobac e aceae
emained app oxima ely cons an h oughou he h ee expe imen al s ages, sugges ing a
balance be ween hem whe eby when one inc eased, he o he dec eased, and ice e sa.
Rhodobac e aceae, which was ini ially highly abundan (70 %) and has p e iously been
linked o imp o ed emo al e iciencies o se e al OMPs (Galla do-Al ami ano e al.,
2019), showed a e y pa icula end. When SMX was spiked a 50 μg L-1, i s abundance
dec eased app oxima ely by hal , bu hen, when he concen a ion was changed o 1000
μg L-1, he ela i e abundance eached alues close o hose o he con ol s age. I is
hypo hesized ha i s ly i was ou compe ed by o he mic oo ganisms ha had a as e
adap a ion o he in oduc ion o SMX. None heless, he capaci y o Rhodobac e aceae
o wi hs and he highes concen a ions o SMX may ha e allowed hem o inc ease hei
abundance again.
Con e sely, Xan hobac e aceae was posi i ely a ec ed by he p esence o SMX a
50 μg L-1 bu i s ela i e abundance d opped when SMX was spiked a 1000 μg L-1. This
sugges s ha i h i ed when he SMX concen a ion was low bu ha i could ha e been
ou compe ed when SMX was added a much highe concen a ions. In his sense, i has
been epo ed be o e ha lowe OMPs concen a ions may imp o e mic obial ichness
and di e si y (Jiang e al., 2017; Vasiliadou e al., 2018; Zhang e al., 2020; G. Zhou e
al., 2019). Howe e , highe concen a ions may cause he opposi e, as obse ed in
hospi al WWTPs, which ypically ha e high pha maceu ical concen a ions and educed
bac e ial di e si y in compa ison o u ban was ewa e s (Vasiliadou e al., 2018).
Chap e 5
110
5.4. CONCLUSION
This chap e highligh s he capaci y o he he e o ophic sludge o bio ans o m SMX
while s ill main aining he high biodeg ada ion le els o he p ima y subs a e. Howe e ,
unde exposu e o inc easing SMX concen a ions, he composi ion o he he e o ophic
mic obio a changed. Fi s ly, Rhodobac e aceae,Comamonadaceae,Xan hobac e aceae,
De osiaceae and Hyphomic obiaceae showed cha ac e is ic ends in he con inuous
bio eac o s. Secondly, he abundance o Co ynebac e ium a ied in he sequen ial ba ch
eac o s, poin ing owa ds i s ele an ole o main ain he i ness o he mic obial
communi y hanks o he ac ion o some enzymes o i s cen al me abolism. Thus, new
insigh s o unde s and he SMX bio ans o ma ion mechanisms and deciphe he key
mic oo ganisms and enzymes unde ae obic he e o ophic condi ions a e p o ided.
Fu he mo e, he ad an ages and use ulness o me ap o eomics o s udy OMPs
bio ans o ma ion mechanisms a e p o ed, p o iding a gumen s o he ex ensi e deba e
aised in he las ew yea s abou he bene i s and limi a ions o omic echniques in
en i onmen al enginee ing applica ions (Ache mann e al., 2020; Fenne e al., 2021;
Da id R. Johnson e al., 2015b; Kennes-Veiga e al., 2021a). Howe e , some enzymes
ha should ha e been p esen in he sludge could no be de ec ed, sugges ing ha u he
echnical de elopmen s o lowe he iden i ica ion and quan i ica ion h esholds a e s ill
equi ed.
