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Treatment of simulated textile wastewater with aerobic granular sludge: efficiency and detoxification potential João Pedro Viana da Silva Master in Biotechnology Supervisor: Dra. Ana Cristina Anjinho Madeira Viegas and Dra. Nídia Dana Mariano Lourenço de Almeida Examination Committee Chairperson: Dra. Leonilde de Fátima Morais Moreira Supervisor: Dra. Ana Cristina Anjinho Madeira Viegas Member of the Committee: Dra. Helena Maria Rodrigues Vasconcelos Pinheiro November 2016
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Page 1: Treatment of simulated textile wastewater with aerobic ... · Treatment of simulated textile wastewater with aerobic granular sludge: efficiency and detoxification potential João

Treatment of simulated textile wastewater with aerobic

granular sludge: efficiency and detoxification potential

João Pedro Viana da Silva

Master in Biotechnology

Supervisor: Dra. Ana Cristina Anjinho Madeira Viegas and Dra. Nídia Dana Mariano

Lourenço de Almeida

Examination Committee

Chairperson: Dra. Leonilde de Fátima Morais Moreira

Supervisor: Dra. Ana Cristina Anjinho Madeira Viegas

Member of the Committee: Dra. Helena Maria Rodrigues Vasconcelos Pinheiro

November 2016

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Acknowledges:

A very special thanks to both Dra. Ana Cristina Anjinho Madeira Viegas and Dra. Nídia Dana Mariano

Lourenço de Almeida for the opportunity to perform this work, the patience and the support

demonstrated towards me.

Also, must be thanked the FCT – Fundação para a Ciência e a Tecnologia for partially financing this

work through the UID/BIO/04565/2013 project.

And lastly but no less important, to my friends and family who have given me strength to live and work

with both energy and a smile in my heart.

“You may have a fresh start any moment you choose, for this thing that we call 'failure' is not the falling

down, but the staying down.” Mary Pickford

“Then there is a still higher type of courage - the courage to brave pain, to live with it, to never let

others know of it and to still find joy in life; to wake up in the morning with an enthusiasm for the day

ahead.” Howard Cosell

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Abstract:

Textile wastewaters are resistant to aerobic treatment and characterised by high organic loads and dyes

with a relevant environmental impact. Previous works have reported a successful biodecolourization of

textile wastewaters containing azo dyes, by using aerobic granular sludge (AGS) technology in

sequencing batch reactors (SBR). The present study compares the performance, at both efficiency and

detoxification levels, of two AGS SBRs with different hydrodynamic regimens in the treatment of

simulated textile wastewater containing the azo dye acid red 14 (AR14). Similar colour and chemical

oxygen demand (COD) removal yields (80%) were obtained for both SBRs, yet higher sludge retention

time was achieved in the slower feeding rate, SBR2. Under dye and organic loading shocks, both SBRs

maintained high colour removal yields, while COD removal yields dropped to 70% due to a lower

anaerobic COD consumption. The detoxification potential of both systems, determined with yeast-based

assays, pointed to a better performance of SBR2 and all of the SBR1 samples were significantly more

toxic. Thus, SBR2 appeared better suited for the treatment of this simulated wastewater. Residual AR14

and the major stable biodegradation product, 4-amino-1-naphthalenesulfonic acid (4A1NS), were not

directly responsible for the toxicity of the samples. Statistical analysis pointed to the existence of

correlations between the levels of two additional unidentified metabolites present in the SBR2 samples

and the cytotoxicity data. Overall results suggest the development of a more diverse microbial

community in the SBR2 able to biodegrade to less harmful metabolites the dye present in the textile

wastewaters.

Key words: Textile wastewater; Azo dye; aerobic granular sludge; sequencing batch reactor;

detoxification; yeast-based toxicity assays

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Resumo:

As águas residuais têxteis são resistentes ao tratamento aeróbico e são caracterizadas pela elevada

carga orgânica e presença de corantes, tendo um elevado impacto ambiental. A descoloração destas

águas contendo corantes azo já tinha sido alcançada em reator sequencial descontinuo (SBR) e com

biomassa granular. Este estudo compara o desempenho, em termos de eficiência e de potencial de

destoxificação, de dois SBRs com regimes hidrodinâmicos distintos no tratamento de uma água residual

artificial têxtil contendo o corante azo acid red 14 (AR14). Em ambos, os rendimentos de remoção de

cor e COD foram semelhantes (80%), contudo o SBR2, com a velocidade de alimentação mais baixa,

atingiu uma idade de lamas superior. O aumento das cargas de corante e orgânica não influenciou a

capacidade de remoção de cor, mas reduziu a capacidade de remoção de COD final. O potencial de

destoxificação obtido com os dois bioreatores foi determinado com base em ensaios de toxicidade com

levedura e revelou o melhor desempenho do SBR2 face ao SBR1, cujas amostras foram

significativamente mais tóxicas. Demonstrou-se que os compostos AR14 e o seu metabolito estável 4-

amino-1-naftaleno-sulfonato (4A1NS), não são responsáveis pela toxicidade detetada. A análise

estatística indicou a existência de dois metabolitos presentes nas amostras do SBR2 que apresentam

correlação com os resultados de toxicidade. Estes resultados sugerem o desenvolvimento de uma

comunidade microbiana capaz de degradar em metabolitos menos nocivos o corante presente nesta

água residual.

Palavras chave: Água residual têxtil; corante azo; grânulos aeróbicos; Reator sequencial descontinuo;

destoxificação; ensaios de toxicidade em levedura.

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Index:

Acknowledges: ..........................................................................................................................................i

Abstract..................................................................................................................................................... ii

Resumo: .................................................................................................................................................. iii

1. Introduction .......................................................................................................................................... 1

1.1. Textile industry and textile wastewaters ....................................................................................... 1

1.1.1. Composition and environmental impact ................................................................................ 1

1.1.2. Azo dyes ................................................................................................................................ 4

1.2. Textile wastewater treatment ....................................................................................................... 9

1.2.1. Physical-chemical treatments ................................................................................................ 9

1.2.2. Biological treatments ........................................................................................................... 10

1.3. Aerobic granular sludge (AGS) .................................................................................................. 14

1.4. Assessment of the toxicity of azo dye wastewaters ................................................................... 15

1.5. Saccharomyces cerevisiae and Caenorhabditis elegans: Relevance in toxicological studies of

xenobiotics ......................................................................................................................................... 17

1.6. Thesis aims and outline .............................................................................................................. 19

2. Materials and methods ...................................................................................................................... 20

2.1. Bioreactor setup and operation .................................................................................................. 20

2.2. Synthetic textile wastewater ....................................................................................................... 21

2.3. Sampling ..................................................................................................................................... 22

2.4. Analytic methods ........................................................................................................................ 22

2.4.1. Total suspended solids and volatile suspended solids ....................................................... 22

2.4.2. Sludge volume index and sludge retention time ................................................................. 23

2.4.3. Chemical oxygen demand removal, colour removal and pH ............................................... 23

2.4.4. High performance liquid chromatography ........................................................................... 24

2.5. Assessment of the toxicity of the collected samples .................................................................. 24

2.5.1. Control solutions and sample processing............................................................................ 24

2.5.2. S. cerevisiae microplate susceptibility assay ...................................................................... 25

2.5.3. S. cerevisiae gene expression assay .................................................................................. 25

2.5.4. C. elegans reproduction assay ............................................................................................ 26

2.5.5. Preliminary work in toxicity assays ...................................................................................... 26

2.5.6. Statistical analysis of toxicity data ....................................................................................... 27

3. Results and discussion ...................................................................................................................... 27

3.1. SBR operation ............................................................................................................................ 27

3.1.1. Biomass properties .............................................................................................................. 28

3.1.2. SBR performance ................................................................................................................ 30

3.1.3. Cycle characterization ......................................................................................................... 32

3.2. Toxicity assessment of the samples from operation period I ..................................................... 38

3.2.1. Sample processing and preliminary studies ........................................................................ 38

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3.2.2. Effects on yeast growth ....................................................................................................... 41

3.2.3. Effects on the transcript levels of the yeast gene GRE2 ..................................................... 44

3.2.4. Effects on C. elegans reproduction ..................................................................................... 46

3.2.5. Wastewater detoxification potential and AR14 metabolite profiles ..................................... 48

3.3. Toxicity assessment of the samples from operation period II .................................................... 53

4. Final discussion and future work ....................................................................................................... 58

5. Bibliography ....................................................................................................................................... 60

6. Annexes: ............................................................................................................................................ 70

Annex I – Toxicity reports on both mono and acid azo dyes ............................................................. 70

Annex II – Chromatograms from samples collected from SBRs during the period I ......................... 80

Annex III – Chromatograms from samples collected from SBRs during the period II ....................... 86

Figure index:

FIGURE 1 - MANUFACTURING TEXTILE PROCESSING LINE REPRESENTED WITH BOTH MAIN RELEVANT

CHARACTERISTICS AND MAJOR POLLUTANTS PRESENT IN THE WASTEWATER GENERATED (ADAPTED FROM

VERMA ET AL. (2012)). .................................................................................................................................. 2

FIGURE 2- SCHEMATIC REPRESENTATION OF THE EFFECT OF TEXTILE WASTEWATER DISCHARGE INTO THE

ENVIRONMENT (ADAPTED FROM VERMA ET AL. (2012)). .............................................................................. 3 FIGURE 3- CHEMICAL STRUCTURE OF AR14. ........................................................................................................ 5 FIGURE 4 - SCHEMATIC DIAGRAM SHOWING INTERACTIONS OF ENVIRONMENTAL FACTORS (EXTRINSIC: OXYGEN,

LIGHT, MOISTURE, ETC.; AND INTRINSIC: MICROBIAL ENZYMES, ETC.) WITH THE AZO DYES (CHEMICAL

STRUCTURE) AND THEIR (BIO)DEGRADATION PRODUCTS, WHICH ALTOGETHER DETERMINE THE

ENVIRONMENTAL FATE AND ECOLOGICAL CONSEQUENCES OF AZO DYES. THE SOLID LINES REPRESENT

PATHWAYS LEADING TO TOXIC METABOLITES, WHEREAS DOTTED LINES REPRESENT PATHWAYS LEADING

TO NON-TOXIC ENVIRONMENTALLY SAFE METABOLITES (ADAPTED FROM RAWAT ET AL. (2016)). .............. 6 FIGURE 5- CHEMICAL STRUCTURES OF THE AZO DYE AR14 AND OF THE TWO AROMATIC AMINES FORMED

DURING THE AZO BOND REDUCTION REACTION, 1-NAPHTHOL-2-AMINO-4-SULFONIC ACID (1N2A4S) AND

4-AMINO-1-NAPHTHALENESULFONIC ACID (4A1NS) (ADAPTED FROM FRANCA ET AL. (2015)). ................. 9 FIGURE 6 -- SCHEMATIC OF SBR OPERATION, WITH THE FILLING, REACTION, SETTLING AND DRAWING WHICH CAN

BE FOLLOWED BY AN IDLE PHASE. ................................................................................................................ 14 FIGURE 7 - TYPES OF TOXICITY TESTING METHODS USED IN THE ASSESSMENT OF THE HAZARD POSED BY AZO

DYES AND THEIR DEGRADATION PRODUCTS THAT MAY BE PRESENT IN TREATED WASTEWATERS (ADAPTED

FROM SOLÍS ET AL. (2012)). ........................................................................................................................ 16 FIGURE 8 - TSS MEASUREMENTS IN THE MIXED LIQUOR OF SBR1 (▪, FULL BLACK LINE) AND SBR2 (●, FULL

GREY LINE) AND IN THE DISCHARGED EFFLUENT OF SBR1(▫, DASH BLACK LINE) AND SBR2 (○, DASH GREY

LINE) DURING PERIOD I (68 UNTIL 103 OPERATIONAL DAY) AND PERIOD II (162 UNTIL 169 OPERATIONAL

DAY). ............................................................................................................................................................. 29 FIGURE 9 - SLUDGE RETENTION TIME (SRT) CALCULATED FOR SBR1 (▪, FULL BLACK LINE) AND SBR2 (●, FULL

GREY LINE) DURING PERIOD I (68 UNTIL 103 OPERATIONAL DAY) AND PERIOD II (162 UNTIL 169

OPERATIONAL DAY). ..................................................................................................................................... 29 FIGURE 10 – TOTAL DISSOLVED COD REMOVAL YIELDS FOR SBR1 (▪, FULL BLACK LINE) AND SBR2 (●, FULL

GREY LINE) DURING PERIOD I, OPERATION DAYS FROM 68 TO 103 (FEED: 500MG O2/L AS COD, 71.5

MGNH4CL/L AND 20 MG AR14/L); AND PERIOD II, OPERATION DAYS FROM 162 TO 173 (DAY 162 WITH

FEED: 500MG O2/L AS COD, 71.5 MGNH4CL/L AND 60 MG AR14/L; DAYS 167 AND 169 WITH FEED:

1500MG O2/L AS COD, 214.5 MGNH4CL/L AND 60 MG AR14/L). ............................................................ 31

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FIGURE 11 - COLOUR REMOVAL YIELDS FOR SBR1 (▪, FULL BLACK LINE) AND SBR2 (●, FULL GREY LINE),

CALCULATED FROM ABSORBANCE READINGS AT THE WAVELENGTH OF 515 NM, DURING PERIOD I,

OPERATION DAYS FROM 68 TO 103 (FEED: 500MG O2/L AS COD, 71.5 MGNH4CL/L AND 20 MG AR14/L),

AND PERIOD II, OPERATION DAYS FROM 162 TO 173 (DAY 162 WITH FEED: 500MG O2/L AS COD, 71.5

MGNH4CL/L AND 60 MG AR14/L; DAYS 167 AND 169 WITH FEED: 1500MG O2/L AS COD, 214.5

MGNH4CL/L AND 60 MG AR14/L). .............................................................................................................. 32 FIGURE 12 – TOTAL DISSOLVED COD, IN FULL LINE, AND COLOUR, IN DASH LINE, REMOVAL PROFILES OF

OPERATIONAL DAY 75 IN BOTH SBR1 (▪) AND SBR2 (●). THE BLACK VERTICAL LINE MARKS THE ONSET OF

AERATION. SBR’S FEEDING SOLUTION WITH 500MG O2/L AS COD, 71.5 MG NH4CL/L AND 60 MG

AR14/L. ....................................................................................................................................................... 33 FIGURE 13 – TOTAL DISSOLVED COD, IN FULL LINE, AND COLOUR, IN DASH LINE, REMOVAL PROFILES OF

OPERATIONAL DAY 82 IN BOTH SBR1 (▪) AND SBR2 (●). THE BLACK VERTICAL LINE MARKS THE ONSET OF

AERATION. SBR’S FEEDING SOLUTION WITH 500MG O2/L AS COD, 71.5 MG NH4CL/L AND 60 MG

AR14/L. ....................................................................................................................................................... 34 FIGURE 14 - TOTAL DISSOLVED COD, IN FULL LINE, AND COLOUR, IN DASH LINE, REMOVAL PROFILES OF

OPERATIONAL DAY 89 IN BOTH SBR1 (▪) AND SBR2 (●). THE BLACK VERTICAL LINE MARKS THE ONSET OF

AERATION. SBR’S FEEDING SOLUTION WITH 500MG O2/L AS COD, 71.5 MG NH4CL/L AND 60 MG

AR14/L. ....................................................................................................................................................... 34 FIGURE 15 - TOTAL DISSOLVED COD, IN FULL LINE, AND COLOUR, IN DASH LINE, REMOVAL PROFILES OF

OPERATIONAL DAY 162 IN BOTH SBR1 (▪) AND SBR2 (●). SBR’S FEEDING SOLUTION WITH 500MG O2/L

AS COD, 71.5 MG NH4CL/L AND 60 MG AR14/L. THE BLACK VERTICAL LINE MARKS THE ONSET OF

AERATION. .................................................................................................................................................... 35 FIGURE 16 - TOTAL DISSOLVED COD, IN FULL LINE, AND COLOUR, IN DASH LINE, REMOVAL PROFILES OF

OPERATIONAL DAY 169 IN BOTH SBR1 (▪) AND SBR2 (●). SBR’S FEEDING SOLUTION WITH 1500MG O2/L

AS COD, 214.5 MG NH4CL/L AND 60 MG AR14/L. THE BLACK VERTICAL LINE MARKS THE ONSET OF

AERATION. .................................................................................................................................................... 36 FIGURE 17 – REPRESENTATIVE PH PROFILES ALONG THE REACTION PHASE OF THREE TREATMENT CYCLES OF

SBR1, NAMELY CORRESPONDING TO THE DAY 75 (FULL LINE, 500MG O2/L AS COD, 71.5 MG NH4CL/L

AND 20 MG AR14/L), DAY 162 (DASH LINE, 500MG O2/L AS COD, 71.5 MG NH4CL/L AND 60 MG

AR14/L) AND DAY 169 (DOT LINE, 1500MG O2/L AS COD, 214.5 MG NH4CL/L AND 60 MG AR14/L). THE

BLACK VERTICAL LINE MARKS THE ONSET OF AERATION. ............................................................................ 37 FIGURE 18 - REPRESENTATIVE PH PROFILES ALONG THE REACTION PHASE OF THREE TREATMENT CYCLES OF

SBR2, NAMELY CORRESPONDING TO THE DAY 75 (FULL LINE, 500MG O2/L AS COD, 71.5 MG NH4CL/L

AND 20 MG AR14/L), DAY 162 (DASH LINE, 500MG O2/L AS COD, 71.5 MG NH4CL/L AND 60 MG

AR14/L) AND DAY 169 (DOT LINE, 1500MG O2/L AS COD, 214.5 MG NH4CL/L AND 60 MG AR14/L). THE

BLACK VERTICAL LINE MARKS THE ONSET OF AERATION. ............................................................................ 37 FIGURE 19 – MASS CONCENTRATION OF AR14 DETERMINED BY HPLC IN THE SOLUTION WWFEED SUBJECTED

TO THREE DIFFERENT STERILIZATION PROCEDURES. FILTRATION THROUGH STERILE WHATMAN™

PURADISC FILTERS WITH 0.2µM PORE; FILTRATION THROUGH STERILE PALL® ACRODISC/SUPOR FILTERS

WITH 0.2µM PORE AND AUTOCLAVED AT 121°C FOR 20 MINUTES. ............................................................. 39 FIGURE 20 – MASS CONCENTRATION OF 4A1NS DETERMINED BY HPLC IN THE SOLUTION WWAMINE

SUBJECTED TO THREE DIFFERENT STERILIZATION PROCEDURES. FILTRATION THROUGH STERILE

WHATMAN™ PURADISC FILTERS WITH 0.2µM PORE; FILTRATION THROUGH STERILE PALL®

ACRODISC/SUPOR FILTERS WITH 0.2µM PORE AND AUTOCLAVED AT 121°C FOR 20 MINUTES. ................ 39 FIGURE 21 – EFFECT ON THE YEAST GROWTH INHIBITION RATIO OF THE SAMPLES COLLECTED AT THE ONSET

OF THE REACTION PHASE (WWFEED) AND THE WWAMINE SOLUTION IN COMPARISON WITH FEED

SOLUTION WITHOUT DYE (WWCONTROL). ERROR BARS REPRESENT ±1 STANDARD DEVIATION. MEANS

FOR WWFEED AND WWAMINE WERE NOT SIGNIFICANTLY DIFFERENT FROM WWCONTROL (TUCKEY´S

TEST; P > 0.999). ......................................................................................................................................... 42 FIGURE 22 – EFFECT ON THE YEAST GROWTH INHIBITION RATIO OF SAMPLES COLLECTED FROM THE SBR1 AT

THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE) FOR THE

OPERATION DAYS 76, 83 AND 90, IN COMPARISON WITH THE FEED SOLUTION WITHOUT DYE

(WWCONTROL). ERROR BARS REPRESENT ±1 STANDARD DEVIATION. “●” DENOTES MEAN VALUES

SIGNIFICANTLY DIFFERENT FROM THE CONTROL WWCONTROL (TUKEY´S TEST; P < 0.0001). ................. 43

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FIGURE 23 – EFFECT ON THE YEAST GROWTH INHIBITION RATIO OF SAMPLES COLLECTED FROM SBR2 AT THE

END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE) FOR THE

OPERATION DAYS 76, 83 AND 90, IN COMPARISON WITH THE FEED SOLUTION WITHOUT DYE

(WWCONTROL). ERROR BARS REPRESENT ±1 STANDARD DEVIATION. “●” DENOTES MEAN VALUES

SIGNIFICANTLY DIFFERENT FROM THE CONTROL WWCONTROL (TUKEY´S TEST; P < 0.0001). ................. 43 FIGURE 24 – TOXICITY ASSESSMENT BASED ON INCREASED EXPRESSION OF THE YEAST STRESS-INDICATOR

GENE GRE2 OF THE SAMPLES COLLECTED AT THE ONSET OF THE REACTION PHASE (WWFEED) AND THE

FEEDING SOLUTION WITH THE STABLE AROMATIC AMINE 4A1NS INSTEAD OF THE AR14 (WWAMINE), IN

COMPARISON WITH THE FEED SOLUTION WITHOUT DYE (WWCONTROL). ERROR BARS REPRESENT ±1

STANDARD DEVIATION. ................................................................................................................................. 44 FIGURE 25 - TOXICITY ASSESSMENT BASED ON INCREASED EXPRESSION OF THE STRESS YEAST INDICATOR

GENE GRE2 OF SAMPLES COLLECTED FROM SBR1 AT THE END OF THE ANAEROBIC REACTION

(WWANAER) AND THE END OF AERATION (WWEFFLUE) FOR THE OPERATION DAYS 76, 83 AND 90 IN

COMPARISON WITH FEED SOLUTION WITHOUT DYE (WWCONTROL). ERROR BARS AND “ND” REPRESENT,

RESPECTIVELY, ±1 STANDARD DEVIATION AND NOT DETERMINED. ............................................................. 45 FIGURE 26 - TOXICITY ASSESSMENT BASED ON INCREASED EXPRESSION OF THE STRESS YEAST INDICATOR

GENE GRE2 OF SAMPLES COLLECTED FROM SBR2 AT THE END OF THE ANAEROBIC REACTION

(WWANAER) AND THE END OF AERATION (WWEFFLUE) FOR THE OPERATION DAYS 76, 83 AND 90 IN

COMPARISON WITH FEED SOLUTION WITHOUT DYE (WWCONTROL). ERROR BARS REPRESENTS ±1

STANDARD DEVIATION. ................................................................................................................................. 46 FIGURE 27 – EFFECTS ON REPRODUCTION OF C. ELEGANS BRISTOL N2 OF SAMPLES COLLECTED FROM SBR1

AT THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF THE AERATION (WWEFFLUE)

FROM THE OPERATION DAYS 76 AND 83, IN COMPARISON WITH THE FEED SOLUTION WITHOUT DYE

(WWCONTROL). ERROR BARS REPRESENT ±1 STANDARD DEVIATION....................................................... 47 FIGURE 28 - EFFECTS ON REPRODUCTION OF C. ELEGANS BRISTOL N2 OF SAMPLES COLLECTED FROM SBR2

AT THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF THE AERATION (WWEFFLUE)

