Master Presentation

Microbial community changes of aerobic granules during the degradation of inhibitory wastewaters after

the application of an optimal control strategy

Presents: Víctor Servando García Rea

Tutorial committee

Dr. Iván Moreno Andrade Dra. Luisa Falcón Álvarez

Dr. Miroslav Macek

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May 2015

Industrial wastewater

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Industrial wastewaters are the residual waters that remain after the industrial processes. Often contain a large number of xenobiotics.

Due of its complex chemical structure, industrial wastewaters are difficult to biodegrade, so that they are environment-persistent.

Images taken from: http://www.greenpeace.org/mexico/es/Noticias/2012/Marzo/Rios-mexicanos-rios-toxicos/

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Sequential batch reactors (SBR)

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Aerobic granules

Microbial origin aggregates which do not coagulate under reduced hydrodynamic shear force and which settle faster than flocculent sludge1.

They provide and increase in the biomass retention and have a higher capacity to resist organic loading rates and toxic compounds.

Can withstand conditions of high biological stress, which other types of biological sludge cannot.

1. de Kreuk, Mc Swain et al. 2005

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4-Clorophenol

Phenolic compounds are produced by: Petrochemical, pharmaceutical, paper mills, etc. They are used as disinfectants, pesticides, herbicides, etc. 4-Chlorophenol (4-CP) used as a model inhibitory compound.

The inhibitory compounds can cause operation problems in the wastewater treatment plants, as well as a diminish in the removal performance. The treatment of industrial wastewaters is characterized by a great variability in the flux and the concentration of the toxics.

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Haldane kinetics (substrate inhibition)

IS

o

KSSK

SS

2)(

S (mg/L)

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FTC or usual operation of a SBR

S*

µ*

Quick fill

S

Volume

Slow reaction

Usual behavior of µ with the fixed time control (FTC) or normal operation of a SBR.

Vf

Vo

Degradation with inhibition

µ

Sf

Modified from Moreno et al. 2006

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Event Driven-Time Optimal Control

The flux is controlled so that, it is only fed the necessary amount of substrate in order to obtain the maximum degradation velocity

The microbial activity estimation is indirectly realized by the measurement of dissolved oxygen in the reactor medium, related with g (the oxygen mass uptake rate).

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S* Toxic concentration (S)

Vf

Vol

ume

Vo

Deg

rad

atio

n r

ate

Maximum

degradation rate

ED-TOC strategy

0

50

100

150

200

250

300

350

400

450

500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Time, h

g (

mg

O2/h

)

0

1

2

3

4

5

6

7

DO

, m

g/L

g DO


10


Vf

Vo

0

50

100

150

200

250

300

350

400

450

500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Time, h

g (

mg

O2/h

)

0

1

2

3

4

5

6

7

DO

, m

g/L

g DO

S* S Toxic concentration

Vol

ume

Deg

rad

atio

n r

ate

Maximum

degradation rate

ED-TOC strategy


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11 S* S

Vf

Vo

0

50

100

150

200

250

300

350

400

450

500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Time, h

g (

mg

O2/h

)

0

1

2

3

4

5

6

7

DO

, m

g/L

g DO

Toxic concentration

Vol

ume

Deg

rad

atio

n r

ate

Maximum

degradation rate

ED-TOC strategy


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12 S* S

Vf

Vo

0

50

100

150

200

250

300

350

400

450

500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Time, h

g (

mg

O2/h

)

0

1

2

3

4

5

6

7

DO

, m

g/L

g DO

Toxic concentration

Vol

ume

Deg

rad

atio

n r

ate

Maximum

degradation rate

ED-TOC strategy


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13 S* S

Vf

Vo

0

50

100

150

200

250

300

350

400

450

500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Time, h

g (

mg

O2/h

)

0

1

2

3

4

5

6

7

DO

, m

g/L

g DO

Toxic concentration

Vol

ume

Deg

rad

atio

n r

ate

Maximum

degradation rate

ED-TOC strategy


14 14


14 S* S

Vf

Vo

0

50

100

150

200

250

300

350

400

450

500

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

Time, h

g (

mg

O2/h

)

0

1

2

3

4

5

6

7

DO

, m

g/L

g DO

Toxic concentration

Vol

ume

Deg

rad

atio

n r

ate

Maximum

degradation rate

ED-TOC strategy


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Background studies

Authors Study Molecular test Time of study Results

Wells y cols. 2011. Evaluation of the microbial population dynamics and the taxa-time relationship in an activated sludge reactor, trying to correlate the operational parameters with the microorganisms.