CHAPTER 6
CHAPTER 6. GENERAL DISCUSSION AND CONCLUSIONS
Chap e 6
112
6.1. MAIN OUTCOMES OF THE THESIS
The conce n abou OMPs emissions in o he en i onmen has been inc easing in he
las decades due o he g owing consump ion o pha maceu icals, pe sonal ca e p oduc s,
indus ial chemicals and many o he o ganic subs ances. These compounds pose nega i e
e ec s o bo h he ecosys em and human heal h, making i necessa y o educe hei
concen a ions in WWTPs e luen s, which a e he main sou ce o OMPs discha ge o he
en i onmen (Ma go e al., 2015). Ye , he elimina ion e iciencies o many OMPs in
WWTPs a e highly a iable o e y low. Fo such eason, esea ch has adi ionally
ocused mos ly on inding he mos app op ia e ope a ional and en i onmen al condi ions
o imp o e OMPs emo als in WWTPs (Al a ino e al., 2018a). None heless, speci ic
changes in he ope a ional pa ame e s ha e led o di e en ou comes in e ms o
elimina ion due o hei a iable e ec on he mic obial popula ions and me abolic
pa hways (Ngo e al., 2020). Fu he mo e, ce ain en i onmen al condi ions may a o
he elimina ion o some OMPs bu , a he same ime, hinde ha o o he s (Al a ino e
al., 2018b; Ha b e al., 2019). The e o e, o es ablish long-las ing solu ions o educe
OMPs concen a ions globally in WWTPs e luen s, i is necessa y o ha e a deep
unde s anding o he bio ans o ma ion mechanisms and o elucida e he bac e ial and
enzyma ic key playe s.
Mos WWTPs consis o an ac i a ed sludge sys em comp ising bo h au o ophic
ni i ying and he e o ophic ac i i ies (Me cal & Eddy, 2014). To da e, mul iple s udies
a e dealing wi h he ole o ni i ie s du ing OMPs bio ans o ma ion, bu he e is sca ce
in o ma ion abou he he e o ophic bio ans o ma ion mechanisms due o hei huge and
complex me abolic ne wo k (Fische and Majewsky, 2014). Howe e , p e ious wo ks
ha e shown hei po en ial o con ibu e o OMPs emo al and educe he accumula ion
o TPs o med by ni i ying bac e ia (T an e al., 2013; Wu e al., 2020). Besides, he
he e o ophic ac i i y is gaining in e es in he de elopmen o inno a i e and mo e
ene gy-e icien WWTPs (Liu e al., 2020).
Thus, his hesis ies o p o ide insigh and inc ease he knowledge in o he OMPs
bio ans o ma ion mechanisms in ae obic he e o ophic condi ions, aiming o sol e he
a o emen ioned ques ions (Chap e 1). In he ollowing sec ions o his chap e , he main
ou comes o his hesis a e in eg a ed and join ly discussed.
Gene al discussion and conclusions
113
6.1.1. A e ae obic he e o ophs e ec i e o bio ans o m OMPs?
The con ibu ion o ae obic he e o ophic mic oo ganisms o he emo al o OMPs
in ac i a ed sludge sys ems has gene ally been unde es ima ed. The esul s shown in
Chap e s 3 and 4 p o e ha he e o ophs can bio ans o m a ange o OMPs wi h b oad
physico-chemical p ope ies o ex en s simila o hose epo ed in he li e a u e o
ac i a ed sludge and pu ely ni i ying sys ems. In ac , o some compounds, such as
SMX, DCF and TCS (Chap e 4), he he e o ophic bio ans o ma ion seems o be be e
han ha o ni i ie s. These esul s sugges ha he A-s ages o no el WWTPs, wo king
a high OLRs and sho SRTs and ope a ing p ima ily unde he e o ophic condi ions
(Jimenez e al., 2015), could be able o e icien ly and ex ensi ely bio ans o m OMPs
be o e hey each he B-s age, which is p oposed o be ope a ed by an anammox
conso ium (Liu e al., 2020). Addi ionally, hese indings ques ion he need o p omo ing
he ac i i y o au o ophic ni i ie s in WWTPs, which, compa ed o he e o ophs, a e
slowe g owe s and possess a less e icien me abolism and a lowe di e si y o o ganisms
(Hol mann and Sell, 2002; K. Kim e al., 2020). Howe e , he combina ion o bo h
mic obial popula ions could s ill be equi ed o educe he accumula ion o some TPs,
ha may be o med by speci ic mic oo ganisms bu equi e o he s o hei comple e
mine aliza ion (Khunja e al., 2011; Wu e al., 2020).