FROM THE OPERATION DAYS 76 AND 83, IN COMPARISON WITH THE FEED SOLUTION WITHOUT DYE

(WWCONTROL). ERROR BARS REPRESENT ±1 STANDARD DEVIATION....................................................... 47 FIGURE 29 - MASS CONCENTRATION OF AR14 (DESCENDANT DIAGONAL STRIPES) AND 4A1NS (ASCENDANT

DIAGONAL STRIPES) IN THE SAMPLES COLLECTED FROM SBR1, IN THE OPERATIONAL DAYS 76, 83 AND

90, AT THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE)

DETERMINED BY HPLC (SPECTROPHOTOMETRIC DETECTION AT 220 NM USING A LICHROCART

PUROSPHER STAR RP-18E COLUMN). “N” REPRESENTS A MASS CONCENTRATION VALUE EQUAL TO

ZERO. ............................................................................................................................................................ 49 FIGURE 30 - MASS CONCENTRATION OF AR14 (DESCENDANT DIAGONAL STRIPES) AND 4A1NS (ASCENDANT

DIAGONAL STRIPES) IN THE SAMPLES COLLECTED FROM SBR2, IN THE OPERATIONAL DAYS 76, 83 AND

90, AT THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE)

DETERMINED BY HPLC (SPECTROPHOTOMETRIC DETECTION AT 220 NM USING A LICHROCART

PUROSPHER STAR RP-18E COLUMN). “N” REPRESENTS A MASS CONCENTRATION VALUE EQUAL TO

ZERO. ............................................................................................................................................................ 49 FIGURE 31 – AREA OF RELEVANT PEAKS DETECTED IN CHROMATOGRAMS OF THE SAMPLES COLLECTED FROM

SBR1 IN OPERATIONAL DAYS 76, 83 AND 90, AT THE END OF THE ANAEROBIC REACTION (WWANAER)

AND THE END OF AERATION (WWEFFLUE). PEAK Α1 (PURPLE BARS), PEAK Β1 (ORANGE BARS) AND PEAK

Γ1 HAD A RETENTION TIME OF 22, 27 AND 29 MINUTES, RESPECTIVELY. SPECTROPHOTOMETRIC

DETECTION AT 220 NM USING A LICHROCART PUROSPHER STAR RP-18E COLUMN. ........................... 51 FIGURE 32 - AREA OF RELEVANT PEAKS DETECTED IN CHROMATOGRAMS OF THE SAMPLES COLLECTED FROM

SBR2 IN OPERATIONAL DAYS 76, 83 AND 90, AT THE END OF THE ANAEROBIC REACTION (WWANAER)

AND THE END OF AERATION (WWEFFLUE). PEAK Α2 (PURPLE BARS), PEAK Β2 (ORANGE BARS) AND PEAK

Γ2 HAD A RETENTION TIME OF 22, 27 AND 29 MINUTES, RESPECTIVELY. SPECTROPHOTOMETRIC

DETECTION AT 220 NM USING A LICHROCART PUROSPHER STAR RP-18E COLUMN. ........................... 52 FIGURE 33 – EFFECT ON THE YEAST GROWTH INHIBITION RATIO OF THE SAMPLES COLLECTED AT THE ONSET

OF THE REACTION PHASE DURING THE PERIOD II AND THE FEED WITH A COD OF 1500 MG O2/L, 214.5 MG

NH4CL/L AND WITHOUT DYE (WWCONTROL3) IN COMPARISON WITH FEED SOLUTION WITHOUT DYE

(WWCONTROL). FEEDING SAMPLES DURING OPERATIONAL DAYS 159 TO 165 (WWFEED+) WITH COD OF

500MG O2/L, 71.5 MG NH4CL/L AND 60 MG AR14/L AND 165 TO 173 (WWFEED3) WITH COD OF

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1500MG O2/L, 214.5 MG NH4CL/L 60 MG AR14/L. ERROR BARS REPRESENT ±1 STANDARD DEVIATION.

MEANS FOR WWFEED+, WWCONTROL3 AND WWFEED3 WERE NOT SIGNIFICANTLY DIFFERENT FROM

WWCONTROL (TUCKEY´S TEST; P > 0.999). .............................................................................................. 53 FIGURE 34 – EFFECT ON THE YEAST GROWTH INHIBITION RATIO OF SAMPLES COLLECTED FROM SBR1 AT THE

END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE) FOR THE

OPERATION DAYS 165 AND 173, IN COMPARISON WITH THE FEED SOLUTION WITHOUT DYE

(WWCONTROL). SBR’S FEEDING SOLUTION AT THE OPERATIONAL DAY 165 WITH 500MG O2/L AS COD,

71.5 MG NH4CL/L AND 60 MG AR14/L; AND AT THE DAY 173 WITH 1500MG O2/L AS COD, 214.5 MG

NH4CL/L AND 60 MG AR14/L. ERROR BARS REPRESENT ±1 STANDARD DEVIATION. ............................... 54 FIGURE 35– EFFECT ON THE YEAST GROWTH INHIBITION RATIO OF SAMPLES COLLECTED FROM SBR2 AT THE

END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE) FOR THE

OPERATION DAYS 165 AND 173, IN COMPARISON WITH THE FEED SOLUTION WITHOUT DYE

(WWCONTROL). SBR’S FEEDING SOLUTION AT THE OPERATIONAL DAY 165 WITH 500MG O2/L AS COD,

71.5 MG NH4CL/L AND 60 MG AR14/L; AND AT THE DAY 173 1500MG O2/L AS COD, 214.5 MG NH4CL/L

AND 60 MG AR14/L. ERROR BARS REPRESENT ±1 STANDARD DEVIATION. ............................................... 55 FIGURE 36 - MASS CONCENTRATION OF AR14 (DESCENDANT DIAGONAL STRIPES) AND 4A1NS (ASCENDANT

DIAGONAL STRIPES) IN THE SAMPLES COLLECTED FROM SBR1, IN THE OPERATIONAL DAYS 165 AND 173,

AT THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE)

DETERMINED BY HPLC (SPECTROPHOTOMETRIC DETECTION AT 220 NM USING A LICHROCART

PUROSPHER STAR RP-18E COLUMN). ...................................................................................................... 56 FIGURE 37 - MASS CONCENTRATION OF AR14 (DESCENDANT DIAGONAL STRIPES) AND 4A1NS (ASCENDANT

DIAGONAL STRIPES) IN THE SAMPLES COLLECTED FROM SBR2, IN THE OPERATIONAL DAYS 165 AND 173,

AT THE END OF THE ANAEROBIC REACTION (WWANAER) AND THE END OF AERATION (WWEFFLUE)

DETERMINED BY HPLC (SPECTROPHOTOMETRIC DETECTION AT 220 NM USING A LICHROCART

PUROSPHER STAR RP-18E COLUMN). ...................................................................................................... 56 FIGURE 38 - AREA OF RELEVANT PEAKS DETECTED IN CHROMATOGRAMS OF THE SAMPLES COLLECTED FROM

SBR1 IN THE OPERATIONAL DAYS 165 AND 173, AT THE END OF THE ANAEROBIC REACTION (WWANAER)

AND THE END OF AERATION (WWEFFLUE). PEAK Α1 (PURPLE BARS), PEAK Β1 (ORANGE BARS) AND PEAK

Γ1 HAD A RETENTION TIME OF 22, 27 AND 29 MINUTES, RESPECTIVELY. SPECTROPHOTOMETRIC

DETECTION AT 220 NM USING A LICHROCART PUROSPHER STAR RP-18E COLUMN. ........................... 57 FIGURE 39 - AREA OF RELEVANT PEAKS DETECTED IN CHROMATOGRAMS OF THE SAMPLES COLLECTED FROM

SBR2 IN THE OPERATIONAL DAYS 165 AND 173, AT THE END OF THE ANAEROBIC REACTION (WWANAER)

AND THE END OF AERATION (WWEFFLUE). PEAK Α2 (PURPLE BARS), PEAK Β2 (ORANGE BARS) AND PEAK

Γ2 HAD A RETENTION TIME OF 22, 27 AND 29 MINUTES, RESPECTIVELY. SPECTROPHOTOMETRIC

DETECTION AT 220 NM USING A LICHROCART PUROSPHER STAR RP-18E COLUMN. ........................... 58 FIGURE 40 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 76 (76 WWANAER). ............................................................................................................................. 80 FIGURE 41 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 76 (76 WWEFFLUE). ............................................................................................................................ 80 FIGURE 42 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 76 (76 WWANAER). ............................................................................................................................. 81 FIGURE 43 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 76 (76 WWANAER). ............................................................................................................................. 81 FIGURE 44 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 83 (83 WWANAER). ............................................................................................................................. 82 FIGURE 45 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 83 (83 WWEFFLUE). ............................................................................................................................ 82 FIGURE 46 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 83 (83 WWANAER). ............................................................................................................................. 83 FIGURE 47 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 83 (83 WWEFFLUE). ............................................................................................................................ 83 FIGURE 48 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 90 (90 WWANAER). ............................................................................................................................. 84 FIGURE 49 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 90 (90 WWEFFLUE). ............................................................................................................................ 84

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FIGURE 50 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 90 (90 WWANAER). ............................................................................................................................. 85 FIGURE 51 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 90 (90 WWEFFLUE). ............................................................................................................................ 85 FIGURE 52 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 165 (165 WWANAER). ......................................................................................................................... 86 FIGURE 53 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 165 (165 WWEFFLUE). ........................................................................................................................ 86 FIGURE 54 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 165 (165 WWANAER). ......................................................................................................................... 87 FIGURE 55 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 165 (165 WWEFFLUE). ........................................................................................................................ 87 FIGURE 56 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 173 (173 WWANAER). ......................................................................................................................... 88 FIGURE 57 – CHROMATOGRAM OBTAIN FROM SBR1 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 173 (173 WWEFFLUE). ........................................................................................................................ 88 FIGURE 58 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF ANAEROBIC PHASE ON THE OPERATIONAL

DAY 173 (173 WWANAER). ......................................................................................................................... 89 FIGURE 59 – CHROMATOGRAM OBTAIN FROM SBR2 AT THE END OF AERATION PHASE ON THE OPERATIONAL

DAY 173 (173 WWEFFLUE). ........................................................................................................................ 89

Tables index:

TABLE 1 - CHEMICAL AND PHYSICAL CHARACTERISTIC OF WASTEWATER GENERATED AT DIFFERENT STAGES OF

THE TEXTILE INDUSTRY PROCESSING (ADAPTED FROM DASGUPTA ET AL. (2015)). ................................. 3

TABLE 2 - SUPPLEMENT 8 OF THE REGULATION CE 1907/2006 OF 18 OF DECEMBER OF 2006. LIST OF TOXIC

AND CARCINOGENIC AROMATIC AMINES GENERATED BY REDUCTIVE CLEAVAGE OF AZO DYES FORBIDDEN

IN THE EUROPEAN UNION. .................................................................................................................. 8

TABLE 3 - SOME EXAMPLES OF RESULTS FROM PHYSICAL AND CHEMICAL TREATMENTS OF SYNTHETIC TEXTILE

INDUSTRY WASTEWATERS CONTAINING AZO DYES. ............................................................................. 10

TABLE 4 - ADVANTAGES AND DISADVANTAGES OF THE DIFFERENT STRATEGIES TYPES OF INTEGRATED

ANAEROBIC-AEROBIC BIOREACTORS. ................................................................................................. 12

TABLE 5 – SOME RESULTS FROM BIOLOGICAL TREATMENTS OF SYNTHETIC TEXTILE INDUSTRY WASTEWATERS

CONTAINING AZO DYES. .................................................................................................................... 13

TABLE 6 -SUMMARY OF THE OPERATING CONDITIONS FOR BOTH SBR1 AND SBR2. .................................... 21

TABLE 7 – SLUDGE VOLUME INDEX (SVI) VALUES FROM SBR1 AND SBR2 MEASURED AFTER 5 MIN SETTLING

(SVI5) AND AFTER 30 MIN SETTLING (SVI30). ................................................................................... 30

TABLE 8 – PERCENTAGE OF DECOLOURIZATION OF THE WWFEED SOLUTION, MEASURED AS ABSORBANCE AT

515NM (THE WAVELENGTH OF MAXIMUM ABSORBANCE OF THE AR14, IN THE VISIBLE REGION), BY S.

CEREVISIAE UPON 14 HOURS’ INCUBATION AT 30°C. SUPPLEMENTATION WITH YPD WHEN PROVIDED WAS

REPRESENTED WITH + YPD. ............................................................................................................. 40

TABLE 9 - PERCENTAGE OF DECOLOURIZATION OF THE WWFEED SOLUTION, MEASURED AS ABSORBANCE AT

515NM (THE WAVELENGTH OF MAXIMUM ABSORBANCE OF THE AR14, IN THE VISIBLE REGION), BY E. COLI

ALONE AND WITH C. ELEGANS UPON 72 HOURS’ INCUBATION AT 20°C. ................................................ 41

TABLE 10 – PEARSON CORRELATION BETWEEN THE VALUES OF THE YEAST GROWTH INHIBITION RATIO AND THE

MASS CONCENTRATIONS OF AR14 AND 4A1NS IN THE SAMPLES COLLECTED FROM BOTH SBR UNITS’. 50

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TABLE 11 - PEARSON CORRELATION BETWEEN THE VALUES OF THE YEAST GROWTH INHIBITION RATIO AND THE

AREA OF RELEVANT PEAKS DETECTED IN CHROMATOGRAMS OF THE SAMPLES COLLECTED FROM SBR1 IN

THE OPERATIONAL DAYS 76, 83 AND 90. PEAK Α1 (PURPLE BARS), PEAK Β1 (ORANGE BARS) AND PEAK Γ1

HAD A RETENTION TIME OF 22, 27 AND 29 MINUTES, RESPECTIVELY. ................................................... 51

TABLE 12 - PEARSON CORRELATION BETWEEN THE VALUES OF THE YEAST GROWTH INHIBITION RATIO AND THE

AREA OF RELEVANT PEAKS DETECTED IN CHROMATOGRAMS OF THE SAMPLES COLLECTED FROM SBR2 IN

THE OPERATIONAL DAYS 76, 83 AND 90. PEAK Α2 (PURPLE BARS), PEAK Β2 (ORANGE BARS) AND PEAK Γ2

HAD A RETENTION TIME OF 22, 27 AND 29 MINUTES, RESPECTIVELY. ................................................... 52

TABLE 13 - TOXICITY REPORTS FOR BOTH MONO AZO DYES. CLASSIFICATION BASED. LEVELS OF TOXICITY

CLASSIFICATION: TOXIC (CARCINOGENICITY CATEGORY 1A/ 1B, ACUTE AND CHRONIC TOXICITY/

SPECIFIC ORGAN TOXICITY); HARMFUL (CHRONIC TOXICITY/ SKIN AND EYE SENSITIZATION); IRRITANT

(SKIN AND EYE SENSITIZATION/ SPECIFIC ORGAN TOXICITY); DANGEROUS FOR ENVIRONMENT (AQUATIC

TOXICITY/ CHRONIC TOXICITY); AND NON-TOXIC (TOXICITY NOT REPORTED IN STUDIES). ...................... 70

TABLE 14 - AVERAGE RETENTION TIMES FOR CHROMATOGRAMS' PEAKS STUDIED. ....................................... 80

Abbreviations list:

1N2A4S – 1-naphtol-2-amino-4-sulfonic acid

4A1NS – 4-amino-1-naphthalenesulfonic acid

AGS – aerobic granular sludge

AOX – absorbable organic halides

AR14 – acid red 14

BOD – biochemical oxygen demand

COD – chemical oxygen demand

DMSO – dimethyl sulfoxide

FAS – ferrous ammonium sulphate

HPLC – high performance liquid chromatography

ISO – International Organization for Standardization

LD50 – 50% lethal dose

OECD – Organization for Economic Co-operation and Development

RT-PCR – reverse transcriptase polymerase chain reaction

SBR – sequencing batch reactor

SRT – sludge retention time

SVI – sludge volume index

TDS – total dissolved solids

TSS – total suspend solids

UASB – up-flow anaerobic sludge blanket reactor

VSS – volatile suspend solids

WWTP – wastewater treatment plant

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1. Introduction

1.1. Textile industry and textile wastewaters

1.1.1. Composition and environmental impact

The textile sector is a 467 billion US dollars’ market (EULER HERMES 2016), with a steady increase

reaching the millions of tonnes of production in 2011 (Ghaly et al. 2014; Muda et al. 2013). Furthermore,

it employs more than 2.2 million workers worldwide (Muda et al. 2013).

This sector is estimated to consume between 50 and 240 m3 of water per ton of finished product in

Europe (Mata et al. 2015), while in India this consumption varies between 200 and 300 m3 of water per

ton of finished product (Dasgupta et al. 2015). The wastewater generation from this industry ranges from

3800 to 7600 million m3 per day (Muda et al. 2013). The wastewater from the textile industry presents

two major polluting components: the high organic load and the colour derived from the dyes used in the

process (Dasgupta et al. 2015; Muda et al. 2013; Khandegar & Saroha 2013), which in turn, in some

extreme cases, can cause eutrophication of the receiving water body and the contamination of soils and

groundwater due to leaching processes (Chan et al. 2009; Saratale et al. 2011; Mathur et al. 2005).

Textile wastewater characteristics greatly depend on the type of raw materials, chemicals, techniques

or specific process operations at the mill, on the equipment used and on the production design of the

textile processes (Muda et al. 2013). Besides the rich colour and organic load in the order of the of tens

of thousands of mg per litre, these wastewaters also contain complex chemicals, inorganic salts in

considerable concentration and high amounts of suspended solids (Dasgupta et al. 2015; Khandegar &

Saroha 2013; Chan et al. 2009). Figure 1 details both the different processing stages typically occurring

in the textile industry and the major pollutants derived from each stage (Verma et al. 2012), while table

1 presents some typical parameters for the wastewaters derived from the different processing stages

(Dasgupta et al. 2015).

Taking in consideration the described characteristics of textile wastewaters in table 1, a negative impact

in the receiving water body can be expected. The discharge of an effluent with these characteristics can

affect both fauna, flora and microbial communities in different ways. For example, by promoting an

abnormal growth of the aquatic flora and, in conjunction with dissolved oxygen depletion, the elimination

or reduction of the fauna. In some extremer cases, the complete eutrophication of the water body can

be the final outcome. Still, the presence of colour and suspended solids can also inhibit the

photosynthetic processes by reducing sunlight penetration, thus eliminating or reducing the primary

producers, which in turn affects the ecosystem trophic network. The highly complex structure, the high

molecular weight and durability give dyes a recalcitrant nature, which lead to prolonged effects in the

environment (Dasgupta et al. 2015; Zablocka-Godlewska et al. 2015; Saratale et al. 2011). There can

also be other effects derived from specific textile effluent components. Dyes, disinfectants, insecticides,

pesticides, surfactants, metals, sulphides, salts, solvents, chlorinated compounds and softeners can all

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be present in the wastewaters and possible effects like the suppression of the immune system or

genotoxicity have been attributed to them (Dasgupta et al. 2015; Verma et al. 2012).

Figure 1 - Manufacturing textile processing line represented with both main relevant characteristics and major pollutants present in the wastewater generated (adapted from Verma et al. (2012)).

Other detectable effects like osmotic and oxidative stress, influence in reproduction and related chronical

exposure can be caused by these wastewaters. Some components of textile wastewaters and/or their

products resulting from microbial and/or physic-chemical degradation can leach to the surrounding soils

and the underground waters, resulting in a broader range of affected organisms (Chan et al. 2009;

Saratale et al. 2011; Mathur et al. 2005; Verma et al. 2012; Lourenço et al. 2001). Most of these

environmental impacts are summarized in the figure 2 (Verma et al. 2012).

As already mentioned, besides the organic load, another concern is the presence of colour. Colour is

manly derived from the dye component, which can be constituted by one or more dyes. Regarding the

total dye industry products, in the order of the millions of tonnes (Ozdemir et al. 2013), two thirds are

consumed by the textile industry. The rest is used in the food, paper, cosmetic, paints, varnishes and

pharmaceutical industries (Lourenço et al. 2001; Pandey et al. 2007; Saratale et al. 2011; Pinheiro et

al. 2004).

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Table 1 - Chemical and physical characteristic of wastewater generated at different stages of the textile industry

processing (adapted from Dasgupta et al. (2015)).

Figure 2- Schematic representation of the effect of textile wastewater discharge into the environment (adapted from Verma et al. (2012)).

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1.1.2. Azo dyes

Since ancient times natural pigments have been used to colourize fabrics. Yet, since 1856, when William

Henry Perkin accidently discovered the first synthetic dye, mauevin, more than 100 thousands new

synthetic dyes have been generated (Asad et al. 2007; Saratale et al. 2011; Robinson et al. 2001;

Zablocka-Godlewska et al. 2015; Chen et al. 2009). Usually dyes are composed by a group of atoms

responsible for colour, the chromophores, and electron donating or withdrawing substituents called

auxochromes, responsible for intensifying the colour of the chromophores (Christie, 2001).

Dyes are categorized according to the chromophoric group and/or the method of application. Some of

the most important chromophores are azo (–N=N–), carbonyl (–C=O), methine (–CH=), nitro (–NO2) and

quinoid groups, while for auxochromes the most important are amine (–NH3), carboxyl (–COOH),

sulfonate (–SO3H) and hydroxyl (–OH) (dos Santos et al. 2007). Regarding the method of application,

synthetic dyes can be reactive, acid, direct, basic, mordant, disperse, pigment, vat, anionic, ingrain,

sulphur, solvent and disperse, and these classes are mainly dependant on the auxochromes present in

the dye (Verma et al. 2012).

Approximately 70% of all dyes used worldwide (by weight) are azo dyes, making them the largest group

of dyes used and, as a result, the larger group being released in the processing and cleaning waters

from the textile industry (Baêta et al. 2015; Saratale et al. 2011; dos Santos et al. 2007; Khouni et al.

2012; Rawat et al. 2016).

The azo dye market dominance is attributed to the more than 3000 different possible varieties of azo

dyes providing a greater variety of colours when compare to natural dyes, as well as to the ease and

cost effectiveness of their synthesis and to their stability (Pereira & Alves 2012; Saratale et al. 2011).

Although azo dyes can potentially cover the entire visible spectrum (Lourenço et al. 2000) they are

mostly used for yellow, orange and red colours (dos Santos et al. 2007). Their characteristic group is

the azo bond (–N=N–) that is usually attached to benzene or naphthalene groups, which in turns contain

different substituents (Pereira & Alves 2012; Saratale et al. 2011).

As seen in table 1, the wastewaters from the textile industry usually are strongly coloured, which can be

attributed to the fact that textile dyeing with azo dyes still remains inefficient, with large amounts of

dyestuff being lost in the dyeing process (Mezohegyi et al. 2008). Several reports point to a dye loss to

the wastewater, by weight, between 10% to 50% (Khandegar & Saroha 2013). Meaning that around

280,000 tons are estimated to be discharged every year worldwide, with each discharge, from dyehouse,

typically containing around 0.6 to 0.8 g of dye per litre (Saratale et al. 2011).