T-RFLP/Clone library

1 year A strong population dynamic, and a sable performance of the reactor.

Fernández y cols.1999. Population dynamics of a functional and stable methanogenic reactor. ARDRA/Clone library

605 days A dynamic community through all the reactor performance. Function stability does not mean a microbial community stability.

Whiteley y cols.2000. Microbial community activity of an industrial reactor degrading phenol. They looked which was the response of the community when was challenged to a change in the pollutant loading.

DGGE/FISH 3 months Highly structured microbial communities

Matsumoto y cols. 2010. Aerobic granulation. Structure of the microbial community of nitrifying granules of an ammonia-fed reactor.

Clone library/FISH 2 months Spatially structured community which evolves in response to the different selective pressures.

Jiang y cols. 2004. Dependent and independent culture methods for studying the microbial community of phenol degrading granules.

DGGE 2 months Changes in the microbial community of the sludge from the “seed” to the granular sludge.

Buitron y cols. 1998. Study of the degradation of a phenol, 4-CP, and 2,4,6-trichlorophenol by activated sludge

Isolation Aeromonas sp, Pseudomonas sp.

Moreno y cols. 2004. Which microorganisms were present in different inocula: domestic WWTP, municipal WWTP and industrial WWTP

Isolation Acinetobacter sp, Brevibacillus sp, Pseudomonas sp.

Moreno-Andrade y cols. 2001.

Study of the 4-CP degradation by activated sludge. DGGE 6 months Suggested at least 11 microbial species, mainly Alphaproteobacteria

Már-Álvarez & Moreno-Andrade 2012.

Study of the microbial community during the acclimation and granulation process for 4-CP degradation.

DGGE/Sequencing

Dynamic community both in the acclimatization process as in the granulation. 10-11 species were sugested

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Hypothesis

The bacterial community of aerobic granules of a 4-Chlorophenol degrading SBR which runs with a conventional operation strategy (FTC) will be specialized when the SBR changes to an operation with an optimal control strategy (ED-TOC) which maintains the microorganisms in close to the maximum growth rate (max).

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Objective

General objective: To evaluate the changes in the bacterial community of aerobic

granules of SBR degrading 4-Chlorophenol when the SBR is changed form the conventional operation strategy (FTC) to an optimal control strategy (ED-TOC) in which the microorganisms are close to the maximum growth rate (max).

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Particular objectives

1. To determine the bacterial community present in aerobic granules of a SBR degrading 4-CP which is operated with the conventional strategy (FTC).

2. To determine the bacterial community present in aerobic granules of a SBR degrading 4-CP which is operated with and optimal control strategy (ED-TOC).

3. To monitor the physicochemical and operational parameters of the granular biomass, in order to determine if there is a change in them after the normal SBR operation (FTC) is switched to an optimal control strategy (ED-TOC), and correlate this changes with the microorganisms present.

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Methodology

Comparación

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Aerobic granular SBR

SBR reactor (1), fed (fill) pump (2), drain pump (3), feeding tank (4), PC (5), data acquisition card (6), temperature and dissolved oxygen sensor, (7) temperature control system (8).

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SBR operation parameters

Parameters Value

Temperature 25°C

Exchange volume 50% (2 L)

H/D ratio 6

Settling time 1 min

Draw time 5 min

Air flux 1 L/min

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Aerobic granules

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Physicochemical results

q= 714 +/- 100 mg4CF/gSSVd

ED-TOC 4-CP degradation kinetic

0 25 50 75 100 125 150 175 200 225 2500

20

40

60

0

2

4

6

O2

4-CF 300 mg/L

tiempo (min)

4-C

F [

mg

/L] O

2 [mg

/L]

Cinética FTC

0 100 200 300 4000

50

100

150

200

250

380 mg/L

450 mg/L

300 mg/L

tiempo (min)

4-C

F [

mg

/L]

FTC 4-CP degradation kinetic

q= 1190 +/- 300 mg4CF/gSSVd

4-CF influent

[mg/L]

4-CF effluent [mg/L]

FTC

Removal

percentage(FTC)

4-CF effluent [mg/L]

ED-TOC

Removal percentage

(ED-TOC)

300-500 0.14 +/- 0.11 99.8 +/- 0.03 % 0.41+/- 0.08 99.8+/-0.09 %

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Molecular biology results

B Primers 46F-534R GC Primers TX9-1391R

DGGE, gradient 40-60%.