So a , ae obic he e o ophs a e he only mic obial popula ion ha has p o en hei
capaci y o me abolize OMPs (T an e al., 2013), aising he ques ion abou he ele ance
o me abolism and come abolism in WWTPs (Al a ino e al., 2018a; Fische and
Majewsky, 2014). Howe e , mos p e ious s udies ha e been pe o med wi h pu e o
en iched cul u es and/o using OMPs concen a ions conside ably highe han hose
ypically epo ed in WWTPs in luen s, making i di icul o ex apola e lab esul s o
eal-scale plan s. In his hesis, seeking o ma ch WWTPs condi ions as much as possible,
con inuous eac o s we e ope a ed long- e m using ac i a ed sludge as he inoculum and
spiking OMPs in he low μg L-1 ange. The esul s p o ed ha come abolism was he
p e e en ial bio ans o ma ion mechanism unde ae obic he e o ophic condi ions and
ha he a e o OMPs is linked o ha o p ima y subs a es (Chap e 3). Acco dingly,
he enzymes wi h low subs a e speci ici y p esen in he eac o a e esponsible o OMPs
bio ans o ma ion hanks o simila i ies in he chemical s uc u es o he OMPs and he
enzyme’s na u al subs a es.
Chap e 6
114
The bio ans o ma ion kine ic cons an (kbiol) p o ides in o ma ion abou he
biodeg adabili y o OMPs (Han T an e al., 2017) and, o he same compound, can
g ea ly a y be ween WWTPs (Helbling e al., 2015; an Be gen e al., 2021). Such
a iabili y is gene ally a ibu ed o changes in ope a ional and en i onmen al condi ions
ha lead o a ia ions in he mic obiome and, consequen ly, in i s bio ans o ma ion
capaci y. In Chap e 3, i is shown ha inc eases in he in ensi y o he he e o ophic
me abolic ac i i y also lead o changes in kbiol, imp o ing OMPs bio ans o ma ion. Thus,
kbiol is no only dependen on he physico-chemical p ope ies o he OMPs and he
condi ions applied o he WWTPs, bu also on he me abolic ac i i y o he biomass. This
inding implies ha he managemen o he o ganic subs a es and he OLR can be
e icien ools o con ol he mic obial ac i i y and, subsequen ly, OMPs
bio ans o ma ion. None heless, he changes obse ed in kbiol p o ed o be compound-
speci ic, sugges ing ha a ia ions in he me abolic ac i i y ca y o e une enly ac oss
he bio ans o ma ion a es o he OMPs. Besides, i he HRT applied is high enough,
imp o ed bio ans o ma ion a es may no ansla e in o be e bio ans o ma ion ex en s.
O e all, ae obic he e o ophic mic oo ganisms can ex ensi ely bio ans o m OMPs
h ough come abolism and in luence he bio ans o ma ion a e h ough changes in kbiol
caused by a ia ions in he me abolic ac i i y.
6.1.2. T ans o ma ion p oduc s as a ool o iden i y key enzyma ic ac i i ies
Bio ans o ma ion eac ions occu hanks o he e sa ile ca aly ic ac i i y o he
enzymes in ol ed in he biodeg ada ion o p ima y subs a es. Iden i ying such key
enzymes is a complex ask ha is s ill in i s ea ly s ages (Ache mann e al., 2020; K ah e
al., 2016; Wicke e al., 2016; Zhou e al., 2015) and, in ae obic he e o ophic condi ions,
he e a e no conclusi e s udies abou he enzyma ic ac i i ies in ol ed in OMPs
bio ans o ma ion. Aiming o ill in ha gap, se e al expe imen s wi h he e o ophic
sludge we e ca ied ou in his hesis h ough wo di e en me hodologies : (i) TPs and
(ii) -omic analyses.
TPs analysis has been adi ionally challenging due o he low OMPs concen a ions
ound in en i onmen al samples and he complex ma ices whe e hey a e p esen (L.