Acid dyes are characterised by the incorporation method based in acid baths and for being one of the

most important group of azo dyes. An example of an azo and acid dye is the acid red 14, also known as

azorubine, carmoisine and food red 3, with a chemical structure detailed in figure 3. This synthetic azo

dye is usually commercialized as a disodium salt with a red to maroon appearance. It is used in the

textile industry and in the food industry, its use being authorized in certain food and beverages as an

additive (E122), for example in dried fruit and some cheeses.

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Figure 3- Chemical structure of AR14.

Textile wastewaters, as already described, present several environmental issues, and some of them,

like for instance the blocking of sun light penetration into water bodies, can be directly related to the dye

components. Nevertheless, these dyes, particularly in the case of the synthetic azo dyes, may cause

other concerns regarding health of ecosystems and humans, due to toxicological potential of both the

parental molecules and their (bio)degradation products. Frequently, assessment of the environmental

risk of azo dyes and regulation of their use have been based solely on the toxicity testing of the dyes

parental molecules using laboratory model species and without taking into account possible deleterious

effects of their degradation products (Rawat et al. 2016).

Although azo dyes possess a persisting and recalcitrant nature, interaction with the environment and

the environmental fate of these compounds are, in general, subjected to both extrinsic and intrinsic

factors. Figure 4 represents a schematic and concise diagram showing both the complex interactions of

environmental factors with the dyes and the possible consequences from the ecological point of view

(regarding environmental fate and ecotoxicity) as described by Rawat et al. (2016).

Intrinsic factors are specific interactions related to the ecosystems’ microbial communities and their

biological products such as secreted enzymes, while extrinsic factors could be more easily understood

as the abiotic factors, such as oxygen availability, light, moisture and temperature. All of these factors

vary in both space and time, thus resulting in the dye molecules undergo different degradation pathways

or remain undegradable (Figure 4). It is worth noting that these types of interactions also apply to the

dye’s (bio)degradation by-products (Rawat et al. 2016). Particularly relevant in the environmental

context seems the possibility that synthetic azo dyes can be transformed and/or broken down by diverse

microorganisms, including aerobic and anaerobic bacteria and fungi in the natural environment (e.g. soil

and water) (Rawat et al. 2016; Pinheiro et al. 2004).

The microbial biodegradation of azo dyes has been reported to involve three main mechanisms: a)

under anaerobic conditions, the cleavage of the azo bond through reduction by the action of

azoreductases, thus originating potentially cytotoxic and mutagenic aromatic amines; b) mainly under

aerobic conditions, the direct oxidation of the azo bond, thus giving rise to highly electrophilic diazonium

salts azo dyes; and c) metabolic oxidation other than the azo bond, specially of free amine groups

(Saratale et al. 2011).

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Figure 4 - Schematic diagram showing interactions of environmental factors (extrinsic: oxygen, light, moisture, etc.; and intrinsic: microbial enzymes, etc.) with the azo dyes (chemical structure) and their (bio)degradation products, which altogether determine the environmental fate and ecological consequences of azo dyes. The solid lines represent

pathways leading to toxic metabolites, whereas dotted lines represent pathways leading to non-toxic environmentally safe metabolites (adapted from Rawat et al. (2016)).

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Several aspects can be directly related to the toxicity of the dyes, namely as follows (Figure 4): a) the

molecular weight and lipophilicity influence the diffusion through the cell membrane, thus controlling

bioavailability; b) the presence of free amines and N-acetylated amines, which may undergo metabolic

activation due to the formation of nitrenium ions, thus being able to bind to DNA and RNA resulting in

genotoxicity; and c) enzymatic or non-enzymatic degradation leading to potentially toxic by-products

(Rawat et al. 2016).

The formation of amines, particularly of aromatic ones, mainly through the anaerobic pathway, can lead

to their conversion into electrophilic groups, which have the ability to covalently bond to DNA, resulting

in DNA damage and possibly into carcinogenic events (Figure 4). The first report related to dye toxicity

was in 1895 by Rehn at the congress of the German Surgical Society. Rehn reported the papillomous

disease of the bladder, more known as the “aniline cancer”, in the Frankfurt’s dye workers during 1890s

due to 2-naphthylamine exposure (Chen 2006). This and other studies led to the discovery of the

carcinogenic potential of at least 22 aromatic amines (Table 2). The use of the azo dyes which by

biological degradation (e.g. azo bond reduction) give rise to the formation of these aromatic amines and

the amines themselves have been banned or restricted by the 19th amendment to Directive 76/769/EEC

(Platzek 2013; Pinheiro et al. 2004).

In summary, the possible toxicological risk of the anaerobic reduction of azo dyes, either in natural

waters and sediments and/or in microenvironments in animals’ bodies (e.g. gut and skin microbiota),

leading to the release of hazardous amines, is of particular concern for scientists, regulators and policy-

makers (Rawat et al. 2016; Platzek 2013; Pinheiro et al. 2004).

In the last decades, the structures of many dyes have been modified by adding certain functional groups

(alkyl, sulphonic acid and carboxyl) in order to increase hydrophilicity, steric hindrance and

intermolecular hydrogen bonding, thus aiming to reducing metabolic activation of the amine groups and

diffusion through cell membranes. These modifications led to a direct toxicity reduction under laboratory

test organisms, but on the other hand may result in the bypassing of currently employed chemical and

physical methods of textile wastewaters treatment while increasing their environmental persistence

(Rawat et al. 2016; Pinheiro et al. 2004).

Several examples of reports from toxicological studies of mono and acid azo dyes and the respective

biodegradation metabolites are summarized in Annex I.

Of concern is the persistent nature of azo dyes and the possible degradation pathways that may lead to

unknown toxicity. For example, dyes like Direct Black 38, Direct Red 28 and Direct blue 1 have some

level of carcinogenic potential since their biodegradation can be predicted to give rise to benzidine, 3,3’-

dimethoxybenzidine and o-toluidine, respectively, the amines number 2, 12 and 18 from the table 2

(Rawat et al. 2016); and dyes like acid orange 7 and reactive black 5, that despite the absence of toxicity

of the parental compounds, the resulting amines were shown to exert phytotoxicity towards Cucumis

sativus, genotoxicity on Escherichia coli and cytotoxicity in Vibrio fischeri (Gottlieb et al. 2003; Bay et al.

2014).

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Table 2 - Supplement 8 of the regulation CE 1907/2006 of 18 of December of 2006. List of toxic and carcinogenic

aromatic amines generated by reductive cleavage of azo dyes forbidden in the European Union.

According to the material safety data sheet by Spectrum®, AR14, the model azo dye chosen to be used

in the present work, shows oral 50% lethal dose (LD50) values higher than 10g/kg and 8g/kg in acute

test preformed in rats and mice, respectively. In addition, it can cause skin, eye and lung irritation in

case of direct contact (Street 2013).

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The breakdown of the azo bond, represented in figure 5, gives rise to two different aromatic amines,

namely the stable 4-amino-1-naphthalenesulfonic acid (4A1NS), commercialized as sodium salt, and

the unstable 1-naphthol-2-amino-4-sulfonic acid (1N2A4S) (Franca et al. 2015). For the 4A1NS product,

the dye producers reported a LD50 value of 1g/kg for rat and negative results in the mutagenicity test

with Salmonella typhimurium and Escherichia coli (JP 2013).

Figure 5- Chemical structures of the azo dye AR14 and of the two aromatic amines formed during the azo bond reduction reaction, 1-naphthol-2-amino-4-sulfonic acid (1N2A4S) and 4-amino-1-naphthalenesulfonic acid

(4A1NS) (adapted from Franca et al. (2015)).

1.2. Textile wastewater treatment

The textile industry produces large amounts of wastewaters which are generally strongly coloured, due

to the still inefficient textile dyeing processes (Mezohegyi et al. 2008), and with a considerable organic

load and several others components (Dasgupta et al. 2015; Chan et al. 2009; Khandegar & Saroha

2013). Thus, in order to both control the environmental impact in the receiving water bodies and allow

water reuse with a reasonable good water quality, several countries have regulated the discharge of

these wastewaters. In the case of Portugal, the discharged effluent must comply with several parameters

described in decree-law 236/98 annex XVIII and ordinance 423/97 (CITEVE 2012). The characteristics

usually evaluated are the organic load, the colour and the presence of solids, both suspended or

dissolved (TEBBUTT & Edition 1983) and considering the typical textile wastewater characteristics,

treatment must be applied.

1.2.1. Physical-chemical treatments

Physical and chemical techniques, like membrane filtration processes, sorption techniques, coagulation

or flocculation, can be highly efficient in colour removal. For example, submerged filtration using a hollow

fibre nanofiltration membrane obtained 99.3% and 91.5% of colour and COD removal, respectively

(Zheng et al. 2013). However, these techniques are usually costly (Lourenço et al. 2000) due to

membrane fouling, intense labour, excessive use of chemicals and energy demand (Baêta et al. 2015;

Dasgupta et al. 2015; Zheng et al. 2013). In addition, it only occurs a transference of the contaminant

to another phase, which generates a highly concentrated stream leading to disposal problems (Pearce

et al. 2003; Pereira & Alves 2012; Van Der Zee & Villaverde 2005; Khandegar & Saroha 2013). Other

physical and chemical methods like ozonation, Fenton’s reagent, electrochemical destruction and

photocatalysis, involve complicated procedures and remain economically unattainable (Muda et al.

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2013; Gao et al. 2012). In summary, although physical and chemical processes allow the necessary

colour and COD removal from textile wastewater, they still: a) are expensive; b) with a low versatility; c)

susceptible to other wastewater constituents interfering with their efficiency; and d) generate secondary

wastes that must be handled, making them better suited to be used as finalization processes in the

wastewater treatment (Van Der Zee & Villaverde 2005; Pereira & Alves 2012). Some result from physical

and chemical treatments of synthetic industrial textile wastewaters containing acid azo dyes, were

condensed in table 3, some extra information like the COD removal is also displayed when available.

Table 3 - Some examples of results from physical and chemical treatments of synthetic textile industry

wastewaters containing azo dyes.

Procedure/treatment Dye COD removal Colour/dye

removal

Reference

Advanced oxidation

with TiO2

Acid Orange 7 27% 80% (Satuf et al.

2011)

Electrocoagulation

80 A/m2

Acid red 14 Not available 93% (Khandegar &

Saroha 2013)

electrocoagulation

102 A/m2

Acid red 14 Not available >91% (Khandegar &

Saroha 2013)

Heterogenous

fenton reaction

Acid red 14 Not available 99.3%

in 6 minutes

(Idel-aouad et

al. 2011)

Ozonation

high-throughput

microporous tube-

in-tube

microchannel

reactor

Acid red 14 45% 98% (Gao et al.

2012)

1.2.2. Biological treatments

Biological wastewater treatment can be a more economically feasible and eco-friendly procedure than

physical-chemical treatment, due to the lower operating cost and the higher versatility (Zablocka-

Godlewska et al. 2015; Baêta et al. 2015; Lourenço et al. 2000; Van Der Zee & Villaverde 2005; Muda

et al. 2013; Pereira & Alves 2012; Dasgupta et al. 2015). These advantages can be especially relevant

when considering countries with very limited water resources (Kassab et al. 2010). Biological systems

are able to reach the desired treatment objectives, like the removal of specific wastewater constituents,

when operating at the appropriated conditions (temperature, carbon and nitrogen sources and oxygen

levels) (Zablocka-Godlewska et al. 2015).

The fate of textile dyes during biological wastewater treatment can be subdivided in two main

categories: a) bioadsorption, where the dye is simply transferred from the wastewater to the biomass,

or b) biodegradation, where the dye is converted into other substances (Mezohegyi et al. 2008; Muda

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et al. 2013; Kalyani et al. 2009; dos Santos et al. 2007). Bioadsorption leads to a secondary waste

disposal problem, since a phase transference occurs (Singh et al. 2015), while biodegradation can reach

the complete mineralization of the organic pollutants and improve the organic load and total suspended

solids (TSS) parameters, if the system is finely tuned (Pereira & Alves 2012; Pandey et al. 2007).

Biodegradation treatments can include the use of enzymes or pure/mixed cultures of either bacteria,

fungi or algae (dos Santos et al. 2007; Baêta et al. 2015; Pearce et al. 2003; Solís et al. 2012; Saratale

et al. 2011; Singh et al. 2015). Several drawbacks can be pointed to these processes. The use of

enzymes can be economically prohibitive due to their production cost and half-life, while the use of pure

cultures rises issues concerning, for example, culture stability. Mixed cultures have been widely studied,

thus resulting in an accumulated knowledge in the treatment process.

When considering mixed cultures, usually mainly composed of bacteria, treatments can be divided in

aerobic, anaerobic and combined anaerobic-aerobic treatments. When comparing solely aerobic and

anaerobic systems, while anaerobic systems can handle high organic loads, have a low sludge

production and have the best environment for azo bond cleavage, aerobic systems can reduce organic

load to low and acceptable levels while having shorter startup periods (Chan et al. 2009). Thus,

considering the already described textile wastewaters, with high colour and high COD, the use of solely

aerobic or anaerobic conditions cannot be considered suitable. The appropriate approach to treat textile

wastewaters should take the advantages of both anaerobic and aerobic metabolic conditions by

combining both treatments (Baêta et al. 2015). In these two-stage combined strategy, microbial

communities would be able: a) under anaerobic conditions, to cleave the azo bond with azoreductase

enzymes, while also reducing the high organic load, and b) under aerobic conditions, to remove the

remaining by-products from the azo dye cleavage and further remove the organic load (Saratale et al.

2011; Chen et al. 2002).

This anaerobic and aerobic approach has already shown to be promising for textile wastewater

treatment, since the anaerobic conditions promote azo bond cleavage with the formation of aromatic

amines, colourless by-products that under aerobic conditions can in some cases reach the complete

mineralization, thus attaining high colour and COD removal levels (Muda et al. 2013; Mezohegyi et al.

2008). The main advantages and drawbacks of the integrated anaerobic-aerobic bioreactor approaches

are displayed in table 4. Both approaches with and without physical separation of the aerobic and

anaerobic zones have high efficiency, yet the design and construction of these systems have a big

weight in terms of costs. Both the combined system, known to have an oxygen separation based on

diffusion, and the SBR must be finely tuned and controlled, in terms of the environmental conditions and

the timings in which they operate, respectively. And although their microbial consortia must also be

controlled, they have a lesser impact in terms of cost due to lower energy requirements and smaller

occupational space (Chan et al. 2009).

Some results from the biological treatment of synthetic textile wastewater containing azo dyes, mainly

of the mono azo acid type, were condensed in table 5, some extra information like the COD removal is

also displayed when available.

Focussing on the SBR, an improved version of the fill and draw activated sludge system and an

integrated anaerobic-aerobic system either as two SBRs or as one anaerobic-aerobic system of high

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rate bioreactors (Chan et al. 2009). Its greater advantage is the incorporation of all the units, processes

and operation of the conventional activated sludge in one tank. It is able to carry in sequence the filling,

reaction, settling, drawing and idling phases (Sperling & Lemos Chernicharo 2005; Kassab et al. 2010),

as seen in figure 6. The biomass is retained in the reactor from cycle to cycle due to its settling properties

(Sperling & Lemos Chernicharo 2005). This solids separation allows for floor space savings, thus

reducing further the costs. Due to its characteristics, there has been an increase in implementing SBR

to wastewater treatment for both industrial and municipal purposes (Schwarzenbeck et al. 2005; Chan

et al. 2009). In the case of continuous wastewaters production, like the domestic wastewaters, the

Wastewater treatment plants (WWTP) usually has two or more SBRs, while in the shorter production

cycles (industrial WWTPs where work shifts can be of 8h or 16h) one can fulfil the work (Sperling &

Lemos Chernicharo 2005).

Table 4 - Advantages and disadvantages of the different strategies types of integrated anaerobic-aerobic

bioreactors.

Integrated bioreactor type advantages disadvantages

With and without physical

separation

High organic removal

efficiency,

Capable of handling high

organic loading rate

Complicated design of

bioreactor,

Relative higher construction

cost

Time separation (SBR) Single tank configuration

without the need of clarifier,

Low capital cost,

Low energy requirement,

Flexibility in operation

Complex control of the

microbial consortia,

Determination of the

microbial residence time

require special attention,

High level of sophistication

required

Oxygen diffusion

separation

Low energy requirements,

Small reactor volume

Complex control of the

microbial consortia,

Sensitive to environmental

changes

Having the solid-liquid separation within the SBR tank can lead to an washout of the slow-settling

biomass, with a consequent increase in the COD and N value of discharged wastewater having a major

impact at the start up periods (Schwarzenbeck et al. 2005).

Wastewater treatment based in SBR is significantly dependent upon two major factors: the metabolic

capabilities of the microbial community and the efficiency of the solid-liquid separation (Schwarzenbeck

et al. 2005). The solid-liquid separation is dependable on the settling properties of the biomass.

Activated sludge can vary its aggregation state, between a disperse form (flocculent biomass) and dense

and well defined granules (granular biomass) (Mata et al. 2015). Flocculent technology is hampered

compared to granular technology when using the SBR unit, since granular sludge has smaller settling

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times and better settling properties leading to shorter cycles and clear discharges (Kreuk et al. 2007;

Bruin et al. 2004).

Table 5 – Some results from biological treatments of synthetic textile industry wastewaters containing azo dyes.

Procedure/treatment Dye COD

removal

Colour/dye

removal

Reference

Pseudomonas sp. SUK1 Reactive Red 2 52% 91% (Kalyani et

al. 2009)

Up-flow stirred packed-

bed reactor with

biological activated

carbon

Acid Orange 7 Not available 96% in 0.5

min

(Mezohegyi

et al. 2008)

Anaerobic digester

sludge and aeration

tank mixed liquor

Acid orange 10,

acid red 14 and

acid red 18

Not available 65-90 (Saratale et

al. 2011)

Four-stage sulfidogenic

anaerobic baffled

reactor

Remazol brilliant

violet 5r

89% 98% (Ozdemir et

al. 2013)

UASB and a continuous

flow stirred-tank reactor

aerobic

acid blue 113,

direct black 22,

sarasit blue

97–83% 87–80% (Işık &

Sponza

2006)

UASB Acid Orange 7 Not available 96% (Carvalho et

al. 2008)

Anaerobic reactor

followed by aerobic

reactor (with powdered

activated carbon)

Remazol

Golden Yellow

85% 90% (Baêta et al.

2015)

Attached growth

bioreactor; suspended

bioreactor

reactive black-5 85% both 92% and

85%

respectively

(Saba et al.

2013)

Granular activated

carbon-biofilm SBR

Acid Orange 7 88% 100% (Ong et al.

2008)

Aerobic granular sludge

SBR

Acid red 14 80% 85% (Mata et al.

2015)

Flocculent and aerobic

granular sludge SBR

Acid red 14 80% and

70%

respectively

Between

75% to 80%

(Lourenço et

al. 2015)

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Figure 6 -- Schematic of SBR operation, with the filling, reaction, settling and drawing which can be followed by an

idle phase.

1.3. Aerobic granular sludge (AGS)

Aerobic granules were defined by many authors as compact microbial aggregates with clear boundary,

differing from biofilms due to the absence of an inert carrier, with much better settling properties and

resilience to chemical toxicity and toxic loads than flocs of activated sludge and that under reduced

hydrodynamic shear do not coagulate (Liu et al. 2010; Lee et al. 2010; Kreuk et al. 2007; Muda et al.

2013).

Although the first report of aerobic granular sludge was in 1991 in a continuous up-flow aerobic sludge

blanket bioreactor, the SBR has shown several advantages in the fast selection of granular sludge (Lee

et al. 2010), being the first patent with aerobic granules in SBR granted to Heijnen and van Loosdrecht

in 1998 (Adav et al. 2008) and extended in 2004 (Kreuk et al. 2007).

In terms of microbial communities, the aerobic granules are quite similar to activated sludge, yet both

cell hydrophobicity and extracellular polymeric substances are twofold higher, which justifies the clearer

separation between the granules and the surroundings (Muda et al. 2011; Adav et al. 2008). Scanning

electron microscopy, light microscopy, and confocal laser scanning microscopy together with

fluorescence in situ hybridization allow us to get a picture of the microbial structure inside the

granules(Adav et al. 2008). Granules have been characterised for having different communities of

bacteria, which are correlated to the culture medium in which they are developed. Heterotrophic,

nitrifying, denitrifying, phosphorous-accumulating bacteria, and glycogen-accumulating bacteria have

been identify (Adav et al. 2008).

In summary, aerobic granules have a dense and strong structure, with regular and smooth shape and

clear surface, making them structures easily separated from the mixed liquor. The settling velocities of

aerobic granules vary between 25 and 70 meters per hour, much higher than activated sludge flocs (7

to 10 m/h), leading to higher biomass retention, which in turn, in combination with their complex

structure, allows aerobic granules to withstand high flow rates and organic loading rates and confers

them less vulnerability to toxic compounds, resulting in an enhanced organic degradation capability (Liu

& Tay 2004; Ni & Yu 2010; Adav et al. 2008; Muda et al. 2013).

In terms of aerobic granule applications, domestic sewage treatment is already a reality with the Nereda

technology applied in the first pilot study in a Dutch wastewater treatment plant in 2012 (Kreuk et al.

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2007). There are currently 10 plants operating or under construction in the Netherlands, Portugal and

South Africa and 20 more projects in Australia, Brazil, UK, Poland, France, Germany, Portugal and

South Africa. Aerobic granules have also demonstrated to be able to remove organic carbon, nitrogen,

phosphorus and even toxic organics like phenol, pyridine, p-nitrophenol and 2,4-dichlorophenol (Chan

et al. 2009; Adav et al. 2008).

Dairy wastewaters have been treated with aerobic granules using SBR. These wastewaters are

characterised by loading rates from 6 to 15 kg COD/(m3.d) and still COD removal of 90%, N-removal of

80% and P-removal of 67% were reported with cycles of 8 hours and a volume exchange ratio of 50%

(Schwarzenbeck et al. 2005; Adav et al. 2008). Food industry with similar wastewaters has also been

treated using aerobic granular sludge (Schwarzenbeck et al. 2005).

There are also some reports on the use of aerobic granular sludge to adsorb toxic metals and nuclear

waste of very low strength solutions by replacing ions such as Na+,K+,Ca+2 and Mg+2 (Adav et al. 2008).

The textile industry wastewater seems to be a promising candidate to be treated with aerobic granular

sludge associated with the SBR configurations, as reported by several researchers, with both COD and

colour removal at high levels (Lourenço et al. 2000; Lourenço et al. 2015; Mata et al. 2015; Chan et al.

2009) and even removing the dye by-products, aromatic amines (Mezohegyi et al. 2008). Starting with

flocculent biomass, granulation was achieved by Mata et al. (2015) treating simulated textile wastewater,

achiving 85% and 80% colour and COD removal, respectively. Yet, comparison between flocculent

biomass and aerobic granular biomass in SBRs treating textile wastewaters pointed to the latter being

an effective alternative with higher tolerance to toxicity and higher organic loads and increased

possibility to establish of a more diverse microbial population (Lourenço et al. 2015).

Thus, SBR in combination with aerobic granular sludge has good operational flexibility, simple running

and compact layout (Lourenço et al. 2001), yet for safety reasons at the start up periods and to prevent

granule disintegration, a sedimentation process of 15-30 minutes of the outlet can prevent the release

of high biomass content into the receiving body (Schwarzenbeck et al. 2005). Finally, in respect to

pathogen removal, these treatments have been reported to have a 1-2 log reduction (Kassab et al.