A B

A B

Denaturing gradient gel electrophoresis (DGGE).

ED-TOC

0

FTC

B

ED-TOC

FTC

16 S rRNA image: Ashby et al , 2007.; Riobsome image taken from: https://es.wikipedia.org/wiki/Ribosoma/

16S rRNA

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OTUs (total genera) 43

OTUs FTC 27

OTUs ED-TOC 21

• Clone library and sequencing

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0

5

10

15C

lon

e p

erc

en

tage

Clones and its genera in the FTC operation

0

5

10

15

20

25

Clo

ne

pe

rce

nta

ge

Clones and its genera in the ED-TOC operation

Phylogeny

Lab ecología bacteriana/Árbol/Árbol para trabajar.pptx#1. Presentación de PowerPoint

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Physicochemical results

Vel Sed FTC vs ED-TOC

0 50 100 1500

20

40

60

80

100FTC

ED-TOC

Día de operación

m/h

Sólidos suspendidos en efluenteFTC vs ED-TOC

FTC ED-TOC0

10

20

30

40

SS

(m

g/L

)n=25, barras 95% de intervalo de confianza

Proteínas Carbohidratos0

50

100

150

200

250 Solubles

Ligadas

EPS FTC

mg

/gS

SV

Proteínas Carbohidratos0

50

100

150

200

250 Solubles

Ligadas

EPS ED-TOC

mg

/gS

SV

FTC n=9, ED-TOC n=10; barras 95% de intervalo de confianza

Respirometrías FTC vs ED-TOC

FTC ED-TOC0

100

200

300

mg

O2/g

SS

Vh

n=10, barras 95% de intervalo de confianza

Granulometría

FTC ED-TOC0.0

0.2

0.4

0.6

0.8

Cen

tím

etr

os

n=500, barras 95% de intervalo de confianza

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Microscopy

A

5X

B C

20X

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Molecular Biology results

0

5

10

15

Clo

ne

s p

erc

en

tage

Clones and its genera in the FTC operation

0

5

10

15

20

25

Clo

ne

s p

erc

en

tage

Clones and their genera in the ED-TOC operation

Phylogeny

Lab ecología bacteriana/Árbol/Árbol para trabajar.pptx#1. Presentación de PowerPoint

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Conclusions

1. The bacterial community of the aerobic granules operating with the ED-TOC strategy showed a specialization compared with the bacterial community of the aerobic granules with the FTC strategy.

The bacterial community of the ED-TOC granules was less diverse (6 phyla and 21 genera) compared with the bacterial community of the FTC granules (7 phyla and 21 genera).

5 genera of the 43 present in both reactors were share between the two communities, representing 12% of the total genera.

2. The phylogenetic analysis showed that both communities are very close related. So even tough there are some changes at bacterial genera level, the granular structure and the biomass metabolic and functional activities do not change.

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3. The 4-CP specific degradation rate (qx) and the specific oxygen uptake rate (SOUR) were higher in the FTC than in the ED-TOC (66% and 101%). The granular settling velocity and the solids concentration in the FTC strategy effluent were 69% lower and 100% higher ,respectively, than in the granules of the ED-TOC strategy, mainly due to the increase of filamentous bacteria. The granule size was 14% bigger in the FTC strategy than in the ED-TOC strategy because of the higher 4-CP concentrations in the FTC strategy. The EPS does not showed any disturbance. The 4-CP removal percentage was the same for the both strategies. Nevertheless it is worth to mention again that the range of 4-CP concentration in which the FTC worked was not deadly for the biomass. If the concentrations would have surpassed 500 mg/L inside of the reactor the reactor operation would have crashed.

Conclusions

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Future perspectives and final remarks

Application of the control strategy in real industrial wastewater with 4-CP

The easiness of move the dynamic of a bacterial community only by the feeding procedure.

Diversity coverage that can be achieved with molecular finger-printing techniques like the DGGE, how close enough is to the real diversity?

Thank you!

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34

Thanks

Master Presentation

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