Zhang e al., 2013). Howe e , TPs s udies aiming a unde s anding hei eco oxici y and
isk o he en i onmen and he bio ans o ma ion limi a ions o OMPs ha e become mo e
equen in ecen imes hanks o majo ad ances in analy ical echniques (Fenne e al.,
Gene al discussion and conclusions
115
2021; Gulde e al., 2016). Addi ionally, TPs s uc u e analysis can be used as a p edic ion
ool o de e mine he main enzyma ic ac i i ies in ol ed in OMPs bio ans o ma ion
(Chap e 1). I p ima y subs a es and TPs a e known, i is possible o pe o m educa ed
guesses abou he enzymes ca ying ou he p ocess.
Applying his me hodology, in his hesis (Chap e 4) i was possible o iden i y
mono- and dioxygenases, dehyd ogenases, hyd olases and ans e ases as some o he
main enzyma ic ac i i ies likely esponsible o OMPs bio ans o ma ion unde
he e o ophic condi ions. The eac ions ca alyzed by hese enzymes in ol e oxida ion,
hyd olysis and conjuga ion s eps. Oxida ion, which is equen ly an ini ial
bio ans o ma ion s ep o many OMPs in ac i a ed sludge sys ems, was he main
bio ans o ma ion eac ion obse ed. Among oxida i e s eps, hyd oxyla ion was he mos
p edominan , being de ec ed o mul iple compounds, al hough oxida ion h ough
deamina ion, deme hyla ion and dehyd ogena ion eac ions was also de e mined.
In e es ingly, o some OMPs, di e en TPs we e iden i ied, sugges ing ha sequences
o ans o ma ion eac ions may ake place a di e en unc ional g oups. Besides, some
o he TPs de ec ed ha e been desc ibed as ac i e me aboli es wi h long-hal li es,
highligh ing he need o elucida e hei a e in WWTPs and pe o m eco oxici y analyses.
Iden i ying TPs can be a challenging ask because OMPs can unde go a huge a ay
o bio ans o ma ion eac ions, leading o a emendously la ge numbe o possible TPs.
The e o e, he applica ion o modeling ools, such as he EAWAG-PPS (EAWAG-
BBD/PPS) (Chap e 4), which a e based on bio ans o ma ion ules suppo ed by
mul iple li e a u e s udies, ha e p o en o be o g ea aid o c ea e lis s o suspec TPs.
Thei use, along wi h lab expe imen s ha se e as a con i ma ion me hod, is
ecommended o build pa hway knowledge and p edic he o ma ion and oxici y o TPs.
O e all, his hesis p o ed ha TPs analysis is a use ul ool o un a el ele an
enzymes aking pa in bio ans o ma ion eac ions. Howe e , his me hodology alone
does no allow o ob ain di ec and comple e con i ma ion o he esponsible enzyma ic
ac i i ies. Thus, i is ad isable o combine he TPs esul s wi h hose om o he analy ical
me hods, such as in i o assays, o omic echniques, as discussed in he ollowing sec ion.
6.1.3. Omic echniques as a ool o iden i y key mic obial and enzyma ic playe s
Me agenomic, me a ansc ip omic and me ap o eomic echniques a e e ol ing
apidly and me a-omics associa ion s udies ha e s a ed o be used o iden i y key
Chap e 6
116
mic obial and enzyma ic playe s in ol ed in OMPs bio ans o ma ion pa hways
(Ache mann e al., 2020; Du e al., 2017; Fenne e al., 2021). Thei po en ial is based on
he ac ha in oducing OMPs in a speci ic en i onmen a ec s he s uc u e o he
mic obiome, leading o a ia ions in he exp ession o genes and gene p oduc s and
allowing o ind bioma ke s ha help unde s and and p edic he a e o OMPs in
WWTPs. Howe e , he applica ion o his app oach s ill aises some doub s; mainly
because (i) many alse posi i es leading o non-causal co ela ions can be easily gene a ed
and (ii) i is s ill necessa y o con i m ha he o e exp ession o gene p oduc s happens
e en when OMPs a e added a ace le el concen a ions (Fenne e al., 2021).