2010).

1.4. Assessment of the toxicity of azo dye wastewaters

Besides decolourisation and organic load removal due to microbial treatment of textile wastewaters,

detoxification of the dye-contaminated wastewaters should also be an important achievement. As

referred above, in many cases, the metabolites formed from azo-dye degradation (for example, aromatic

amines) are more toxic than the parental molecule. Therefore, it is critical to assess the toxicity of the

dyes and of the metabolites formed after dyes (bio)degradation (and/or of the treated effluent samples)

in order to assess the efficacy of each particular textile wastewater treatment process and thus predict

risks associated to the release of the treated wastewaters in natural water courses (Rawat et al. 2016;

Solís et al. 2012).

Since ecosystems involve important trophic interactions between diverse microbial, animal and plant

species, ecotoxicity is trophic level related. Importantly, in general, the magnitude and type of toxic

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effects can be highly different from species to species and, for each organism species, it depends greatly

on the level of biological organization analysed (Figure 4; Annex I). Therefore, different methodologies

have been used to assess the toxicity of azo dyes and their degradation products, as summarized in

Figure 7. Several organisms and endpoints have been used to assess the impact of azo dye degradation

products on the different trophic levels and at different levels of biological organization, such as

cytotoxicity, mutagenicity, genotoxicity, acute ecotoxicity in invertebrates and fish, microbial toxicity, etc.

(Solís et al. 2012). Deleterious effects ranging from DNA-damage and oxidative stress to inhibition of

growth or reproduction have been observed in several organisms like micro-organisms, plants, animals

and even human cells lines (reviewed in Sollis et al. 2012 for products from azo dye degradation).

Figure 7 - Types of toxicity testing methods used in the assessment of the hazard posed by azo dyes and their

degradation products that may be present in treated wastewaters (adapted from Solís et al. (2012)).

Given the impossibility of carrying out the more complex and expensive (but environmentally more

relevant) ecosystem-based studies, (eco)toxicity testing have involved mostly laboratory-based assays

with standard model organisms, at least in a preliminary screening phase. These test organisms are, in

general, selected based on one or more of the following criteria: a) to be representative of the impacted

ecosystems and of different trophic levels, b) to be well studied and easy to maintain and breed in the

laboratory, c) if possible, to indirectly allow to predict possible effects on humans and commercially

important organisms (Knight 2008). Thus, an array of endpoints in several model organisms have been

used in the toxicity testing of azo dye wastewater samples, to assess their potential risk for the

environment. Some examples of commonly applied standard model organisms in ecotoxicity tests

involving endpoints at different categories of biological organization, are described below: a) he Vibrio

fischeri strain NRRL B-11177 to evaluate the disruption of the respiratory process thus reducing

luminescence, in the Microtox® test (Mendonça et al. 2011); b) the algae Pseudokirchneriella

subcapitata and the freshwater aquatic plant Lemna minor to assess the effects on growth of aquatic

organisms from the autotrophic trophic level, by following the Organization for Economic Co-operation

and Development (OECD) guideline 201 and the International Organization for Standardization (ISO)

20079:2005 protocol, respectively. The inhibition in germination of seeds of several plants like Sorghum

vulgare, Phaseolus mungo, Triticum aestivum, Sorghum bicolor, Oryza sativa, Lepidium sativum,

Cucumis sativus, Cajanus cajan and Cicer arietinum (Anjaneya et al. 2011; Solís et al. 2012). The

freshwater crustacean Daphnia magna, widely used and recommended at regulatory level, to assess

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inhibition of reproduction and/or effects on mobility or lethality, using OECD guideline 211 and ISO

6341:2012 (Solís et al. 2012; Kim et al. 2010;)(OECD (Organization for Economic Cooperation and

Development) 2012; Solís et al. 2012; Kim et al. 2010). The fish species Danio rerio, Salmo gairdneri,

Gambusia affinis, Leuciscus idus, Lepomis macrochirus, Cyprinidae, Pimephales promelas, in growth

development and reproduction assays (Brooks 2008).

A special interest has been given to the detection of the mutagenic potential of azo dyes and their

degradation products in several works (Rawat et al. 2016). For instance, the Ames test, the SOS

chromotest and the umu-test, which were developed in 1970’s and 1980’s, have been among the most

frequently applied tests for this purpose (Schmitt et al. 2005; Afanassiev et al. 2000; Lichtenberg-Fraté

et al. 2003; Jia et al. 2002; Knight et al. 2004; Zhang et al. 2008). Yet, these tests do present some

drawbacks since: a) several different strains must be used in order to detect different mutagens; b) some

reports indicated the failure in detecting some known DNA-damaging carcinogens, and c) being based

in prokaryotes such as the bacterium Salmonella tiphimurium, no direct effects in eukaryotes can be

inferred (Afanassiev et al. 2000; Jia et al. 2002; Zhang et al. 2008; Solís et al. 2012). The use of cell

lines from higher eukaryotes, like for example lymphocytes or hepatocytes from human or other animal

origin and Allium cepa root cells, have been used in genotoxicity studies related with chromosomal

damage detection, by using the comet assay. Yet, this type of cultures require more time and labour

and are more prone to contaminations, lacking the advantages of the microbial assays (Knight 2008;

Solís et al. 2012; Kolekar et al. 2012; Waghmode et al. 2012).

1.5. Saccharomyces cerevisiae and Caenorhabditis elegans: Relevance in toxicological

studies of xenobiotics

As already mentioned, regulatory assessment of the ecological risk of environmental samples, in

general, and textile wastewaters, in particular, has relied mainly on toxicity data obtained with standard

model organisms representative of diverse ecosystems trophic levels and taxa and using mostly

phenotypic endpoints. Nevertheless, short-term in vitro bioassays based on the assessment of

deleterious effects on microbial cultures (e.g. bacteria, yeast, fungi, protozoa), animal cell lines or

enzyme activity, are being developed and increasingly used, to meet regulatory guidelines (EU directive

86/609/EEC) to reduce, refine and replace (3Rs) whole animals in toxicity testing of xenobiotics and

environmental samples (Gil et al. 2015; Knight et al. 2004; Knight 2008).

The yeast Saccharomyces cerevisiae is a non-pathogenic experimental eukaryote model system. It is

an easily cultivated microorganism, with a fully annotated and sequenced genome, and has been used

as test-organism in both toxicity assessment and in studies to unravel toxicity mechanisms of xenobiotic

compounds (Knight 2008; Papaefthimiou et al. 2004). Relatively easy endpoints like growth and cellular

viability have been successfully used in assessment of toxicity in several works (Kasemets et al. 2009;

Gil et al. 2015; Knight et al. 2004; Papaefthimiou et al. 2004; Gil et al. 2014; Ivask et al. 2014; Lourenço

et al. 2015; Mendes et al. 2011). Interestingly, the yeast shares strong conservation, at metabolic and

regulatory levels, with experimentally less accessible higher eukaryotes (Knight, 2008).

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Yet, to try to enlighten the type of stress the organism is being subjected as well as possible particular

types of effects, more specific assays must be applied. For example, specific types of stress like

osmotic, oxidative, damage to DNA (genotoxicity) and other biological structures, hypoxia and others,

can be caused by particular compounds or mixtures (Simmons et al. 2009). Measurement of alterations

in the expression of specific genes is a promising tool in (eco)toxicity and environmental risk

assessment, because: (a) it is sensitive and reflects organism responses to environmental challenges,

even before measurable cytotoxic effects or reductions in vitality may occur; (b) it can provide

information on stressor mechanism of action. Among diverse ecotoxicological studies based on gene

expression profiling in diverse organisms, a number have used the yeast S. cerevisiae as a bioassay

in the assessment of environmental samples and to disclose toxicity biomarkers and predict toxicity

mechanisms of xenobiotics (Sirisattha et al. 2004; Gil et al. 2011; Gil et al. 2014). Recently, the

usefulness of a S. cerevisiae gene expression assay for the rapid screening of the toxicity of

environmental samples (e.g. eluates from fungicide-sprayed soil) was shown by comparing yeast data

with data from assays with standard soil and freshwater organisms (Gil et al 2015). This yeast-based

assay measured changes in the expression of selected genes previously reported as indicators of the

fungicide toxicity reflecting physiological effects of the xenobiotic (Gil et al. 2014).

There are a number of yeast genes whose transcription has been reported to be enhanced upon cells

exposure to DNA-damaging xenobiotics, being for example involved in the repair of damaged DNA.

Therefore, damage in DNA can be detectable and measurable by monitoring alterations in the

expression of those genes, which were validated as genotoxicity biomarkers in toxicity testing with S.

cerevisiae (Lichtenberg-Fraté et al. 2003; Knight et al. 2004; Afanassiev et al. 2000; Schmitt et al. 2005).

Examples of these yeast genes are as follows: the gene RAD54 (Lichtenberg-Fraté et al. 2003; Knight

et al. 2004; Afanassiev et al. 2000; Schmitt et al. 2005) encoding for a DNA-dependent ATPase involved

in the recombinational repair of double-strand breaks in DNA during vegetative growth and meiosis

(Saccharomyces Genome Database, 2016); the genes RNR3 and the RNR2 (Afanassiev et al. 2000;

Jia et al. 2002; Zhang et al. 2008) encoding for respectively the minor isoform of large subunit and the

small subunit of the ribonucleotide-diphosphate reductase responsible for regulating DNA replication

and DNA damage checkpoints (Saccharomyces Genome Database, 2016) and the gene MAG1 (Jia et

al. 2002) encoding for the 3-methyl-adenine DNA glycosylase involved in DNA protection against

alkylating agents and responsible for the excision of damaged bases to create abasic sites for further

repair (Saccharomyces Genome Database, 2016). Recently, a yeast-based gene expression assay

based on the measurement of changes in the number of RDA54 and RNR3 transcripts was successfully

used to assess the potential detoxification of a textile wastewater during SBR operation (Lourenço et

al., 2015).

Besides genotoxicity, xenobiotics can provoke other types of specific effects (e.g. oxidative, osmotic,

etc.) in the yeast and other organisms. In several cases, a general stress response can be instead

provoked. The yeast gene GRE2 is an example of a general stress responsive gene whose expression

modification has been used as indicator of the toxicity level of pesticide solutions (Gil et al., 2011, 2014)

and of azo dye wastewaters (Lourenço et al. 2015). GRE2 encode a NADPH-dependent methylglyoxal

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reductase (Guo et al. 2014) and has been reported to be induced also due to heat shock, exposure to

heavy metals and osmotic, ionic and oxidative stress (Saccharomyces Genome Database, 2016).

Caenorhabditis elegans, a bacterivorous soil nematode that inhabits the soil liquid phase, has been

used as an eukaryotic model in toxicity testing being representative for both water, sediment and soil

(Sochová et al. 2006; Shen et al. 2009). The recognized relevance of C. elegans in this respect can be

attributed to several advantageous characteristics, as follows: a) it is a simple animal model, with a short

life cycle, b) its genome is fully sequenced, c) it has rudimentary organs similar to those found in

mammals, d) it has cellular signalling pathways highly conserved among eukaryotic organisms, e) its

physiology and developmental system is well characterised, f) it is easy to culture, and its growth and

reproduction are easy to follow up, and g) it is usable in high-throughput toxicological studies since it

can be cultured in multi-well plates using small amounts of medium and sample volume (Boyd et al.

2011; Hitchcock et al. 1997; Leung et al. 2008; Corsi 2006; Bianchi et al. 2015; Rui et al. 2013).

Several endpoints have been used on C. elegans nematodes for the assessment of the toxicity of

xenobiotics and environmental samples (reviewed in Leung et al. 2008). Besides lethality, several sub-

lethal endpoints can be considered such as: a) body length, b) locomotion behaviour, c) feeding

behaviour, d) reproduction, e) life-span, f) neuronal development and function, g) stress response,

oxidative damage and reactive oxygen species production, h) defecation and permeable state of

intestinal barrier and j) expression of stress-responsive genes and other specific genes (Anderson et al.

2001; Sochová et al. 2006; Zhao et al. 2014; Wu et al. 2014; Rui et al. 2013; Bianchi et al. 2015; Gil et

al. 2015; Leung et al. 2008).

In summary, both S. cerevisiae cells and C. elegans worms have been used as eukaryotic models to

assess the potential toxicity not only of compounds and mixtures, but also of industrial wastewaters

(Hitchcock et al. 1997; Hitchcock et al. 1998; Lourenço et al. 2015; Leung et al. 2008; Mendes et al.

2011). It is worth noting that toxicity data obtained with the worm model may complement the data

obtained with the microbial yeast model, since the former can integrate effects not only at subcellular

and cellular levels but also at tissue and organ levels comprising xenometabolism as well as the

reproduction, developmental, neurological and digestive systems (Leung et al. 2008).

1.6. Thesis aims and outline

Previous works have pointed that textile wastewater treatment may generate cytotoxic and mutagenic

toxicity during operation, which cannot be overlooked. Regulatory guidelines have pushed to the

development and application of more short-term in vitro bioassays in order to improve wastewater

treatments. Taking in consideration that toxicity may derive from dye components or its by-products,

beside the evaluation of efficiency parameters, the detoxification potential must also be taken into

consideration. Lourenço et al. (2015) compared the treatment of flocculent and AGS in the treatment of

a simulated textile wastewater containing the model azo dye AR14. Both biomass types had similar

colour removal yields (75%-80%), but a higher anaerobic and overall COD removal yield (80%) were

obtained in the AGS SBR. Also, the comparison of toxicity of samples collected from both SBR using

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yeast based assays revealed the better performance of the AGS reactor, with respect to detoxification

potential. Thus, highlighting the better performance of the AGS.

The present work aims to compare two hydrodynamic regimens in the treatment of the same simulated

textile wastewater using the AGS in SBR. For this, two SBR units with different feeding rate strategies,

were compared not only in terms of the efficiencies of organic load and AR14 removal, but also their

potential to detoxify the simulated textile wastewater. The two SBRs, named SBR1, with the same

configuration as the AGS SBR used by Lourenço et al. (2015), and SBR2, with a slower feeding rate,

were fed with a simulated textile wastewater containing AR14, and samples were collected along several

cycles of operation in order to characterize operational parameters and compare the potential toxicity of

the wastewater at three different cycle stages (namely, the feeding phase, at the end of the anaerobic

reaction and at the end of the aeration reaction (i.e. the end of the treatment cycle)). For the toxicity

testing bioassays, the eukaryotic microbial model S. cerevisiae was chosen to detect responses to

stress and/or genotoxicity using a gene expression assay and to measure potential cytotoxicity using a

growth inhibition microplate assay. Effects of the samples on the reproduction of the simple animal

model C. elegans were also tested. Aiming to understand the toxicity data, Pearson correlations were

attempted between toxicity results and aromatic metabolites detected in SBR samples by HPLC.

Finally, in this work, the impact of first an increase of the AR14 concentration followed by an increase

of COD and NH4Cl concentrations, were used as loading shocks to evaluate the resiliency of both SBRs.

2. Materials and methods

2.1. Bioreactor setup and operation

The experimental system comprised two 1.5-litre bubble-column anaerobic/aerobic SBRs (SBR1 and

SBR2) with a height/diameter ratio of 2.5. These bioreactors were operated during 173 days with 6-hour

cycles consisting of five discrete sequential phases, namely: the filling, the reaction (anaerobic and

aerobic), the settling, the discharge and the idle. Both reactors were inoculated with aerobic granules (2

g TSS/L) previously stored for 2.5 months at 4°C. The granules were originally harvested from the

Nereda® demonstration SBR at Frielas WWTP (Portugal) and used in a similar laboratory-scale SBR

run for 80 days prior to storage.

SBR1 was operated with a static feeding stage and a mechanically-mixed anaerobic reaction followed

by aeration. SBR2 was operated with a slow non-mixed anaerobic feeding stage, from the bottom and

through the granular sludge bed, followed by a mechanically-mixed anaerobic phase and a subsequent

aeration phase.

Both SBR units were operated with a volumetric exchange ratio of 50%, thus with a hydraulic retention

time of 12 hours. The sludge retention time was not controlled, i.e., biomass loss was limited to sampling

and to discharge within the treated wastewater resulting from low settleability. The feed solution was

inserted at the bottom of both reactors.

The reaction phase included an anaerobic mixed stage followed by an aerobic stage, with 1.5 and 3.5

hours, respectively. In the anaerobic stage, mixing was provided by magnetic stirring in SBR1 and by a

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motor in SBR2. In the aerobic stage, aeration was supplied by air compressors through membrane

diffusers with an air flow of approximately 3.0 L/min for each reactor, leading to an aeration rate of 2

vvm (volume per volume per minute).

After a settling phase of 5 minutes, the treated wastewater was pumped out of the SBRs through a

probe located at the liquid’s mid-height. SBR operation, with the exception of sampling, was

automatically controlled by a computer with a dedicated software. The different cycle phases for each

reactor are summarized in detail in table 6.

Table 6 -Summary of the operating conditions for both SBR1 and SBR2.

Cycle phase SBR1 SBR2

Filling

(no mixing; no aeration)

25 min 50 min

Anaerobic reaction

(with stirring)

1.5 h

Aerobic reaction

(no stirring)

3.5 h

Settling 5 min

Discharge 1 min

Idle 29 min 4 min

Total 6 h

Regarding pH and temperature, these parameters were not controlled throughout the operation.

However, the phosphate buffer present in the feed solution kept the pH of the mixed liquor between 5.7

and 7.0 pH units. Moreover, according to the room temperature variations, the reactors were operated

within the [17.4; 28.2] ºC range.

SBRs operation was subdivided in two periods: period I, comprising the operational days 1 to 158; and

period II, comprising the operational days 158 to 173, where system resilience to two loading shocks

was studied.

2.2. Synthetic textile wastewater

A synthetic textile wastewater was prepared to feed both reactors. The feeding solution was prepared

with a starch-based sizing agent (Emsize E1, Emsland-Stärke GmbH, Germany) as carbon source

(1000 mg O2/L as COD corresponding to 1.15 g/L Emsize E1), which was pre-hydrolysed by adding

NaOH, mixed overnight and finally neutralized with HCl to pH 7, supplemented with phosphorus,

nitrogen, macronutrients and micronutrients in the following concentrations: 2310 mg/L

Na2HPO4·12H2O, 762 mg/L KH2PO4, 143 mg/L NH4Cl, 22.5 mg/L MgSO4·7H2O, 27.5 mg/L CaCl2, 250

g/L FeCl3·6H2O, 40 µg/L MnSO4·4H2O, 57 µg/L H3BO3, 43 µg/L ZnSO4·7H2O and 35 µg/L

(NH4)6Mo7O24·4H2O. The model azo dye Acid Red 14 (AR14, Chromotrope FB, Sigma-Aldrich, 50% dye

content) was added to the feeding solution, with a final concentration of 40 mg/L. This solution was used

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as the synthetic textile wastewater during the period I of operation. Regarding period II, two feeding

solutions were used. In order to apply the loading shocks, these solutions had the same composition as

the previously described one with some differences. Briefly: a) between days 158 and 165 of operation,

the feeding solution had a three-fold increase in AR14 concentration (120 mg/L) than the feeding

solution of the period I; b) from day 165 until the end of the operation, the feeding solution had a three-

fold increase in AR14, Emsize and NH4Cl concentrations (respectively, 120 mg/L AR14, 3000 mg O2/L

as COD and 429 mg/L NH4Cl) than the feeding solution of the period I.

2.3. Sampling

Samples collected during selected SBR cycles were clarified by centrifugation (at 4 000 rpm for 10 min)

prior to both treatment efficiency and toxicity assessment. The samples for treatment efficiency

evaluation (COD, colour and pH determination) were collected at specific time points of the reaction

phase. For the anaerobic stage: a) at the start, b) after 30 minutes and c) at the end. For the aerobic

stage: a) after 30 minutes and b) at the end. Samples for suspended solids assessment were collected

during the last 30 minutes of the aerobic stage using a solids pipet. Samples were then conserved in a

freezer at -20°C in 50 mL plastic falcons.

Samples for toxicity assessment were collected, 24 h after those used in the SBRs operational

characterization, at three points of each analysed cycle: a) at the end of the feeding phase

(corresponding to the simulated dye-laden textile wastewater prior to treatment) being labelled as

WWfeed (during the first 158 operational days), WWfeed+ (between operational days 159 and 165) or

WWfeed3 (from operational day 166 until the end of the reported experimental period); b) at the end of

the anaerobic stage, labelled as WWanaer and c) at the end of the aerobic reaction stage, i.e. from the

discharged effluent, labelled as WWefflue. The collected samples were stored at -20°C until used in

toxicity analysis.

2.4. Analytic methods

2.4.1. Total suspended solids and volatile suspended solids

Total suspended solids (TSS) and volatile suspended solids (VSS) were measured on mixed liquor

samples taken from both reactors and respective effluents, according to standard procedures (American

Public Health Association et al. 1999). Glass microfiber filters (GF/C, Whatman of 1.2 µm pore) were

washed with distilled water and treated in a muffle furnace. The drying-combustion cycle included a

temperature increase up to 550ºC during 30 minutes followed by 60 minutes at that temperature. The

empty filters were then weighted and used to retain suspended solids by filtration. After filtration, filters

were dried at 106°C in a moisture balance for TSS determination and were subsequently submitted to

a drying-combustion cycle in a muffle furnace for VSS determination.

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2.4.2. Sludge volume index and sludge retention time

Sludge volume index (SVI) was determined by measuring the volume occupied by the sludge settled

from 1 L of mixed liquor, after 5 and 30 min of settling (SVI5 and SVI30, respectively), and dividing it by

the corresponding mixed liquor TSS value. This measurement was carried out offline in an Imhoff cone,

and the mixed liquor sample was promptly returned to the reactor.

Sludge retention time (SRT) was assessed based in equation 1, where Vr corresponds to the volume of

the reactor (L), Ve to the total volume of effluent discharged from the reactor in one operational day

(L/day), Vs to the total volume of samples collected from the reactor during a complete week (L/week),

TSSr and TSSe correspond to the total suspended solids, respectively, in the reactor and in the effluent

(g/L), and W to number of days in the week (day/week).

𝑆𝑅𝑇 (𝑑𝑎𝑦) = 𝑇𝑆𝑆𝑟 ∗ 𝑉𝑟

𝑇𝑆𝑆𝑒 ∗ 𝑉𝑒 + 𝑇𝑆𝑆𝑟 ∗ 𝑉𝑠/𝑊 (𝑒𝑞𝑢𝑎𝑡𝑖𝑜𝑛 1)

2.4.3. Chemical oxygen demand removal, colour removal and pH

The Chemical oxygen demand (COD), dye concentration and pH were monitored during selected SBR

cycles. Dissolved COD was determined according to a standard procedure (American Public Health

Association et al. 1999). Digestion of the organic matter with potassium dichromate was conducted in a

thermoreactor (TR 420, Spectroquant, Merk) and the digested samples were titrated using ferrous

ammonium sulphate solution (FAS) with a concentration of 0.125 M. COD was quantified according to

equation 2, with VFAS-B corresponding to the volume of FAS solution spent in titration of the blank, VFAS-

samp the volume of FAS solution spent in titration of the sample (mL), and the [FAS] to the molar

concentration of FAS solution.

𝐶𝑂𝐷 (𝑚𝑔 𝑂2

𝐿) =

(𝑉𝐹𝐴𝑆−𝐵 − 𝑉𝐹𝐴𝑆−𝑠𝑎𝑚𝑝) ∗ [𝐹𝐴𝑆] ∗ 8000

𝑉 𝑠𝑎𝑚𝑝

(𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 2)

Colour was determined spectrophotometrically, in a UV-Vis spectrophotometer (Specord 200, Analytik

Jena, Germany; Aspect Plus software, Zeiss, Germany), by reading the sample absorbance at 515 nm

(wavelength of maximum absorbance of the dye AR14, in the visible region), against distilled water,

using quartz cells of 1 cm path length. Based on a calibration curve (equation 3) obtained through

absorbance measurements of AR14 solutions of known concentrations (Lourenço et al. 2015), the

concentration of dye in terms of colour equivalents was determined for each sample.

[𝐴𝑅14](𝑚𝑔

𝐿) = 0,034 ∗ 𝐴𝑏𝑠(515𝑛𝑚) + 0,007 (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 3)

The pH was measured using a Metrohm 6.0262.100 glass electrode connected to a Metrohm

1.691.0010 potentiometer (pH meter 691, Metrohm, Switzerland).

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2.4.4. High performance liquid chromatography

The azo dye AR14 and the sub-products of its degradation were followed by High Performance Liquid

Chromatography (HPLC) with spectrophotometric detection at 220 nm using a LiChroCART Purospher

STAR RP-18e column (250 mm × 4 mm). The eluent consisted of phosphate buffer (25 mM, pH 5.5)

and acetonitrile. A gradient elution (0–50% acetonitrile for 30 min) was used with a flow of 0.7 mL/min.

Calibration curves of AR14 and of the sub-product 4A1NS were established (equations 4 and 5,

respectively) to determine the concentration of these compounds in the samples collected for toxicity

assessment:

[AR14] (mg

L) = 101981 ∗ peak area + 31019 (Equation 4)

[4𝐴1𝑁𝑆] (𝑚𝑔

𝐿) = 211420 ∗ peak area + 13849 (𝐸𝑞𝑢𝑎𝑡𝑖𝑜𝑛 5)

2.5. Assessment of the toxicity of the collected samples

2.5.1. Control solutions and sample processing

Solutions prepared with the same components as the simulated dye-laden textile wastewater prior to

treatment (WWfeed and WWfeed3) but without the dye component were used as control samples.

These control solutions were denominated as WWcontrol and WWcontrol3; the main difference between

them is that the concentration of both Emsize E1 and NH4Cl is the triple in the last one when compared

with the first. Thus, WWcontrol was used for samples collected in the first 165 operational days and the

WWcontrol3 for the samples collected from 165 until the end of operation.

Also, a solution denominated WWamine was prepared with 6.101 mg/L of 4N1AS (Sigma-Aldrich, 80%

purity) plus the same components as the WWcontrol. This solution was prepared assuming the complete

breakdown of the azo bond of the AR14 molecules present in the WWfeed (20 mg AR14/L).

Since all test-samples and control-solutions should be subjected to sterilization prior to being used in

the toxicity assays, preliminary work was carried out to determine what could be the influence of three

different sterilization methods (described below) in the concentrations of AR14 and 4N1AS; the solutions

WWfeed and WWamine were used for this purpose. Therefore, samples of both solutions were either:

a) filtrated through a 0.2 µm pore Supor® membrane filter (hydrophilic polyethersulfone membrane) in a

modified acrylic housing, from Pall® Life Sciences (Pall® acrodisc/supor filters) , or b) filtrated through a

0.2 µm pore membrane filter (polyethersulfone membrane) in a polypropylene housing, from

Whatman™ GE Healthcare Life Sciences (Whatman™ puradisc filters), or c) autoclaved at 121°C for

20 minutes. Each sample was then run in the HPLC for determination of AR14 and 4N1AS

concentrations as described above (2.5.4.). A comparison with the initial concentrations was done to

determine which method was best for sterilization.

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All collected samples and control solutions were then sterilized by the chosen sterilization method

(filtration with filters from Whatman™ GE Healthcare Life Sciences) prior to toxicity testing with S.

cerevisiae or C. elegans.

2.5.2. S. cerevisiae microplate susceptibility assay

In the yeast-based toxicity assays, the S. cerevisiae double deletion mutant Δcwp1Δcwp2 (Zhang et al.

2008) was used (kindly offered by Professor Wei Xiao, University of Saskatchewan, Canada). This

mutant was constructed in the genetic background of S. cerevisiae strain BY4741 through the deletion

of two yeast genes, CWP1 and CWP2, encoding two cell wall mannoproteins (Zhang et al. 2008). This

mutant was chosen to be used as test organism in the present work because these deletions proved to

contribute to improve the sensitivity of the yeast-based assay by altering the outer cell wall structure and

increasing cell wall permeability to a broad range of genotoxic xenobiotics (Zhang et al. 2008).

The inhibitory effects of the filter-sterilized samples and solutions on the growth of S. cerevisiae BY4741

Δcwp1Δcwp2 were measured in a microplate susceptibility assay as previously described (Gil et al.

2015; Mendes et al. 2011; Pereira et al. 2009; Papaefthimiou et al. 2004) with adaptations. Briefly, a

standardized population of yeast cells grown to mid-exponential phase (optical density at 640nm

(OD640nm) equal to 0.50 ± 0.05) in liquid YPD growth medium (glucose 20 g/L, bactopeptone 10 g/L,

yeast extract 5 g/L; pH 6.2) was collected by centrifugation at 10000 rpm for 10 min at 25 °C. Cellular

pellets were resuspended in a total of 2 mL mixture comprising 1933 µL of each test-sample (WWfeed,

WWfeed+, WWfeed3, WWanaer or WWefflue) or of control-solution (WWcontrol, WWcontrol3 and

WWamine) plus 67µL of thirty times-strength YPD medium previously sterilized by filtration, in order to

attain an OD640nm approximately equal to 0.05 (this way dilution of the test-samples or the control-

solutions was minimized, being always around 3.5%). Immediately after the mixtures preparation, they

were distributed in the wells (150 µL per well with triplicates for each mixture) of polystyrene 96-well

microplates (Greiner bio-one). Wells containing reconstituted YPD medium with 6% (v/v) dimethyl

sulfoxide (DMSO) or without any toxicant were also included in the microplate (in triplicate) as positive

and negative toxicity controls respectively (denominated Cont-DMSO and Cont-YPD respectively).

Microplates were then covered with a breathseal and a polystyrene lid (Greiner bio-one) and incubated

at 30 °C with a constant agitation (approximately 250 rmp) for 14 hours. Subsequently, yeast growth

was assessed by counting colony-forming-units (cfu) in 0.1 mL of culture serial dilutions spread-plate

onto YPD agarized medium (agar 20 mg/L). The potential toxicity of the samples was indicated by the

yeast growth inhibition ratio which was estimated using the ratio cfuX/cfu0, where cfuX and cfu0 represent

the concentration of culturable yeast cells (in cfu/mL) attained after the incubation for 14 hours in the

presence of the test-samples or the control-solution respectively. The inhibitory yeast growth ratios for

the Cont-DMSO and Cont-YPD were 0.19 ± 0.11 and 1.09 ± 0.55 respectively.

2.5.3. S. cerevisiae gene expression assay

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The gene expression assay was based on the evaluation of changes in the transcript levels of the yeast

toxicity-indicator genes GRE2 and RAD54 (Schmitt et al. 2005; Garay-Arroyo & Covarrubias 1999;

Lourenço et al. 2015) by quantification reverse transcriptase real-time PCR (qRT-PCR) in an Applied

Biosystems’ 7500 RT-PCR device (Carlsbad, CA, USA). Total RNA extracted, by hot-phenol method

(Kohrer, et al., 1991) from standardized S. cerevisiae BY4741 Δcwp1Δcwp2 cells upon two-hour

exposure (to either the test-samples or the control solution), was used as template. The qRT-PCR assay

was carried out as described elsewhere using the following specific primers: GRE2-F

(GGTGCTAACGGGTTCATTGC), GRE2-R (CGATGACCT- TATAGTCTTCCTTCAACA), RAD54-F

(GAAGCTGAGGCGTTCAACACT) and RAD54-R (GTGCCTTCTCGTCGCTCTTT) (Gil et al. 2015;

Lourenço et al. 2015). For each gene, fold changes in the transcript levels were calculated using relative

quantification (ΔΔCT method, according to manufacturer’s instructions) with mRNA levels of the

housekeeping gene ACT1 as endogenous control. All fold-change values are relative to the control feed

solution without dye (WWcontrol).

2.5.4. C. elegans reproduction assay

The collected test-samples (WWanaer and WWefflue) and control solution (WWcontrol) were also

tested regarding their inhibitory effects on the reproduction of C. elegans Bristol N2 upon a 72-h

exposure period, as previously described (Mendes et al. 2011; Gil et al. 2015) with some modifications.

Briefly, the assay was conducted, with four replicated wells for each sample/solution tested, in 48-well

polystyrene microplates (Greiner Bio-one, Frickenhausen, Germany). In each well, about 2 worms from

age-synchronized cultures (L4 larval stage) were added to a total of 500 µL of a mixture consisting of

440 µL of test-sample (WWanaer, WWefflue) or control-solution (WWcontrol) and 50 µL of a

concentrated freshly grown E. coli OP50 cells (OD640nm=1.5) suspended in K medium (2.098 g/L KCl

and 2.98 g/l NaCl, pH 7.1) and a 10 µL of a fifty times-strength K medium. Wells with K medium (labelled

as Cont-K) and wells with a 6% DMSO solution in K medium (labelled as Cont-DMSO) were used as

toxicity negative and positive controls, respectively, to assess the nematodes heath status and

response. After incubation at 20 °C in the darkness for 72 hours in a FITOCLIMA S600 (Aralab)

incubator, the number of offsprings at all stages beyond the egg stage was scored visually using a

stereomicroscope (50× magnification; Zeiss Stemi 2000-C). The relative toxicity of the test-samples and

the control-solutions was estimated using the ratio OfsX/Ofs0, where OfsX and Ofs0 represent the number

of descendants per parent obtained after the 72 hours’ incubation respectively for the test-samples

(WWanaer and WWefflue) and the control solution (WWcontrol).

2.5.5. Preliminary work in toxicity assays

Some preliminary assays were conducted to check whether the S. cerevisiae cells could remove the

AR14 from the samples/solutions, through for instance absorption or biodegradation, under the test

conditions. For this purpose, a microplate was set up where the solution WWfeed (feed solution with

AR14 at 20 mg/L) was used in two ways: a) as described above (2.5.2) and b) with the mixture

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preparation but missing the yeast cells (denominated YEAST and NOYEAST, respectively). Incubation

was performed for 14 hours at 30ºC, as in the yeast microplate susceptibility assay. After incubation, a

given volume (150 l) was removed from each well and transferred to 1.5mL eppendorfs (one per well)

and centrifuged at 10000 rpm for 10 minutes at room temperature. The colour of the cellular pellets was

observed visually and the supernatants were transferred to a new 96 well microplate for determination

of the absorbance at 515 nm. Absorbance values were compared between the YEAST-wells and the

NOYEAST-wells in order to determined colour reduction, since as, already mentioned, 515nm is the

wavelength of maximum absorbance of the dye AR14, in the visible region. The same was done with

and without supplementation with YPD medium.

As above, C. elegans and E. coli ability to remove AR14 under the test conditions used in the C. elegans

reproduction assay were also assessed. For this purpose, a microplate was prepared only with the

WWfeed solution as described in 2.5.4 with two types of wells, the ones as already described and wells

that did not receive the worms. After incubation for 72 hours at 20°C, the volume of each well was

centrifuged at 10 000 rpm for 2 minutes at room temperature. Pellets colour was observed visually, while

supernatant was transferred to a new 96 well-microplate for determined absorbance at 515 nm.

Absorbance values were compared with the initial solution that did not receive either C. elegans nor E.

coli and was kept in the dark at 4°C.

2.5.6. Statistical analysis of toxicity data

Toxicity data was subject to different statistical analysis using GraphPad Prism 6.01 (© 1992-2012

GraphPad Software, Inc.). Results from toxicity assessment from samples were compared with

WWfeed’s toxicity results using a one-way ANOVA with Tukey’s multiple comparison test (α value of

0.05). Pearson correlations between toxicity assessment results from samples and the peaks detected

in the correspondent samples with HPLC were attempted (α value of 0.05).

3. Results and discussion

3.1. SBR operation

Two anaerobic/aerobic SBR units, inoculated with aerobic granules, were fed with a synthetic textile

wastewater containing AR14 as azo dye model. The SBR units were operated in parallel in 6-hour cycles

and both biomass properties and system efficiency were evaluated weekly. The current work has been

focused in two operational periods. Period I, from day 68 to day 103, corresponded to the start of the

SBR reaction phase with 500mg O2/L as COD, 71.5 mgNH4Cl/L and 20 mg AR14/L. Period II, from day

158 to day 173, comprised the feed loading shocks. A first load increase of AR14 to 60 mg/L, from day

158 to day 165; and a second load increase of the carbon and nitrogen sources and of AR14 to 1500mg

O2/L, 214.5 mgNH4Cl/L and 60 mg AR14/L, respectively from day 165 to 173.

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3.1.1. Biomass properties

Biomass within the SBR units started at the TSS concentration of 2 g/L and increased rapidly in the first

two weeks of operation. This rapid increase can be explained by the rich nutrient availability in the feed

solution and the good settling properties of the aerobic granules leading to low biomass loss in the final

effluent.

A comparison of the TSS in the mixed liquor and in the discharged effluent from both SBR units is

presented in figure 8 for both periods in study. Figure 9 and table 7 comprise the calculated SRT values

and the SVI values measured for both reactors during the analysed periods, respectively. Although VSS

was also assessed, the corresponding data is not shown, since the values corresponded to 90-91% of

TSS in all the analysed samples. Thus, not indicating any difference between either the SBR units, and

the mixed liquor and the discharged effluents.

During period I, both SBRs maintained high levels of biomass within the reactor system, reaching TSS

values of around 5.1 and 8.0 g/L for SBR1 and SBR2, respectively, which were similar to the values

reported in Mata et al. (2015). On the other hand, TSS levels remained low in the discharged effluent,

especially for SBR2, confirming the good settling properties of aerobic granular sludge. Yet, SBR1

conditions seems to be less suitable to grow dense aerobic granules than those of SBR2, which is

supported by the lower SRT and the higher SVI values obtained for SBR1 when compared to the ones

obtained for SBR2.

There was an accidental biomass loss on operational day 82 during the SVI assessment of SBR2, from

the bottom of the Imhoff cone. This event had a special impact on the microbial population of SBR2,

leading to an increase in SVI values and a decrease in the SRT, which suggests that denser granules,

with longer retention times were lost.

Although SRT values from SBR1 were low and decreased in SBR2 resulting from particular accidents,

the biomass remained in the granular form in both reactors, as indicated by the low SVI5 and SVI30

values, and also by the small difference between the SVIs, which were similar to previous works (Mata

et al. 2015). Nonetheless, SBR2 slower feeding rate led to a higher nutrient concentrated solution in

contact with the biomass, which in turn allowed better diffusion within the granular structure and

presumably provided better growing conditions than SBR1.

Within period II, the two loading shocks had different results in the biomass properties. The increase of

the AR14 concentration from 20 to 60 mg/L, and the further increase of the COD to 1500mg O2/L and

NH4Cl to 214.5 mg/L led to different responses by the SBR units. In SBR2, the AR14 concentration

increase resulted in a reduction in its TSS values in the mixed liquor, while the further increase of the

COD and NH4CL increased TSS in the mixed liquor and more than doubled the values in the discharged

effluent to 0.5 g/L. As for SBR1, biomass properties were maintained similar to period I. As a result, the

SRT and SVI values were maintained for SBR1, while SBR2 decreased and increased respectively,

showing that the slow feeding rate exposed more the biomass to these loading shocks. Yet, SBR2

seems to start recovering as indicated by the SVIs reduction observed in the day 169.

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Figure 8 - TSS measurements in the mixed liquor of SBR1 (▪, full black line) and SBR2 (●, full grey line) and in the

discharged effluent of SBR1(▫, dash black line) and SBR2 (○, dash grey line) during period I (68 until 103

operational day) and period II (162 until 169 operational day).

Figure 9 - Sludge retention time (SRT) calculated for SBR1 (▪, full black line) and SBR2 (●, full grey line) during

period I (68 until 103 operational day) and period II (162 until 169 operational day).

0

1

2

3

4

5

6

7

8

9

68 75 82 89 103 162 167 169

TS

S (

g/L

)

Operation day (day)

Period IIPeriod I

0

2

4

6

8

10

12

14

16

18

20

68 75 82 89 103 162 167 169

SR

T (

d)

Operation time (d)

Period IIPeriod I

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Table 7 – Sludge volume index (SVI) values from SBR1 and SBR2 measured after 5 min settling (SVI5) and after

30 min settling (SVI30).

Period Operation

day

SBR1 SBR2

SVI5 (mL/g TSS) SVI30 (mL/g TSS) SVI5 (mL/g TSS) SVI30 (mL/g TSS)

I

68 106 64 76 48

75 103 62 73 45

82 113 67 92 56

89 126 74 88 52

103 111 69 84 50

II

162 105 72 121 71

167 91 59 93 56

169 107 71 89 58

3.1.2. SBR performance

The total dissolved COD and colour removal yields attained in both reactors were similar, with some

exception points, and were depicted in figures 10 and 11, respectively. Regarding period I, the COD

removal levels obtained in both units were above 80%, which is consistent with previous works

(Lourenço et al. 2015; Mata et al. 2015). Although the slower feeding rate promoted higher biomass

levels, this was not reflected in the overall final COD removal yields, probably because enough biomass

was present inside both reactors to consume the COD. When considering period II, the impact of the

increased AR14 concentration showed that COD removal was reduced to less than 70%, revealing a

slight impact of AR14 in the COD removal. This does not contradict the already described null impact

on COD removal of AR14 in a 20 mg/L concentration (Lourenço et al. 2015), since AR14 is three fold

higher (60 mg/L), detonating a concentration dependence. Still in period II, the further increase of COD

and NH4Cl kept the COD removal levels below 70%. SBR1 was the most affected one by this loading

shock, yet a slight recovery can be observed in the day 169.

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Figure 10 – Total dissolved COD removal yields for SBR1 (▪, full black line) and SBR2 (●, full grey line) during

period I, operation days from 68 to 103 (feed: 500mg O2/L as COD, 71.5 mgNH4Cl/L and 20 mg AR14/L); and

period II, operation days from 162 to 173 (day 162 with feed: 500mg O2/L as COD, 71.5 mgNH4Cl/L and 60 mg

AR14/L; days 167 and 169 with feed: 1500mg O2/L as COD, 214.5 mgNH4Cl/L and 60 mg AR14/L).

In terms of colour removal, both SBR units kept high yields, above 80%, during both experimental

periods. These colour readings can be overestimated since readings done spectrophotometrically at

515 nm do not reflect entirely the residual dye concentration. This overestimation is the result of the

brownish colour resulting from reactions involving reduction of dye metabolites, such as the unstable 1-

naphtol-2-amino-4-sulfonic acid (1N2A4S), which in the presence of oxygen can suffer auto-oxidation

and form coloured oligomeric structures (Mata et al. 2015).

Regarding period I, it should be noticed that the yields obtained were also in accordance with previous

works (Lourenço et al. 2015; Mata et al. 2015). The only exception, below 80% of colour removal yield,

was registered in operational day 89 in SBR2, possibly as a result of the biomass loss during the SVI

measurement, as already described. This event could indicate that, although colour removal has been

suggested to be independent from biomass concentration (Lourenço et al. 2015), the type of biomass

have an influence on it, since the loss of denser biomass lead to a reduction of the colour removal yield.

Yet, since accumulation of coloured oligomeric structures can interfere with the spectrometric

quantification of AR14 at low concentrations, no direct conclusion can be assumed.

In contrast with the COD, colour removal yields remained unchanged during period II, showing that the

part of the population responsible for azo bond reduction either was not directly affected or was

protected inside the granular structure from the increased concentrations. Yet, the absolute quantity of

colour removed was three times higher. When comparing the performance of SBR2 resulting from the

loss of the denser biomass, the behaviour in terms of colour and COD removal diverged, colour removal

yield decreased, while COD removal yield increased in day 89 when comparing to day 82. This can be

50%

60%

70%

80%

90%

100%

110%

68 75 82 89 103 162 167 169

% C

OD

rem

oval

Operation day

Period IIPeriod I

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a possible indication that the denser the aerobic granules have a bigger population of either facultative

or anaerobic organisms, able to reduce the azo bond of AR14, than less dense ones.

Figure 11 - Colour removal yields for SBR1 (▪, full black line) and SBR2 (●, full grey line), calculated from

absorbance readings at the wavelength of 515 nm, during period I, operation days from 68 to 103 (feed: 500mg

O2/L as COD, 71.5 mgNH4Cl/L and 20 mg AR14/L), and period II, operation days from 162 to 173 (day 162 with

feed: 500mg O2/L as COD, 71.5 mgNH4Cl/L and 60 mg AR14/L; days 167 and 169 with feed: 1500mg O2/L as

COD, 214.5 mgNH4Cl/L and 60 mg AR14/L).

3.1.3. Cycle characterization

Profiles of COD and colour removal from both periods and SBR units were analysed and depicted in

figures 12 to 16. Figures 12, 13 and 14 correspond to operational days 75, 82 and 89, respectively,

being representative of period I. From period II, figure 15 depicted the operational day 162, with a shock

load of 60 mg/L of AR14, and figure 16 depicted operational day 169, with the feed concentrations

altered to 60 mg AR14/L, 1500mg O2/L as COD and 214.5 mgNH4Cl/L.

In both periods it can be observed that the colour removal, depicted in dashed lines, only occurred

during the anaerobic phase, as demonstrated by previous works (Lourenço et al. 2015; Mata et al.

2015). On the other hand, the COD removal, in full lines, occurred to some extent in both phases.

However, as in Lourenço et al. 2015, during the first 30 minutes a faster consumption can be observed,

suggesting the presence of polyphosphate accumulating organisms, which have been considered key

organisms both in the formation and stability of aerobic granules (Kreuk et al. 2007; Adav et al. 2008;

Lourenço et al. 2015). Yet, anaerobic COD consumption in SBR1 and SBR2, was reduced to less than

10% and 20%, respectively, after the second feed alteration (AR14, COD and NH4Cl levels changed to

60 mg/L, 1500mg O2/L and 214.5 mgNH4Cl/L, respectively).

In period I, as already showed, SBR2 had a higher biomass content in the mixed liquor when compared

with the SBR1. Yet, this did not result in different COD removal yields in the anaerobic phase, with

values between 40 and 50%. COD removal profiles were similar to those presented by Lourenço et al.