In Chap e 5, he cha ac e is ic ends ollowed by i e bac e ial amilies belonging
o he phylum P o eobac e ia and he key ole o Co ynebac e ium du ing he
he e o ophic bio ans o ma ion o SMX could be de e mined hanks o a ia ions in hei
abundance measu ed h ough he applica ion o DNA me aba coding and me ap o eomic
echniques. Besides, a di e en ial exp ession o i e enzymes in ol ed in cen al
me abolic unc ions o Co ynebac e ium was also obse ed, sugges ing i s ele ance
du ing SMX bio ans o ma ion. In e es ingly, hese indings poin owa ds he
impo ance o he TCA cycle enzymes du ing OMPs bio ans o ma ion, which has no
been epo ed be o e. I is unlikely ha hese enzymes pa icipa e in he ini ial
bio ans o ma ion s eps gi en he usual size and s uc u e o OMPs, bu hey could be
in ol ed in he con e sion o smalle TPs o med a e se e al bio ans o ma ion s eps,
such as hose belonging o he SMX-p e in conjuga ion pa hway.
The e o e, esul s highligh ha omic echniques a e use ul o un a el
bio ans o ma ion pa hways and iden i y ele an mic oo ganisms and enzyma ic
ac i i ies. None heless, some enzymes ha should ha e been p esen in he sludge could
no be de ec ed h ough me ap o eomics (Chap e 5), possibly due o hei low
abundance compa ed o ha o housekeeping o cen al me abolism p o eins, which
shows ha echnical de elopmen s o imp o e enzyme iden i ica ion and quan i ica ion
a e s ill equi ed. Addi ionally, in Chap e 5,Co ynebac e ium abundances di e ed
be ween he esul s om DNA me aba coding and me ap o eomic analyses. Thus, omic
echniques ep esen ing exp essed gene p oduc s a a gi en ime, such as
me a ansc ip omics o me ap o eomics, may be mo e sui able o ind causal links
be ween OMPs bio ans o ma ion and mic obiological da a.
Gene al discussion and conclusions
117
6.2. MAIN GAPS AND FUTURE PERSPECTIVES
The e a e se e al esea ch a eas and knowledge gaps ha need o be add essed o
be e unde s and biological p ocesses in WWTPs and imp o e OMPs emo al.
Come abolism has been iden i ied as he main mechanism d i ing OMPs
bio ans o ma ion; howe e , u he esea ch is equi ed o de e mine whe e he bounda y
be ween me abolism and come abolism lies (Nsenga Kumwimba and Meng, 2019; T an
e al., 2013). To his end, expe imen s wi h inc easing OMPs doses, along wi h -omics
and TPs analyses, could be ca ied ou . These expe imen s would also allow de e mining
he h eshold whe e inhibi o y e en s appea and p o ide in o ma ion abou changes in
bio ans o ma ion ou es a highe OMPs concen a ions (Jia e al., 2020; Wegne e al.,
2015), which could be pa icula ly use ul o highly concen a ed s eams, such as
hospi al o indus ial was ewa e s. Mo eo e , he con ibu ion o seconda y biological
ac i i ies o he sludge du ing he come abolic bio ans o ma ion o OMPs emains
unclea (Gonzalez-Gil e al., 2021). The mo e ecen use o adiolabeling-based
app oaches, which consis o he addi ion o a labeled subs a e o he expe imen al
en i onmen along wi h an analysis o label inco po a ion in o TPs, bioma ke s, enzymes
o cells, has been poin ed ou as a p omising echnique o sol e hese ques ions. Ye , i s
applica ion wi h OMPs p esen a low le els emains challenging (Falås e al., 2018;
Fenne e al., 2021).