50%

60%

70%

80%

90%

100%

110%

68 75 82 89 103 162 167 169

% c

olo

ur

rem

oval

Operation day

Period I Period II

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(2015), both with an anaerobic phase able to remove around 40% of the initial COD and an aerated

phase with a first stage of rapid COD consumption, followed by an increasingly slower stage. As for

colour removal, AR14 concentrations got below 5 mg/L, as in previous works (Franca et al. 2015;

Lourenço et al. 2015). Yet, as already described, these concentration values can be overestimated since

the oxidation of the unstable 1N2A4S can lead to the development of a brownish colour, thus masking

the real AR14 concentration (Franca et al. 2015).

The only unexpected event occurred in SBR1, with deceleration of the COD consumption near 300 mg

O2/L, and further consumption in the aeration phase. This can be related to the biomass in anaerobic

systems although able to cope with high organic load cannot consume it to very low concentrations,

needing a further aerobic phase to consume it. Still, operational day 75 in SBR1seems differ from the

remaining days. COD removal during the anaerobic phase reached a level slightly below 300 mg O2/L

and the final removal was 96.7%, this could indicate that the facultative/anaerobic population had

somehow changed between this and the next days.

Figure 12 – Total dissolved COD, in full line, and colour, in dash line, removal profiles of operational day 75 in

both SBR1 (▪) and SBR2 (●). The black vertical line marks the onset of aeration. SBR’s feeding solution with

500mg O2/L as COD, 71.5 mg NH4Cl/L and 60 mg AR14/L.

0

4

8

12

16

20

24

28

0

100

200

300

400

500

600

700

0.5 1.0 2.0 2.5 3.5 5.5

AR

14 (

mg/L

)

CO

D (

mg O

2/L

)

Cycle time (h)

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Figure 13 – Total dissolved COD, in full line, and colour, in dash line, removal profiles of operational day 82 in

both SBR1 (▪) and SBR2 (●). The black vertical line marks the onset of aeration. SBR’s feeding solution with

500mg O2/L as COD, 71.5 mg NH4Cl/L and 60 mg AR14/L.

Figure 14 - Total dissolved COD, in full line, and colour, in dash line, removal profiles of operational day 89 in both

SBR1 (▪) and SBR2 (●). The black vertical line marks the onset of aeration. SBR’s feeding solution with 500mg

O2/L as COD, 71.5 mg NH4Cl/L and 60 mg AR14/L.

Period II had two loading shocks, as already described, the first being the AR14 concentration increase

to 60 mg/L, and the second with 60 mgAR14/L and an increase in COD and NH4Cl to 1500 mg O2/L and

214.5 mg/L, respectively. The cycle from operational day 162 (Figure 15) represents the SBR response

during the first loading shock. As in the period I, decolourization only occurred during the anaerobic

phase, as expected. Yet, the SBR units presented different removal patterns. While SBR1 presented a

colour removal profile directly associated to the COD removal, SBR2 was still able to remove 20% of

0

4

8

12

16

20

24

28

0

100

200

300

400

500

600

700

0.5 1 2 2.5 3.5 5.5

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14 (

mg/L

)

CO

D (

mg O

2/L

)

Cycle time (h)

0

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8

12

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0.5 1 2 2.5 3.5 5.5

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Cycle time (h)

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the initial dye concentration during the last hour of the anaerobic phase, while consuming less than 1%

of the COD. This may suggest that azo bond reduction can occur through different metabolic pathways,

yet further cycles should be characterized to support this indication.

Figure 15 - Total dissolved COD, in full line, and colour, in dash line, removal profiles of operational day 162 in

both SBR1 (▪) and SBR2 (●). SBR’s feeding solution with 500mg O2/L as COD, 71.5 mg NH4Cl/L and 60 mg

AR14/L. The black vertical line marks the onset of aeration.

The cycle from operational day 169 represents the SBR response during the second loading shock

(Figure 16). Colour removal was again observed only in the anaerobic phase and, as described before,

with similar removal yields as other operational days characterized, yet the apparent association

between COD consumption and colour removal observed in SBR1 on the day 162, could no longer be

seem. In SBR1, COD consumption was inhibited during the anaerobic phase, passing from the 27%

removal during the 162 day to 2% removal during the 169 day. This inhibition took some days to be

clearly observed, since COD consumption during day 167 was only reduced to 7%. Since cellular death

can most likely be exclude due to the steady TSS values in the discharged effluents, this inhibition could

be an indication that some alterations in either composition or metabolism of the facultative/anaerobic

population were taking place.

Of interest, would be the increase of the aeration phase to observe whether a longer aeration time would

lead to a COD reduction to reach the levels measured in the period I, around 100 mg O2/L, or would

stabilized before reaching them.

0

10

20

30

40

50

60

70

0

100

200

300

400

500

600

700

0.5 1 2 2.5 3.5 5.5

AR

14 (

mg/L

)

CO

D (

mg O

2/L

)

Cycle time (h)

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Figure 16 - Total dissolved COD, in full line, and colour, in dash line, removal profiles of operational day 169 in

both SBR1 (▪) and SBR2 (●). SBR’s feeding solution with 1500mg O2/L as COD, 214.5 mg NH4Cl/L and 60 mg

AR14/L. The black vertical line marks the onset of aeration.

Regarding the evolution of pH, cycles from operational days 75, 162 and 169 were chosen to represent

the effect of the different feeding compositions. Figure 17 and 18 depicts the representative pH profiles

for SBR1 and SBR2, respectively. The variation of pH values was limited by the phosphate buffer

present in the feeding solution. Despite the presence of phosphate buffer, it was possible to observe a

trend in both SBR units, corresponding to a reduction of the pH during the anaerobic phase, followed by

an increase in the beginning of the aeration phase and a stabilization until the end of the cycle. The

exceptions to this profile were cycles from the second loading shock, as example the operational day

169. In these day pH was lower than other characterized cycles and decreased after the initial increase

at the begging of the aeration phase. The same was observed for the day 167 (data not showed). This

may point that the increased concentration of the carbon source lead to an increased in the

fermentations processes. Also, the final decrease observed in the aerated phase seems the result of

further fermentation, which could be an indication that the aeration was not sufficient.

0

10

20

30

40

50

60

70

0

400

800

1,200

1,600

2,000

2,400

2,800

0.5 1 2 2.5 3.5 5.5

AR

14 (

mg/L

)

CO

D (

mg O

2/L

)

Cycle time (h)

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Figure 17 – Representative pH profiles along the reaction phase of three treatment cycles of SBR1, namely

corresponding to the day 75 (full line, 500mg O2/L as COD, 71.5 mg NH4Cl/L and 20 mg AR14/L), day 162 (dash

line, 500mg O2/L as COD, 71.5 mg NH4Cl/L and 60 mg AR14/L) and day 169 (dot line, 1500mg O2/L as COD,

214.5 mg NH4Cl/L and 60 mg AR14/L). The black vertical line marks the onset of aeration.

Figure 18 - Representative pH profiles along the reaction phase of three treatment cycles of SBR2, namely

corresponding to the day 75 (full line, 500mg O2/L as COD, 71.5 mg NH4Cl/L and 20 mg AR14/L), day 162 (dash

line, 500mg O2/L as COD, 71.5 mg NH4Cl/L and 60 mg AR14/L) and day 169 (dot line, 1500mg O2/L as COD,

214.5 mg NH4Cl/L and 60 mg AR14/L). The black vertical line marks the onset of aeration.

In summary, both SBR units preformed with similar results in terms of both colour (80%) and COD

removal (83%) during period I, though different biomass levels were present within the reactors. Period

II saw a loss of COD removal efficiency in the anaerobic phase, with a shift of the removal to the aeration

phase, especially for SBR1. It is also important to notice that SBR1 revealed a higher loss of biomass

in the discharged effluent and an almost complete loss of COD removal capacity during the anaerobic

phase, reducing to 2% after the second loading shock.

4.0

4.5

5.0

5.5

6.0

6.5

7.0

7.5

8.0

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Cycle time (h)

75 162 169

SBR1

4

4.5

5

5.5

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6.5

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pH

Cycle time (h)

75 162 169

SBR2

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3.2. Toxicity assessment of the samples from operation period I

Two types of assay based on the eukaryotic model S. cerevisiae were used to compare the toxicity of

samples collected from the SBRs units at different cycle stages. The microplate susceptibility assay

measured their inhibitory effects on yeast growth as an indication of cytotoxicity. The expression assay

measured modifications on the transcription of yeast genes GRE2 and RAD54, related with general

response to stress and specific response to DNA damage, respectively, in the yeast (Schmitt et al. 2005;

Garay-Arroyo & Covarrubias 1999). And a reproduction assay based on the worm model C. elegans

was introduced in order to complement the data obtained with the microbial yeast model, since the

former can integrate effects not only at subcellular and cellular levels but also at tissue and organ levels.

As already referred, the samples for toxicological assessment were collected 24 h after those used in

the SBRs operational characterization (section 3.1). In particular, within the operation period I, samples

were taken at the operational days 76, 83 and 90.

3.2.1. Sample processing and preliminary studies

Before toxicity assessment of the samples collected from SBRs operational, it was necessary to sterilize

these samples. Three sterilization procedures were selected to be tested, using two solutions already

described, WWfeed and WWamine, which contained, respectively, the dye AR14 (20mg/L) and the

stable amine 4A1NS (6.104mg/L). These solutions were chosen to assess the modifications, resulting

from thermal instability or adsorption to filtration material, on the parental dye and/or its transformation

products in the samples.

Two types of disposable membrane filtration systems were chosen to be tested, the Whatman™

Puradisc 25 AS filter device and the Pall® Life Sciences Acrodisc syringe filter, both with 0.2 m

polyethersulfone membranes but with different housing materials (namely, polypropylene or modified

acrylic in the former or the later device, respectively). Also, an autoclave cycle with 20 minutes at 121°C

was chosen. Concentrations of both AR14 and 4A1NS in the model solutions before and after being

subjected to the three indicated sterilization processes were assessed by HPLC and the obtained data

from the single assay, are depicted on figures 19 and 20, respectively. As observed in the figures 19

and 21, the PALL® Life Science Acrodisc/Supor filter device and the autoclave led to the alteration of

the concentrations of AR14 or 4A1NS respectively, while the Whatman™ Puradisc device seemed not

to affect these concentrations. Thus, the sterilization method chosen to proceed with samples

sterilization was the filtration using the Whatman™ Puradisc syringe filters with the polypropylene

housing.

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Figure 19 – Mass concentration of AR14 determined by HPLC in the solution WWfeed subjected to three different

sterilization procedures. Filtration through sterile Whatman™ Puradisc filters with 0.2µm pore; filtration through

sterile Pall® Acrodisc/Supor filters with 0.2µm pore and autoclaved at 121°C for 20 minutes.

Figure 20 – Mass concentration of 4A1NS determined by HPLC in the solution WWamine subjected to three

different sterilization procedures. Filtration through sterile Whatman™ Puradisc filters with 0.2µm pore; filtration

through sterile Pall® Acrodisc/Supor filters with 0.2µm pore and autoclaved at 121°C for 20 minutes.

As preliminary work in the toxicity test, was also important to verify whether or not the model organisms

selected for toxicity testing could contribute for the removal and/or biodegradation of the AR14 in the

tests conditions. This was relevant since several organisms, like filamentous fungi, bacteria and yeast,

have been described as able to carry out transformation and/or biodegradation reactions of azo dyes

(Pereira & Alves 2012; Singh et al. 2015; Ramalho et al. 2005). It seems particularly relevant in this

0

5

10

15

20

25

no treatment Whatman™ Puradisc

Pall®Acrodisc/Supor

autoclave

[AR

14]

(mg/l)

0

1

2

3

4

5

no treatment Whatman™ Puradisc

Pall®Acrodisc/Supor

autoclave

[4A

1N

S]

(mg/l)

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context that growing cells of the yeast species S. cerevisiae were able to decolorize the azo dye m-[(4-

dimethylamino) phenylazo] benzenesulfonic acid as a result of the extracellular reduction of the dye

molecule by a plasma membrane ferric reductase (Ramalho et al. 2005). Thus, a model solution of the

azo dye used in the present study (WWfeed, containing AR14 at 20 mg/L) was incubated in the presence

of a S. cerevisiae cells population or a C. elegans population under the same conditions to be used in

the respective toxicity tests, namely the yeast microplate susceptibility assay and the nematode

reproduction assay, respectively. Data on the degree of decolourization of the AR14 solution, assessed

based on the measurement of the absorbance at 515 nm (wavelength of maximum absorbance of the

AR14) of the respective supernatants, are displayed in tables 8 and 9 for yeast microplate susceptibility

assay and the nematode reproduction assay respectively.

In the conditions of the yeast microplate susceptibility assay, cellular pellets were completely white,

which lead to exclude the bioadsorption as a removal hypothesis. Nevertheless, the absorbance at 515

nm of the respective supernatants decreased after 14 h incubation (Table 8), suggesting that the yeast

cells may have the ability to somehow transform the azo dye in colourless products. In addition, the

yeast cells without the supplementation of YPD were not able neither to grow (data not showed) nor to

biodegrade AR14 (Table 8). These results are consistent with a previous report for a different azo dye

(Ramalho et al. 2005) which pointed to the existence of azo reductase activity in intact actively growing

S. cerevisiae cells (Ramalho et al. 2005). These results indicate that caution should be considered when

using the yeast-based toxicity assay for the assessment of azo dyes. Yet, regarding the samples

collected along the SBRs operation, whose potential toxicity towards yeast cells is compared in the

present work, it should be noted that those samples are expected to have very low levels of AR14 and,

besides that, as far as we are aware, there are no evidences that the yeast cells could be able to further

degrade the presumably formed aromatic amines.

In the C. elegans reproduction assay conditions, both the impact of C. elegans and its food source, E.

coli, in the AR14 concentration were tested (Table 9). Neither the nematodes nor the E. coli cells seemed

to have any effect in the AR14, since absorbance remained as in the start of the test (Table 9) and

pellets also did not present red colour.

Table 8 – Percentage of decolourization of the WWfeed solution, measured as absorbance at 515nm (the

wavelength of maximum absorbance of the AR14, in the visible region), by S. cerevisiae upon 14 hours’

incubation at 30°C. Supplementation with YPD when provided was represented with + YPD.

Reduction of absorbance at 515 nm (%)

average standard deviation

WWfeed + YPD 83.1 2.2

WWfeed 0.0 0.9

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Table 9 - Percentage of decolourization of the WWfeed solution, measured as absorbance at 515nm (the

wavelength of maximum absorbance of the AR14, in the visible region), by E. coli alone and with C. elegans upon

72 hours’ incubation at 20°C.

Reduction of absorbance at 515 nm (%)

E. coli 0.0

E. coli + C. elegans 0.0

3.2.2. Effects on yeast growth

The yeast microplate susceptibility assay measured the inhibitory effects of the samples on yeast growth

as a potential indicator of cytotoxicity. This assay had already been applied in several works, in the

assessment of the potential toxicity of several xenobiotic pesticides or of different synthetic textile dyes

compared with their degradation products (Mendes et al. 2011; Lourenço et al. 2015; Papaefthimiou et

al. 2004; Gil et al. 2014; Pereira et al. 2009).

First, we intended to compare the potential toxicity of the sample collected at the feeding stage

(containing the intact dye AR14; named WWfeed) and the solution WWamine with that of the WWcontrol

(the control solution without both AR14 and its by-products) to assess the inhibitory effect resulting from

the parental dye and its more stable derived amine (4A1NS). Figure 21 shows the yeast growth inhibition

ratio values (relative to the WWcontrol) caused by these two test solutions. The ratio values were close

to 1 not revealing any growth inhibitory effect of neither the AR14 dye nor the aromatic amine 4A1NS

(Figure 21). Statistical analysis with Tukey’s test confirm that the values of the yeast growth inhibition

ratio obtained for both the WWfeed and the WWamine were not significantly different from the

WWcontrol (p>0.9999). A similar result was obtained before using the same solution as WWfeed by

Lourenço et al. (2015), thus supporting the low or absent toxicity of the dye AR14. However, it is worth

noting that the yeast cells may chemically modify the azo dye, as observed above (Table 8), and

therefore this result is not completely conclusive. When considering the result obtained with the

WWamine, i.e. a yeast growth inhibition ratio close to 1, this result suggest that the major stable amine

formed as a result of AR14 biodegradation in the SBRs seems to be not toxic towards yeast growth.

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Figure 21 – Effect on the yeast growth inhibition ratio of the samples collected at the onset of the reaction phase

(WWfeed) and the WWamine solution in comparison with feed solution without dye (WWcontrol). Error bars

represent ±1 standard deviation. Means for WWfeed and WWamine were not significantly different from

WWcontrol (Tuckey´s test; p > 0.999).

The potential toxicity towards the yeast growth of samples collected at the two cycle stages, namely

WWanaer and WWefflue at the end of, respectively, the anaerobic phase and the aeration phase, were

compared for operational days 76, 83 and 90 in SBR1 (Figure 22) and SBR2 (Figure 23).

In contrast with the WWfeed, all samples collected from both SBRs (Figure 22 and 23), with the

exception of the end of aeration phase in SBR2 (Figure 23), inhibited significantly the yeast growth

suggesting that they may have considerably higher potential cytotoxicity than the WWfeed (Tukey’s test,

p≤0.0001). Therefore, it seems that the SBR1 during the operational period I (towards day 90) did not

result in the abatement of the potential cytotoxicity of the simulated textile wastewater towards the yeast

(Figure 22). On contrary, the samples from SBR2 presented a pattern of increased cytotoxic potential

in the end of the anaerobic phase (around 60% growth inhibition relative to control), with a clear

decrease in the toxicity of the samples collected upon the aeration phase (WWefflue) that reached less

20% growth inhibition relative to the control (Figure 23). Indeed, the yeast growth inhibition ratio values

of the WWefflue samples collected from the SBR2 during period I were not significantly different from

that of the WWcontrol and the WWfeed (Tukey’s test, p values 0.1522, 0.9219 and 0.6182 respective

for days 76, 83 and 90) and the same samples from the days 83 and 90 were significant different from

the end of the anaerobic phase (Tukey’s test, p values 0.0013 and 0.006 respective for days 83 and

90). Therefore, this analysis suggested that the aerobic treatment in the SBR2 was able to almost

completely remove the cytotoxic potential presumably generated during the anaerobic phase. Results

of increase toxicity during anaerobic phases were already expected and described by Lourenço et al.

(2015), with a similar result with aerobic granular biomass after aeration during their 71 day of operation.

0

0.2

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1

1.2

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1.6

1.8

WWcontrol WWfeed WWamine

Yeast

gro

wth

(r

ela

tive t

o W

Wcontr

ol)

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Figure 22 – Effect on the yeast growth inhibition ratio of samples collected from the SBR1 at the end of the

anaerobic reaction (WWanaer) and the end of aeration (WWefflue) for the operation days 76, 83 and 90, in

comparison with the feed solution without dye (WWcontrol). Error bars represent ±1 standard deviation. “●”

denotes mean values significantly different from the control WWcontrol (Tukey´s test; p < 0.0001).

Figure 23 – Effect on the yeast growth inhibition ratio of samples collected from SBR2 at the end of the anaerobic

reaction (WWanaer) and the end of aeration (WWefflue) for the operation days 76, 83 and 90, in comparison with

the feed solution without dye (WWcontrol). Error bars represent ±1 standard deviation. “●” denotes mean values

significantly different from the control WWcontrol (Tukey´s test; p < 0.0001).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8Y

east

gro

wth

(r

ela

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o W

Wcontr

ol)

SBR1

●●

● ●

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Yeast

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SBR2

● ● ●

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3.2.3. Effects on the transcript levels of the yeast gene GRE2

With the yeast gene expression assay, we intended to measure the modifications on the transcription

of yeast genes GRE2 and RAD54, related with general response to stress and specific response to DNA

damage, respectively, in the yeast (Schmitt et al. 2005; Garay-Arroyo & Covarrubias 1999). The

rationale was that if exposure of a standardized yeast cells population to a sample collected from the

SBRs units would lead to increased expression of these genes, it would suggest that that sample could

cause general stress conditions and genotoxicity respectively, in the yeast cells. Results regarding

expression of gene RAD54 were not conclusive and thus they are not presented. Data regarding

modification of the expression of the gene GRE2 will be presented and discussed below.

In a similar way, as for the yeast microplate susceptibility assay (3.2.2.), the effects of the WWfeed or

WWamine solutions on the expression of the general stress-responsive gene GRE2 were measured;

the respective fold change values relative to WWcontrol are depicted in figure 24. Both the WWfeed and

WWamine solutions did not modify significantly the transcript levels of GRE2 relative to the WWcontrol

(Figure 24), suggesting that neither the AR14 nor the 4A1NS exerted a general stress response in the

yeast cells. This observation is consistent with the absence of yeast growth inhibition by these two

compounds (Figure 21). On what concerns the 4A1NS, this result (Figure 24) together with the lack of

inhibitory effects on yeast growth (Figure 21) suggest that the presence of this major aromatic amine

product released from AR14 biodegradation in the AGS SBRs (Lourenço et al. 2015) may not contribute

significantly for the potential toxicity of the SBRs’s samples under study in the present work. With regard

with the AR14, this result confirms the reports of Lourenço et al. (2015).

Figure 24 – Toxicity assessment based on increased expression of the yeast stress-indicator gene GRE2 of the

samples collected at the onset of the reaction phase (WWfeed) and the feeding solution with the stable aromatic

amine 4A1NS instead of the AR14 (WWamine), in comparison with the feed solution without dye (WWcontrol).

Error bars represent ±1 standard deviation.

0

0.5

1

1.5

2

2.5

3

3.5

4

WWcontrol WWfeed WWamine

GR

E2

expre

ssio

n f

old

change

(rela

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o W

Wcontr

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Fold change relative to WWcontrol in the expression of the gene GRE2 in the yeast cells exposed to the

samples collected from the SBR1 and the SBR2 are depicted in figures 25 and 26 respectively.

Considering the SBR1 samples, in general, they all increased GRE2 transcription between 1.5- and 3.5-

fold relative to WWcontrol (Figure 25). Interestingly, an apparent pattern of transcription modification

seems to be detected, i.e. slightly higher increase in expression for samples collected at the end of

anaerobic phase (WWanaer) that in the end of aeration phase (WWefflue), at least for the samples from

days 76 and 83. Despite this apparent slight decrease in sample toxicity from the end of anaerobic

reaction to the end of effluent treatment, all the tested samples seem to induce a considerable stress

response in the yeast cells compared with the WWcontrol solution (Figure 25), which is consistent with

their potential cytotoxicity towards the yeast.

Regarding the SBR2 samples, an increased expression of GRE2 was also observed for the WWanaer

samples from the begging of the period I (days 76 and 83) as well as for the sample collected in the end

of the cycle treatment (WWefflue) at day 76 (Figure 26); however, the fold change values for these

samples (between 1.5 and 2) were almost always lower (Figure 26) than for the corresponding samples

collected at SBR1 (Figure 25), denoting a higher potential cytotoxicity of the later. Interestingly, the

transcription fold change values decreased to just one for WWefflue from day 83 and for both samples

from day 90 (Figure 26). These results together with the reduced toxicity of the SBR2 WWefflue samples

towards yeast growth (Figure 23) suggest the success of the SBR2 in achieving detoxification of the

simulated textile wastewater upon 83 and 90 days of operation. This may possibly be due to changes

in the SBR2 population leading to the ability to convert the aromatic amines and other metabolites

formed during anaerobic azo dye reduction in less toxic products, as suggested before for AGS SBR

(Lourenço et al. 2015).

Figure 25 - Toxicity assessment based on increased expression of the stress yeast indicator gene GRE2 of

samples collected from SBR1 at the end of the anaerobic reaction (WWanaer) and the end of aeration (WWefflue)

for the operation days 76, 83 and 90 in comparison with feed solution without dye (WWcontrol). Error bars and

“nd” represent, respectively, ±1 standard deviation and not determined.

0

0.5

1

1.5

2

2.5

3

3.5

4

GR

E2

expre

ssio

n f

old

change

(rela

tive t

o W

Wcontr

ol)

SBR1

nd

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46

Figure 26 - Toxicity assessment based on increased expression of the stress yeast indicator gene GRE2 of

samples collected from SBR2 at the end of the anaerobic reaction (WWanaer) and the end of aeration (WWefflue)

for the operation days 76, 83 and 90 in comparison with feed solution without dye (WWcontrol). Error bars

represents ±1 standard deviation.

3.2.4. Effects on C. elegans reproduction

We also intended to assess the toxicity of the same samples collected from the SBR1 and SBR2 based

on the measurement of the inhibitory effects on the reproduction of the simple animal model C. elegans.

Reproduction of C. elegans in the K medium confirmed the health of the population used in the assays,

with the generation of 61 21 offsprings per parent, in agreement with values previously reported (Gil

et al. 2015; Anderson et al. 2001).

Like before, the WWcontrol (feed without dye) solution was used for comparison purposes in the

reproduction test. The effect was expressed as the ratio between the offsprings generated per worm

exposed to the test samples relative to the control after a 72h exposure; the obtained values are depicted

in figures 27 and 28 for the SBR1 and the SBR2, respectively.

The worm reproduction was slightly inhibited (less than 40% inhibition of reproduction) upon exposure

to the WWanaer samples collected at days 76 and 83 from SBR1 (Figure 27) and to the WWanaer

sample collected at day 83 from SBR2 but slightly stimulated (~1.4-fold) by the 76-day sample (Figure

29). In general, the WWefflue samples from both SBRs exerted no inhibitory effects on nematode

reproduction (Figures 27 and 28), suggesting a pattern of reduction of the inhibitory effect generated

during the anaerobic phase. This SBR2’s results are in agreement with the detoxification potential

observed during period I suggested by toxicity data obtained with the yeast-based tests (discussed

above, 3.2.1.). However, in the case of SBR1, there is no clear correlation between the toxicity data for

the yeast and the nematode. The observed differences between the two testing systems are probably

related to the different complexity of the microbial and the worm biological organization and physiology.

0

0.5

1

1.5

2

2.5

3

3.5

4

GR

E2

expre

ssio

n f

old

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(rela

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o W

Wcontr

ol)

SBR2

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However, it is worth of noting that the data presented in figures 27 and 28 represent one sole

independent assay (with four replicas).

Figure 27 – Effects on reproduction of C. elegans Bristol N2 of samples collected from SBR1 at the end of the

anaerobic reaction (WWanaer) and the end of the aeration (WWefflue) from the operation days 76 and 83, in

comparison with the feed solution without dye (WWcontrol). Error bars represent ±1 standard deviation.

Figure 28 - Effects on reproduction of C. elegans Bristol N2 of samples collected from SBR2 at the end of the

anaerobic reaction (WWanaer) and the end of the aeration (WWefflue) from the operation days 76 and 83, in

comparison with the feed solution without dye (WWcontrol). Error bars represent ±1 standard deviation.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

WWcontrol 76 WWanaer 76 WWefflue 83 WWanaer 83 WWefflue

C. ele

gans r

epro

duction

(rela

tive t

o W

Wcontr

ol)

SBR1

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

WWcontrol 76 WWanaer 76 WWefflue 83 WWanaer 83 WWefflue

C. ele

gans r

epro

duction

(rela

tive t

o W

Wcontr

ol)

SBR2

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3.2.5. Wastewater detoxification potential and AR14 metabolite profiles

During period I, results from yeast microplate susceptibility assay (Figures 22 and 23) and GRE2

expression assay (Figure 25 and 26) present the relevant differences between both SBRs, with apperant

no relation with the results from SBRs performances (3.1 chapter). Thus, in order to clarify these results,

the samples collected from the SBR1 and the SBR2 and subjected to toxicological assessment (days

76, 83 and 90) were also analysed by HPLC and the respective chromatograms obtained (presented in

Annex II) were compared.

From the peaks detected at the wavelength of 220 nm (Annex II), only 4A1NS and AR14, respectively

with a retention time around 14 and 26 minutes, had been previously identified and the respective

concentrations in the SBR1 and SBR2 samples under study were determined based on calibration

curves. Mass concentration values determined this way for the samples collected from SBR1 and SBR2

were depicted respectively in figures 29 and 30.

AR14 concentrations, determined spectrophotometrically, that varied between 4 and 5 mg/L in the end

of the aeration phase for operational days 75, 82 and 89 (Figures 12, 13, 14), where showed to be

higher than the concentration values determined with HPLC in the samples collected at 76, 83 and 90.

Indeed SBR1 samples presented less than 1 mg AR14/L (Figure 29), while SBR2 revealed a greater

variance yet not higher than 5 mg AR14/L (Figure 30). These results show that AR14 degradation was

possibly higher than the 80% reported previously (Figure 11). Results from SBR performance chapter

and the HPLC analyses showed here may indicate that reactions to form coloured oligomeric structures

described by Mata et al. (2015) occurred preferably in the SBR1, since differences between the days

where higher.

The concentration values of the stable amine 4A1NS, in the samples under analysis, were surprisingly

lower than expected (Figures 29 and 30), since concentrations were lower than 6.101 mg/L, the

concentration predicted to be present if assuming a complete cleavage of the azo bond in the AR14 dye

fed to the reactor. These concentration values suggest that the stable amine was to some level either

degraded or adsorbed by the biomass. Yet the last option seems less likely to occur due to the high

water solubility of 4A1NS (1×106 mg/L at 25 °C (JP 2013)).

To verify whether or not a correlation existed between the concentrations of AR14 and its stable

degradation product 4A1NS in the samples collected from the SBR1 and the SBR2 and the potential

cytotoxicity determined for these same samples (3.2.2. chapter), a Pearson correlation test was applied

to the AR14 and the 4A1NS concentration values (Figures 29 and 30, respectively) versus the yeast

growth inhibition ratio values determined for these samples (Figures 22 and 23, respectively); the

obtained results are summarized in Table 10. No correlation was observed between the toxicity data

and the AR14 and 4A1NS concentrations (Table 10). A similar analysis was performed with the fold

change values of the transcription of gene GRE2 (Figures 25 and 26, respectively) and no correlation

was found also (result not showed). These observations suggest that the potential toxicity of these

samples towards the yeast was not directly related with the concentrations of either AR14 or 4A1NS.

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Figure 29 - Mass concentration of AR14 (descendant diagonal stripes) and 4A1NS (ascendant diagonal stripes) in

the samples collected from SBR1, in the operational days 76, 83 and 90, at the end of the anaerobic reaction

(WWanaer) and the end of aeration (WWefflue) determined by HPLC (spectrophotometric detection at 220 nm

using a LiChroCART Purospher STAR RP-18e column). “n” represents a mass concentration value equal to zero.

Figure 30 - Mass concentration of AR14 (descendant diagonal stripes) and 4A1NS (ascendant diagonal stripes) in

the samples collected from SBR2, in the operational days 76, 83 and 90, at the end of the anaerobic reaction

(WWanaer) and the end of aeration (WWefflue) determined by HPLC (spectrophotometric detection at 220 nm

using a LiChroCART Purospher STAR RP-18e column). “n” represents a mass concentration value equal to zero.

0

1

2

3

4

5

6

7

8

9

10[X

] (m

g/L

)

4A1NS AR14

SBR1

n

0

1

2

3

4

5

6

7

8

9

10

[X] (m

g/L

)

4A1NS AR14

SBR2

n

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Table 10 – Pearson correlation between the values of the yeast growth inhibition ratio and the mass

concentrations of AR14 and 4A1NS in the samples collected from both SBR units’.

SBR1 SBR2

AR14 4A1NS AR14 4A1NS

Pearson r

[-1;1]

0.1007 0.2353 0.2254 0.3427

95%

confidence

interval

[-0.7743;0.8434]

[-0.7124;0.8791]

[-0.7175;0.8767]

[-0.6496;0.9031]

R square [0;1] 0.01013 0.05535 0.05079 0.1175

P (two-tailed) 0.8495 0.6536 0.6677 0.506

Significant?

(alpha = 0.05)

No No No No

Since no correlations were observed between the toxicity data and AR14 and 4A1NS concentrations,

chromatograms were analysed in more detail. In general, three additional peaks could be detected in

the chromatograms of all samples, with retention times around 22, 27 and 29 minutes, which were

denominated α, β and γ, respectively, followed by the number of the corresponding SBR. Even though

both SBR1 and SBR2 samples had these peaks with the same retention times, there is no conclusive

indication that these peaks correspond to the same metabolites in both reactors. Thus, correlations were

done separately for each SBR. Peaks area are depicted in figure 31 and 32 for the SBR1 and the SBR2

samples, respectively.

Regarding the samples collected from the SBR1 during the period I, table 11 shows the results of the

correlation test between the α1, β1 and γ1 peaks’ areas (Figure 31) and data obtained with the yeast

microplate susceptibility assay (Figure 22). Although moderated Pearson r coefficients, which may

indicate a correlation between the peak and the yeast growth inhibition data, this could be attributed to

chance due to small R square values. Thus, the apparent correlations were not significant in the test

(Table 12). Correlation test with the GRE2 expression results (Figure 25) did not reveal any conclusive

result (data not showed).

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Figure 31 – Area of relevant peaks detected in chromatograms of the samples collected from SBR1 in operational

days 76, 83 and 90, at the end of the anaerobic reaction (WWanaer) and the end of aeration (WWefflue). Peak α1

(purple bars), peak β1 (orange bars) and peak γ1 had a retention time of 22, 27 and 29 minutes, respectively.

Spectrophotometric detection at 220 nm using a LiChroCART Purospher STAR RP-18e column.

Table 11 - Pearson correlation between the values of the yeast growth inhibition ratio and the area of relevant

peaks detected in chromatograms of the samples collected from SBR1 in the operational days 76, 83 and 90.

Peak α1 (purple bars), peak β1 (orange bars) and peak γ1 had a retention time of 22, 27 and 29 minutes,

respectively.

SBR1

α1 peak β1 peak γ1 peak

Pearson r [-1;1] 0.5177 0.6244 0.5389

95% confidence interval [-0.5070;0.9360] [-0.3797;0.9530] [-0.4848;0.9396]

R square [0;1] 0.268 0.3898 0.2904

P (two-tailed) 0.2928 0.1852 0.2699

Significant? (alpha = 0.05) No No No

Regarding the samples collected from the SBR2 during the period I, table 12 shows the results of the

correlation test between the α2, β2 and γ2 peaks’ areas (Figure 32) and data obtained with the yeast

microplate susceptibility assay (Figure 23). For the samples from this SBR, two strong correlations were

detected, with R square closer to 1 (0.896 and 0.679 respective for peaks α2 and γ2) and with P values

lower than 0.05 acceptance limit (Table 12). Peak α2 has a pearson r coefficient of -0.947 meaning that

if the area increases the ratio obtained in the growth assay should decrease, thus increasing the

magnitude of toxicity detected. A similar result for the peak γ2, although a weaker correlation

demonstrated by the person r coefficient (-0.824), closer to 0 of no correlation; a lower R square and a

higher P value. There was also observed a correlation between both peaks (person r coefficient of 0.956,

0

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peak's

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peak α1 peak β1 peak γ1

SBR1

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52

R square of 0.9145 and a P value of 0.0028) revealing either they are by-products of the same reaction

or interconverted forms of the same molecule. Although the results obtained from the correlation test

(Table 12) may indicate that the potential abatement of toxicity detected in the WWefflue samples of the

SBR2 may be associated with the decrease in the concentration of the metabolites represented by these

two peaks (α2 and γ2), the correlation between the peaks may point to only one of the peaks being

responsible for sample potential toxicity rather than both.

Figure 32 - Area of relevant peaks detected in chromatograms of the samples collected from SBR2 in operational

days 76, 83 and 90, at the end of the anaerobic reaction (WWanaer) and the end of aeration (WWefflue). Peak α2

(purple bars), peak β2 (orange bars) and peak γ2 had a retention time of 22, 27 and 29 minutes, respectively.

Spectrophotometric detection at 220 nm using a LiChroCART Purospher STAR RP-18e column.

Table 12 - Pearson correlation between the values of the yeast growth inhibition ratio and the area of relevant

peaks detected in chromatograms of the samples collected from SBR2 in the operational days 76, 83 and 90.

Peak α2 (purple bars), peak β2 (orange bars) and peak γ2 had a retention time of 22, 27 and 29 minutes,

respectively.

SBR2

α2 peak β2 peak γ2 peak

Pearson r [-1;1] -0.9466 0.4582 -0.8243

95% confidence interval [-0.9943; -0.5823] [-0.5628;0.9256] [-0.9802; -0.03816]

R square [0;1] 0.896 0.2099 0.6794

P (two-tailed) 0.0042 0.3608 0.0436

Significant? (alpha = 0.05) Yes No Yes

0

100000

200000

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600000

peak's

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a

peak α2 peak β2 peak γ2

SBR2

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3.3. Toxicity assessment of the samples from operation period II

Period II comprise the loading shock operational days’ (3.1. chapter). The first loading shock started on

the operational day 158, where dye concentration was increased from 20 to 60 mg AR14/L. After the

first shock, dye concentration was maintained and second loading shock was performed. This last shock

started at the operational day 165, after toxicity samples were taken, and lasted until operational day

173. The second loading shock, as already mentioned, had a dye and NH4Cl concentrations and COD

of 60 mg/L, 214.5 mg/L and 1500 mg O2/L respectively.

For samples collected during this period, only the microplate assay in S. cerevisiae was performed.

Resembling what was done for the period I, an assessment of the potential cytotoxicity of the feeding

solutions and controls (WWfeed+; WWfeed3; WWcontrol3) was done. The differences from the WWfeed

of the period I were the AR14, NH4Cl and COD concentrations. Yeast growth inhibition ratio values

between these solutions and the WWcontrol obtained were depicted in figure 33.

The three times increase in AR14 concentration from the WWfeed+, when compared with WWfeed, had

no impact on the yeast growth, indicating that 60 mg/L as the 20 mg/L previously tested had no cytotoxic

potential in the yeast model. Similar conclusion can be assumed also for the three times increased COD

and NH4Cl, without dye (WWcontrol3) and with dye (WWfeed3), since no significant alteration was

observed. Thus, supporting the conclusion that these wastewater constituents in these concentrations

cannot be considered toxic. To confirm these conclusion, the Tukey’s test was used and p values above

0.9999 were obtained, confirming that WWfeed, WWfeed+, WWfeed+, WWfeed3, WWcontrol and

WWcontrol3 results were indistinguishable.

Figure 33 – Effect on the yeast growth inhibition ratio of the samples collected at the onset of the reaction phase

during the period II and the feed with a COD of 1500 mg O2/L, 214.5 mg NH4Cl/L and without dye (WWcontrol3)

in comparison with feed solution without dye (WWcontrol). Feeding samples during operational days 159 to 165

(WWfeed+) with COD of 500mg O2/L, 71.5 mg NH4Cl/L and 60 mg AR14/L and 165 to 173 (WWfeed3) with COD

of 1500mg O2/L, 214.5 mg NH4Cl/L 60 mg AR14/L. Error bars represent ±1 standard deviation. Means for

WWfeed+, WWcontrol3 and WWfeed3 were not significantly different from WWcontrol (Tuckey´s test; p > 0.999).

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

WWcontrol WWcontrol3 WWfeed+ WWfeed3

Yeast

gro

wth

(r

ela

tive t

o W

Wcontr

ol)

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As for period II, the potential toxicity of samples collected from the two SBRs, namely WWanaer and

WWefflue at the end of, respectively, the anaerobic phase and the aeration phase, were compared for

SBR1 in figure 34 and SBR2 in figure 35.

As can be observed in these figures, the samples from this period presented high levels of yeast growth

inhibition which increased between the 60% to 80% relative to WWcontrol. Thus, an almost complete

loss of the detoxification potential can be pointed out. Even though both the SBRs were able to slightly

reduce the toxicity generated during anaerobic phase, all the yeast growth inhibition ratio values were

very low and there was no statistical distinguish (Tukey’s test, p>0.9999) between the end of anaerobic

phase (WWanaer) and the end of aeration phase (WWefflue).

Comparing both SBR units, operational day 165 results suggest that apparently SBR2 retained more of

detoxification potential than SBR1 as a result from the first loading shock, with the last having no

apparent toxicity removal capacity during the aeration phase (Figures 34 and 35). Yet when looking to

operational day 173, during the second loading shock, SBR1 slightly preforms better (Figures 34 and

35) this could be related with the SRT results during the period II (Figure 9), where SRT for SBR1 is

stable while for SBR2 is decreasing.

Figure 34 – Effect on the yeast growth inhibition ratio of samples collected from SBR1 at the end of the anaerobic

reaction (WWanaer) and the end of aeration (WWefflue) for the operation days 165 and 173, in comparison with

the feed solution without dye (WWcontrol). SBR’s feeding solution at the operational day 165 with 500mg O2/L as

COD, 71.5 mg NH4Cl/L and 60 mg AR14/L; and at the day 173 with 1500mg O2/L as COD, 214.5 mg NH4Cl/L and

60 mg AR14/L. Error bars represent ±1 standard deviation.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Yeast

gro

wth

(r

ela

tive t

o W

Wcontr

ol)

SBR1

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Figure 35– Effect on the yeast growth inhibition ratio of samples collected from SBR2 at the end of the anaerobic

reaction (WWanaer) and the end of aeration (WWefflue) for the operation days 165 and 173, in comparison with

the feed solution without dye (WWcontrol). SBR’s feeding solution at the operational day 165 with 500mg O2/L as

COD, 71.5 mg NH4Cl/L and 60 mg AR14/L; and at the day 173 1500mg O2/L as COD, 214.5 mg NH4Cl/L and 60

mg AR14/L. Error bars represent ±1 standard deviation.

As previously done for period I, period II samples were also run in HPLC and chromatograms obtained

were presented in the annex III. Figures 36 and 37 represent the mass concentration of both AR14 and

4A1NS determined in samples from SBR1 and SBR2 respectively. As in period I toxicity samples the

AR14 determined by HPLC reveal lower AR14 concentration than the 80% colour removal determined

spectrophotometrically for cycle characterization (3.1.2 chapter). From toxicity samples the predicted

AR14 removal was around the 99% and 95% on the day 165 and 90% and 99% on the day 173,

respectively for SBR1 and SBR2. These differences between the methods may indicate the presence

of the oligomer structures in the samples, which could be responsible for toxic results.

With respect to the stable amine 4A1NS, although also demonstrated to have no influence on the growth

inhibition (Figure 21), the predicted concentration should be 18 mg/L assuming only the complete

cleavage of the azo bond in AR14, yet the observe concentration vary on both reactors either above or

below this value (Figures 36 and 37). This could indicate an accumulation or degradation of this amine,

yet no conclusive behaviour can be observed. Still as already demonstrated AR14 and 4A1NS had no

direct effect of the growth inhibition detected.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

Yeast

gro

wth

(r

ela

tive t

o W

Wcontr

ol)

SBR2

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Figure 36 - Mass concentration of AR14 (descendant diagonal stripes) and 4A1NS (ascendant diagonal stripes) in

the samples collected from SBR1, in the operational days 165 and 173, at the end of the anaerobic reaction

(WWanaer) and the end of aeration (WWefflue) determined by HPLC (spectrophotometric detection at 220 nm

using a LiChroCART Purospher STAR RP-18e column).

Figure 37 - Mass concentration of AR14 (descendant diagonal stripes) and 4A1NS (ascendant diagonal stripes) in

the samples collected from SBR2, in the operational days 165 and 173, at the end of the anaerobic reaction

(WWanaer) and the end of aeration (WWefflue) determined by HPLC (spectrophotometric detection at 220 nm

using a LiChroCART Purospher STAR RP-18e column).

0

5

10

15

20

25

30

[X] (m

g/L

)

4A1NS AR14

SBR1

0

5

10

15

20

25

30

[X] (m

g/L

)

4A1NS AR14

SBR2

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The three peaks detected in previous chromatograms from period I were also observed in the

chromatograms of samples from period II. Figure 38 and 39 depicted the area observed in the

chromatograms from SBR1 and SBR2 respectively.

As in period I, SBR1 did not reveal any pattern between peaks observed and yeast inhibition growth

results. Although during period I correlation between peaks α2, γ2 and the yeast growth inhibition data

were observed, this was not observed for period II. Yet, this could be attributed to the higher cytotoxicity

detected that can result also from other sample components not detected.

The hypothesis of another component being generated during SBR treatment seems more reliable when

observing results from samples collected, from SBR2 at the end of aeration phase during day 165 and

the end of anaerobic phase during day 173, where both peaks α2 and γ2 have the same order of

magnitude area as in the previous period I, yet growth inhibition is much greater. One possible

component responsible could be the oligomeric structures that could be in higher amounts. Even

thought, they were not directly measured, they could have originated from the other amine resulting from

the azo bond (1N2A4S), which during the loading shock would be generated in higher amounts. This

amine would form these oligomeric structures when in contact with oxygen, which happen during both

yeast assay and the sample removal from the reactors.

Figure 38 - Area of relevant peaks detected in chromatograms of the samples collected from SBR1 in the

operational days 165 and 173, at the end of the anaerobic reaction (WWanaer) and the end of aeration

(WWefflue). Peak α1 (purple bars), peak β1 (orange bars) and peak γ1 had a retention time of 22, 27 and 29

minutes, respectively. Spectrophotometric detection at 220 nm using a LiChroCART Purospher STAR RP-18e

column.

0

200000

400000

600000

800000

1000000

1200000

1400000

peak's

are

a

peak α1 peak β1 peak γ1

SBR1

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Figure 39 - Area of relevant peaks detected in chromatograms of the samples collected from SBR2 in the

operational days 165 and 173, at the end of the anaerobic reaction (WWanaer) and the end of aeration

(WWefflue). Peak α2 (purple bars), peak β2 (orange bars) and peak γ2 had a retention time of 22, 27 and 29

minutes, respectively. Spectrophotometric detection at 220 nm using a LiChroCART Purospher STAR RP-18e

column.

4. Final discussion and future work

The anaerobic/aerobic SBR systems’ performance and detoxification potential in the treatment of a

simulated dye-laden textile wastewater were compared for both periods of operation. In summary, SBR1

and SBR2 differed in the hydrodynamic regimens, resulting from the slower feeding rate performed in

the last one. Also, period I comprised characterised cycles fed with simulated dye-laden textile

wastewater (500mg O2/L as COD, 71.5 mg NH4Cl/L and 20 mg AR14/L), and period II comprised the

cycles where the loading shocks were applied to test both systems resilience. The first shock was done

by increasing the AR14 concentration from 20 to 60 mg/L and occurred between days 158 to 165, while

the second shock beside the 60 mg AR14/L had an increase in both COD and NH4Cl/L, respectively to,

1500mg O2/L and 214.5 mg NH4Cl/L.