Insigh on he ole and o igin o he enzymes esponsible o OMPs
bio ans o ma ion, as well as on he condi ions ha inc ease hei exp ession, is necessa y
o implemen ope a ional s a egies in WWTPs ha enhance hei abundances and
ac i i ies (Fische and Majewsky, 2014; K ah e al., 2016). Such knowledge could
e en ually allow o selec enzymes as a bio emedia ion echnique o a ge speci ic
compounds, pa icula ly o he ea men o well-de ined was e s eams. I would o e a
mo e di ec , con olled and de ined al e na i e han con en ional WWTPs, which
comp ise a huge conso ium o mic oo ganisms and enzymes and equi e he con ol o
cell g ow h, subs a e anspo o he cell and so p ion, among o he s (S adlmai e al.,
2018). None heless, he applica ion o his echnique in eal sys ems is s ill a long way
o o se e al easons: (i) he isola ion o a ge enzymes om WWTPs has no been
achie ed ye , (ii) he pu chase o enzymes is oo cos ly and p esen s limi a ions o la ge-
scale p oduc ion and (iii) se ing up coope a ion be ween mul iple enzymes in WWTPs
Chap e 6
118
and dealing wi h enzyma ic inac i a ion, s abili y and speci ici y is s ill a complex ask
(Feng e al., 2021; Langbehn e al., 2021; S adlmai e al., 2018).
To da e, al hough some app oaches ha e been de eloped and applied o iden i y he
enzymes in ol ed in OMPs bio ans o ma ion, hey s ill p esen mul iple limi a ions. Fo
ins ance, enzyma ic in i o assays wi h cell- ee lysa es equi e u he esea ch on he
ex ac ion p ocedu e o p ese e he indigenous enzyma ic ac i i ies p esen in he sludge,
including he applica ion o p e ea men s and bu e addi i es (K ah e al., 2016).
Mo eo e , i is necessa y o imp o e he unde s anding o he ci cums ances in which in
i o assays a e ully compa able o eal biological p ocesses and o e alua e he adequacy
o applying co ac o s o enzyma ic inhibi o s, ha may boos o hinde speci ic enzymes
and help o con i m hei ac i i y owa ds OMPs (Gonzalez-Gil e al., 2019b). To
acili a e he link be ween enzymes and bio ans o ma ion pa hways, he de ec ion,
quan i ica ion and assessmen o TPs equi e he con inuous imp o emen o modeling
ools and analy ical echniques ha allow sensi i e and un a ge ed TPs cha ac e iza ion
in sho ime ames e en when e e ence s anda ds a e no a ailable (Nguyen e al.,
2021). In he pas decades, high- esolu ion mass spec ome y has esul ed in no o ious
ad ances o de e mine low OMPs and TPs concen a ions e en in complex mix u es.
Mo e ecen ly, compound-speci ic iso ope analysis is being de eloped, al hough i s
applica ion o en i onmen al OMPs concen a ions emains a challenge. This echnique
can po en ially help o de e mine he bio ans o ma ion ex en and eac ion mechanisms
o OMPs and elucida e di e en o igins o he same TPs hanks o he usual enzyma ic
p e e ence o molecules wi h ligh iso opes, which leads o changes in he iso opic a ios
o he bio ans o med compounds (Fenne e al., 2021). Finally, omics echniques need
o educe hei cos s o b oaden hei accessibili y and hei esul s mus be suppo ed wi h
powe ul s a is ical ools o acili a e he sea ch o causal ela ionships be ween enzymes
and OMPs bio ans o ma ion (Ache mann e al., 2020; Da id R. Johnson e al., 2015b).
As a esul o he abo emen ioned limi a ions, unde s anding he in luence o
en i onmen al and p ocess condi ions and con iden ly ob aining mechanis ic insigh s
abou he bio ans o ma ion o OMPs in WWTPs by di ec ly and exclusi ely applying
any o he well-es ablished app oaches is s ill no possible. Howe e , a combina ion o
all me hodological app oaches is he bes way o o e come some o he indi idual
d awbacks and o mula e sound hypo heses abou he mic oo ganisms and enzymes
pa icipa ing in OMPs bio ans o ma ion, in line wi h p e ious s udies (Ache mann e al.,
Gene al discussion and conclusions
119
2018b, 2020; Zhao e al., 2020) and he esul s o his hesis (Chap e 5). Thus, o
maximize OMPs emo al is essen ial an in e disciplina y con ibu ion om he scien i ic
communi y, co e ing he ields o en i onmen al enginee ing, analy ical chemis y,
molecula biology and da a sciences, as well as alida ing labo a o y indings in ull-scale
mixed cul u es wi h a wide ange o OMPs p esen a en i onmen ally ele an
concen a ions.
Re e ences
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