Considering period I, although a slower feeding rate led to higher TSS levels in the mixed liquor of SBR2

and thus higher SRT, compared with SBR1, this did not result in a higher performance in terms of COD

and colour removal yields. Both SBR1 and SBR2 presented high COD and colour removal yields above

80%. Toxicity data obtained with the yeast-based assays (3.2.2 and 3.2.3 chapters) did show a smaller

toxicity level in samples collected from the SBR2. Yeast growth inhibition results (figure 22 and 23)

showed the capacity of SBR2 during the aerobic phase to remove the toxicity originated during the

anaerobic phase to the level of the control solution (feed without dye), while SBR1 was only able to

slightly reduce it in the day 83. Also, the expression changes in GRE2 gene seems to indicate that

exposure to the SBR2 samples provoked a less intense stress response in yeast cells than exposure to

0

200000

400000

600000

800000

1000000

1200000

1400000

peak's

are

a

peak α2 peak β2 peak γ2

SBR2

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the SBR1 samples. Both AR14 and 4A1NS were shown to have no direct correlation with the toxicity

results in the yeast model as pointed by the low Pearson correlations coefficients (table 10) and the

absence of both inhibitory effects on growth and GRE2 expression when exposed to WWfeed and

WWamine. From the sterilized mixed liquor sample chromatograms, three unidentified peaks were

chosen to be studied. Strong correlations between peak areas and each toxicity data was only obtained

with yeast growth inhibition data from SBR2 samples, namely with peaks α2 and γ2. Both the difference

in SRT and toxicity results seems to indicate that different microbial communities have developed in

each SBR, most likely due to the different hydrodynamic regimens.

The two loading shocks, during period II, led to the loss of detoxification capacity of SBR2, possibly

because of the high amount of AR14 by-products, since the higher organic load and its by-products can

be excluded based on the toxicity results obtained for WWcontrol3 and WWfeed3. The increased dye

concentration led to a reduction of the COD removal yield to below 70% in both SBRs. This was due to

the low COD consumption during the anaerobic phase, passing from the 46% to 27% and from 60% to

38%, respectively in SBR1 and SBR2. The further increase in COD and NH4Cl led to a further reduction

of the COD removal ability during the anaerobic phase, which in the case of SBR1 can be considered

an almost complete loss. Although COD removal was affected, colour removal yields were kept above

80%. Toxicity results shown that the toxicity generated by dye biodegradation during the anaerobic

phase was higher than in the previous period and that the SBR units were not able to reduce it

significantly during the aeration phase, on the contrary to what was observed in SBR2 during period I.

In summary, although both SBR seem to remove well colour and COD during period I, SBR2 showed a

better performance in terms of detoxification potential. Yet, loading shocks applied reduced the

efficiency of the bioreactors in terms of both COD removal and the detoxification’s potential. Still remains

the question of whether the prolongation of the loading shock would lead to a recovery or the complete

loss of the treatment system. Besides the prolonged loading shocks, it would be interesting to confirm if

the unidentified peaks derived all from the biodegradation of AR14. For this purpose, a control reactor

operated in the same way, but without dye, could be used. Also, as future work, should be interesting

the identification of the metabolites present in the chromatograms, specially peaks α2 and γ2, in order

to further elucidated both the mechanism of toxicity involved and the AR14 degradation performed within

the SBRs.

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6. Annexes:

Annex I – Toxicity reports on both mono and acid azo dyes Table 13 - Toxicity reports for both mono azo dyes. Classification based. Levels of toxicity classification: Toxic (Carcinogenicity Category 1A/ 1B, Acute and Chronic toxicity/

Specific organ toxicity); Harmful (Chronic toxicity/ Skin and eye sensitization); Irritant (Skin and eye sensitization/ Specific organ toxicity); Dangerous for environment (Aquatic toxicity/ Chronic toxicity); and Non-toxic (Toxicity not reported in studies).

Dye tested Toxicity

status (as

per EU

Directives)

Chemical moiety

responsible for

potential toxicity

Microbe used

for dye

degradation

Metabolite identified Biological organization

level or species assessed

(single vs multilevel; single,

two vs multispecies)

Reference

Reactive

Blue 13

Not available 2-amino-1-naphthol

(Irritant)

Proteus mirabilis

LAG

sodium-2 (2-formyl-2-

hydroxyvinyl) benzoate

and sodium-6-chloro-4-

(chloroamino)-1-3-5-

triazinoxide

Organismic: Phytotoxicity on

Zea mays and Phaseolus

vulgaris

(Olukanni et

al. 2010)

Reactive

Red 195/

Synozol Red

HF-6BN

Not available β-aminonaphthalene

(Toxic/ Dangerous to

environment)/ Aniline

(Toxic/ Irritant/ Dangerous

for environment)

Enterococcus

faecalis strain

YZ66

Phthalic acid, Trihydroxy-

1-naphthalene and an

unidentified product

Organismic: Phytotoxicity on

Sorghum vulgare and

Phaseolus mungo

(Mate &

Pathade

2012)

Aspergillus niger

and Nigrospora

sp.

Not identified Organismic: Microbial toxicity

on Bacillus cereus and

Azotobacter sp. and

Phytotoxicity on Vigna radiata

(Ilyas &

Rehman

2013)

Pleurotus

ostreatus

Not identified Organismic: Microbial toxicity

on Bacillus cereus and

(Ilyas et al.

2012)

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Azotobacter sp. and

Phytotoxicity on Vigna radiata

Reactive

Orange 16

Harmful Aniline (Toxic/ Irritant/

Dangerous for

environment)

Ganoderma sp.

En3

Not identified Organismic: Phytotoxicity on

Triticum aestivum and Oryza

sativa

(Ma et al.

2014)

Pseudomonas

species SUK1,

LBC2 and LBC3

6-

(acetylamino)naphthalene-

2-sulfonic acid and Aniline

Molecular: Genotoxicity and

Comet assay on Allium cepa

Cellular: Cytotoxicity on

Allium cepa

Organismic: Phytotoxicity on

T. aestivum and P. mungo

(Jadhav et

al., 2010a)

Lysinibacillus sp.

RGS

(methylsulfonyl)benzene

and sodium 4-hydroxy-

5,8-dihydronaphthalene-2-

sulfonate

Molecular: Cytogenotoxicity

and Comet assay on Allium

cepa

Organismic: Phytotoxicity on

Sorghum vulgare and

Phaseolus mungo

(Bedekar et

al. 2014)

Activated Sludge

and Irpex lacteus

Not identified Molecular: Salmonella

reversion assay

(Malachová

et al. 2006)

Providencia

rettgeri strain

HSL1 and

Pseudomonas

sp. SUK1

Not identified Organismic: Acute toxicity

test with Daphnia magna

(Lade et al.

2015)

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Direct

orange 39

Not available Aniline (Toxic/ Irritant/

Dangerous for

environment)/ Sulfanilic

acid (Irritant)/ p-

phenylenediamine (Toxic/

Irritant/ Dangerous for

environment)

Pseudomonas

aeruginosa strain

BCH

Not identified Organismic: Phytotoxicity on

Phaseolus mungo and

Triticum aestivum

(Jadhav et

al., 2010b)

Reactive

Yellow 107

Not available Aniline (Toxic/ Irritant/

Dangerous for

environment)

Staphylococcus

arlettae

Not identified Organismic: Toxicity assay on

Daphnia magna

(Elisangela et

al. 2009)

Reactive

Red 2/

Procion Red

MX-5B

Harmful Aniline (Toxic/ Irritant/

Dangerous for

environment)/ 2-

aminonaphthol (Irritant)/

2-Amino-4,6-dichloro-

1,3,5-triazine (Harmful)

Pseudomonas

sp. SUK1

Aniline, 2-naphthol and

1,3,5-triazine 2,4 diol.

Organismic: Phytotoxicity on

S. vulgare and P. mungo

(Kalyani et al.

2009)

Aspergillus niger

and Aspergillus

terreus

Organismic: Acute toxicity on

L. sativa seeds and A. salina

larvae

(Almeida &

Corso 2014)

Reactive

Red 198/

Red RBN/

Remazol

Red

Not available Aniline (Toxic/ Irritant/

Dangerous for

environment)/ 2-

aminonaphthol (Irritant)/

Benzene (Toxic)

Brevibacterium

sp. strain VN-15

4-chloro-N-o-tolyl-1,3,5-

triazin-2-amine; sodium 4-

aminonaphthalene-2-

sulfonate and 3,6-

dimethyl-7- (o-

tolyldiazenyl) naphthalen-

1-amine

Organismic: Toxicity on

Daphnia magna

(Franciscon

et al. 2012)

Mixed Fungal

Culture

Not identified Organismic: Toxicity on

Daphnia pulex

(Nascimento

et al. 2011)

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Penicillium

simplicissimum

INCQS 40211

Not identified Organismic: Toxicity on

Daphnia pulex

(Bergsten-

Torralba et

al. 2009)

Cyathus bulleri Not identified Molecular: Ames test on

Salmonella typhimurium

Organismic: Respiratory

toxicity of Pseudomonas

putida

(Chhabra et

al. 2008)

Lysinibacillus sp.

RGS

1,3,5-triazine,

methanesulfinic acid,

Benzenesulphonate,

Benzene and

Naphthalene-2,7-

disulphonate

Organismic: Phytotoxicity

assay on Sorghum vulgare

and Phaseolus mungo

(Saratale et

al. 2013)

Pseudomonas

aeruginosa BCH

Aniline, Naphthalene-1-ol,

2-[ (3-diazenylphenyl)

Sulfonyl] ethanesulfonate

and 2-Chloro-1,2,3 triazine

Molecular: Genotoxicity on A.

cepa

Cellular: Cytotoxicity, protein

oxidation and lipid

peroxidation on A. cepa

Organismic: Phytotoxicity on

S. vulgare and P. mungo

(Jadhav et al.

2011)

Brevibacillus

laterosporus

(ethylsulfonyl)

Benzene, 3-(1,3,5-triazin-

2-ylamino)

Benzenesulfonate and

Naphthol

Organismic: Phytotoxicity on

P. mungo and S. vulgare

(Kurade et al.

2013)

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Reactive

Orange 1

Non-toxic

(limited

studies)

Aniline (Toxic/ Irritant/

Dangerous for

environment)/ 2-

Aminonaphthol (Irritant)/

2-Amino-4,6-dichloro-

1,3,5-triazine (Harmful)

Cyathus bulleri

Not identified Molecular: Ames test on

Salmonella typhimurium

Organismic: Respiratory

toxicity of Pseudomonas

putida

(Chhabra et

al. 2008)

Reactive

Yellow 84A

Not available Aniline (Toxic/ Irritant/

Dangerous for

environment)/ 2-

aminobenzoic acid

(Irritant)

Galactomyces

geotrichum

4 (5-hydroxy, 4-amino

cyclopentane)

sulfobenzene and 4 (5-

hydroxy cyclopentane)

sulfobenzene

Organismic: Phytotoxicity on

P. mungo and S. vulgare

(Govindwar

et al. 2014)

Reactive

Orange 7

Not available Aniline (Toxic/ Irritant/

Dangerous for

environment)

Cyathus bulleri

Not identified Molecular: Ames test on

Salmonella typhimurium

Organismic: Respiratory

toxicity of Pseudomonas

putida

(Chhabra et

al. 2008)

Acid Orange

52/Methyl

Orange

Toxic Aniline (Toxic/ Irritant/

Dangerous for

environment)/ Sulfanilic

acid (Irritant)/ N,N-

Dimethyl-benzene-1,4-

diamine (Toxic)

Pseudomonas

putida mt-2

N,N′-dimethyl-p-

phenylenediamine and 4-

aminobenzenesulfonic

acid as intermediates

(Further Reduced)

Cellular:

Butyrylcholinesterase

inhibition assay on Human

RBCs

(Mansour et

al. 2011)

Kocuriarosea

MTCC 1532

N,N′-dimethyl-p-

phenylenediamine and 4-

aminobenzenesulfonic

acid

Organismic: Zone inhibition

study on Kocuria rosea,

Pseudomonas aeruginosa

and A. vinelandii and

(Parshetti et

al. 2010)

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Phytotoxicity on T. aestivum

and P. mungo

Acid Orange

7

Irritant 1-amino-2-naphthol

(Harmful/ Suspected

Carcinogen)/ Sulfanilic

acid (Irritant)

Mixed bacterial

culture BAC-ZS

1,2 naphthoquinone and

1,4 benzaquinone (Further

mineralised to carboxlic

acids)

Organismic: Phytotoxicity on

C. sativus

(Bay et al.

2014)

Enterococcus

faecalis and

Clostridium

butyricum

Not identified Molecular: Genotoxicity on

Escherichia coli

Organismic: Toxicity on Vibrio

Fischeri

(Gottlieb et

al. 2003)

Acid Violet 7 Non-toxic

(limited

studies)

4’-Aminoacetamlide

(Irritant)

Pseudomonas

putida mt-2

4’-aminoacetanilide, 5-

acetamido-2-amino-1-

hydroxy-3,6-naphtalene

disulfonic acid

Molecular: Mutagenicity

assay on S. typhimurium

(Mansour et

al. 2009)

Pseudomonas

putida mt-2

4’-aminoacetanilide, 5-

acetamido-2-amino-1-

hydroxy-3,6-naphtalene

disulfonic acid

Molecular: Chromosome

aberrations

Cellular: lipid peroxidation

and acetylcholinesterasic

activity inhibition on mice

(Mansour et

al. 2010)

Acid Red 2/

Methyl Red

Dangerous for

environment

2-aminobenzoic acid

(Irritant)/ N,N-Dimethyl-

benzene-1,4-diamine

(Toxic)

Pseudomons

nitroreducens

and Vibrio logei

Not identified Cellular: Cytotoxicity assay

on COS-7 cells

(Adedayo et

al. 2004)

Sphingomonas

paucimobilis

Not identified

Organismic: Acute toxicity

test on Artemia salina,

(Ayed et al.

2011)

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Phytotoxicity on Sorghum

bicolor and Triticum aestivum,

and Microbial toxicity on S.

paucimobilis

Acid Red 88

Non-toxic

(limited

studies)

1-Aminonaphthalene

(Toxic/ Harmful/

Dangerous for

environment)/ 1-amino-2-

naphthol (Harmful/

Suspected Carcinogen)

Mixed culture 1-isopropyldiaziridine, 3-

methyl piperidine,

piperazine, 1,2-

benzenedicarboxylic

acid, bis (1-methylethyl)

ester and pyrazolo [5, 1-

c][1,2,4]

benzotriazin-8-ol

Organismic: Phytotoxicity on

Phaseolus

mungo, Triticum aestivum,

and Sorghum bicolor

(Anil Kumar

et al. 2015)

Reactive

Orange 4

Not available 2-aminonaphthalene

(Toxic/ Dangerous to

environment)/ 2-

aminonaphthol (Irritant)

Lysinibacillus sp. Naphthalene,

Naphthalene diazonium

and 1,3,5-triazine 2,4 diol

Organismic: Microbial toxicity

on multiple species and

phytotoxicity on Sorghum

vulgare and Phaseolus

mungo

(Saratale et

al. 2015)

Reactive

Violet 1/

Procion

Violet H3R

Not available 2-Amino-1-naphthol

(Irritant)/ Aniline (Toxic/

Irritant/ Dangerous for

environment)

Aspergillus

oryzae

Not identified Organismic: Trimmed

Spearman–Karber method

with growth inhibition of

Daphnia similis

(Corso &

Maganha De

Almeida

2009)

Remazol

Orange

Not available 1-Amino naphthalene

(Toxic/ Harmful/

Pseudomonas

aeruginosa BCH

Naphthalene-1-carboxylic

acid and Benzene

Cellular: Oxidative Stress

studies on A. cepa

(Jadhav et al.

2012)

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Dangerous for

environment)

Acid Red

266

Irritant 4-Chloro-2-

trifluoromethyl-

phenylamine (Harmful)

White Rot Fungi Not identified

Molecular: Genotoxicity test

on Salmonella typhimurium

Cellular: Toxicity

measurement on Caco-2 cells

Organismic: Toxicity

assessment on Vibrio fischeri

(Vanhulle et

al. 2008)

Acid Red

27/Amaranth

Irritant 4-

aminonaphthalenesulfonic

acid (Irritant)/ 1-amino-2-

naphthol (Harmful/

Suspected Carcinogen)

Shewanella

decolorationis

strain S12

4-aminonaphthalene

sulfonic acid

Molecular: Ames test on

Salmonella typhimurium

(Hong et al.

2007)

Acid Orange

10

Non-toxic

(limited

studies)

Aniline (Toxic/ Irritant/

Dangerous for

environment)/ 1-amino-2-

naphthol (Harmful/

Suspected Carcinogen)

Dichomitus

squalens

Not identified

Organismic: Inhibition assay

on Lemna minor

(Eichlerová et

al. 2007)

Acid Red

183

Non-toxic

(limited

studies)

3-Amino-5-chloro-2-

hydroxybenzenesulfonic

acid (Toxic/ Corrosive)

Penicillium

oxalicum

Not identified

Molecular: Differential study

Organismic: Inhibitory effects

on the growth of S. cerevisiae

(Saroj et al.

2014)

Rubine GFL/

Disperse

Red 78

Not available p-phenylenediamine

(Toxic/ Irritant/ Dangerous

for environment)/ 3- (N-

Galactomyces

geotrichum

MTCC 1360

1-3- (nitrophenyl)

methananime and N- (3-

aminopropyl) benzene-1,

4-diamine

Molecular: Genotoxicity on A.

cepa

(Waghmode

et al. 2012)

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Ethylanilino) propionitrile

(Harmful)

Cellular: Cytotoxicity, Cell

death, and Antioxidant

enzymes assay on A. cepa

Organismic: Phytotoxicity on

Phaseolus mungo and

Sorghum vulgare

Aspergillus

ochraceus NCIM-

1146 fungi and

Pseudomonas

sp. SUK1

bacterium

2-methyl-4-nitrophenol,

Ethylamine and Benzene

Organismic: Phytotoxicity on

P. mungo and S. vulgare

(Lade et al.

2012)

Providencia

rettgeri strain

HSL1 and

Pseudomonas

sp. SUK1

Not identified

Organismic: Acute toxicity on

Daphnia magna

(Lade et al.

2015)

Brevibacillus

laterosporus

1-(2-

methylphenyl)-2-

phenyldiazene

Organismic: Phytotoxicity on

P. mungo and S. vulgare

(Kurade et al.

2013)

Disperse

Red 73

Not available 3- (N-Ethylanilino)

propionitrile (Harmful)

Consortium-

RARB

Not identified

Molecular: Comet assay on

A. cepa

Organismic: Phytotoxicity on

Sorghum vulgare and

Phaseolus mungo

(Kadam et al.

2014)

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79

Disperse

Red 1

Irritant 4-Amino-N-(2-

hydroxyethyl)-N-

ethylaniline (Toxic)/ 4-

Nitroaniline (Toxic)

Microbial

consortium

Multiple products Molecular: Mutagenicity test

on Salmonella typhimurium

Organismic: Toxicity on

Daphnia similis and Hydra

attenuate

(Franciscon

et al. 2015)

Basic Blue

159

Not available p-phenylenediamine

(Toxic/ Irritant/ Dangerous

for environment)

Funalia trogii

Not identified

Organismic: Growth inhibition

test on F. trogii and

Staphylococcus aureus

(Apohan &

Yesilada

2005)

Basic Red

46

Not available N-methyl-N-benzylaniline

(Irritant)

Funalia trogii

Not identified

Organismic: Growth inhibition

test on F. trogii and

Staphylococcus aureus

(Apohan &

Yesilada

2005)

Navy blue

2GL

Not available 2-Bromo-4,6-dinitroaniline

(Non-toxic)

Bacillus sp. VUS

4-Amino-3- (2-bromo-4, 6-

dinitro-phenylazo)-phenol

and acetic acid 2- (-

acetoxy-ethylamino)-ethyl

ester

Organismic: Phytotoxicity on

Triticum aestivum and

Sorghum bicolor

(Dawkar et

al. 2009)

Sudan

Orange G

Irritant Aniline (Toxic/ Irritant/

Dangerous for

environment)/ 4-

aminoresorcinol (Non-

toxic)

CotA-laccase

from Bacillus

subtilis

Benzene radical

Organismic: Growth inhibitory

on Saccharomyces cerevisiae

(Pereira et al.

2009)

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Annex II – Chromatograms from samples collected from SBRs during the period I

Table 14 - Average retention times for chromatograms' peaks studied.

Compound/ peak Retention time (min)

4A1NS 14

α 22

AR14 26

β 27

γ 29

Figure 40 – Chromatogram obtain from SBR1 at the end of anaerobic phase on the operational day 76 (76 WWanaer).

Figure 41 – Chromatogram obtain from SBR1 at the end of aeration phase on the operational day 76 (76 WWefflue).

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Figure 42 – Chromatogram obtain from SBR2 at the end of anaerobic phase on the operational day 76 (76 WWanaer).

Figure 43 – Chromatogram obtain from SBR2 at the end of aeration phase on the operational day 76 (76 WWanaer).

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82

Figure 44 – Chromatogram obtain from SBR1 at the end of anaerobic phase on the operational day 83 (83 WWanaer).

Figure 45 – Chromatogram obtain from SBR1 at the end of aeration phase on the operational day 83 (83 WWefflue).

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Figure 46 – Chromatogram obtain from SBR2 at the end of anaerobic phase on the operational day 83 (83 WWanaer).

Figure 47 – Chromatogram obtain from SBR2 at the end of aeration phase on the operational day 83 (83 WWefflue).

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Figure 48 – Chromatogram obtain from SBR1 at the end of anaerobic phase on the operational day 90 (90 WWanaer).

Figure 49 – Chromatogram obtain from SBR1 at the end of aeration phase on the operational day 90 (90 WWefflue).

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Figure 50 – Chromatogram obtain from SBR2 at the end of anaerobic phase on the operational day 90 (90 WWanaer).

Figure 51 – Chromatogram obtain from SBR2 at the end of aeration phase on the operational day 90 (90 WWefflue).

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Annex III – Chromatograms from samples collected from SBRs during the period II

Figure 52 – Chromatogram obtain from SBR1 at the end of anaerobic phase on the operational day 165 (165 WWanaer).

Figure 53 – Chromatogram obtain from SBR1 at the end of aeration phase on the operational day 165 (165 WWefflue).

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Figure 54 – Chromatogram obtain from SBR2 at the end of anaerobic phase on the operational day 165 (165 WWanaer).

Figure 55 – Chromatogram obtain from SBR2 at the end of aeration phase on the operational day 165 (165 WWefflue).

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Figure 56 – Chromatogram obtain from SBR1 at the end of anaerobic phase on the operational day 173 (173 WWanaer).

Figure 57 – Chromatogram obtain from SBR1 at the end of aeration phase on the operational day 173 (173 WWefflue).

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Figure 58 – Chromatogram obtain from SBR2 at the end of anaerobic phase on the operational day 173 (173 WWanaer).

Figure 59 – Chromatogram obtain from SBR2 at the end of aeration phase on the operational day 173 (173 WWefflue).


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