Presentacion3

Contributions of CINVESTAV to the study of bioreactors with

simultaneous electron acceptors

Removal of Trichlorophenol under partially-aerated methanogenic using a Fluidized Bed bioreactor

Removal of Perchloroethylene in partially-aerated methanogenic regime using a Fluidized Bed bioreactor

Removal of Perchloroethylene under M-D conditions

M-A and M-D bioreactors coupled to zero valent iron for PCE degradation

Removal of perchloroethylene in two methanogenic-denitrifying

continuous systems

Héctor M. Poggi-Varaldo

CINVESTAV-IPN, Dept. Biotechnology and Bioengineering, Environmental Biotechnology R&D Group, México

Contents

Acknowledgements Abbreviations Introduction

– Perchloroethylene– Simultaneous Electron Acceptor (SEA)

systems Methodology

– Reactors set-up and operation– Methods

Results and discussion Conclusions

Acknowledgements

Orgazing Committee and ENCB-IPN CINVESTAV, CONACYT Mr. Claudio Garibay-Orijel,

Mr. Rafael Hernández-Vera, Ms Paola Zárate-Segura

Prof. Elvira Ríos-Leal Dr. Jaime García-Mena

Abbreviations

Bv organic loading rate per unit volume

CM complete mix reactor

DCE dichloroethylene

FBBR anaerobic fluidized bed biological reactor

Ig biogas productivity

PCE perchloroethylene

TCE trichloroethylene

VC vinyl chloride

alpha factor

net increase

removal efficiency

loading ratio of organic matter as COD to nitrogen-nitrate

v PCE loading rate per unit volume

Introduction

Perchloroethylene

•potentially hazardous

•included in the priority list of hazardous pollutatns of USA and other countries (EPA, 1993)

•widely used as a solvent in dry-cleaning industry as and a degreasing solvent in the metal and machinery industries for more than 50 years (HSIA, 1999)

•in the 90’s, a world demand between 160 000 and 520 000 tonnes/yr has been recorded (EPA, 1993; WHO, 2000).

•PCE is recalcitranr (not biodegradable) in aerobic conditions (Vogel and McCarty 1985). Its biological transformation is generally carried out in anaerobic environments (van Eekert, 1999).

•Previous works on biological treament and bioremediation of PCE have used anaerobic consortia that mediated the sequential reductive dehalogenation of PCE, presumably by cometabolism (Vogel y McCarty, 1985; Prakash y Gupta, 2000; Cope et al., 2001; López-Navarrete et al.,

2003).

•In most of these studies, accumulation of dichloroethylene (DCE) and vinyl chloride (VC) has been observed. A few works have been able to show the full transformation of PCE to ethene, after a long enrichment of anoxic consortia or using pure cultures of dehalorespiring bacteria (Mayor et al., 2002).

Series reactorsand

Simultaneous Electron Acceptor (SEA) systems

High chlorine content

Penta-, tetra-, tri-

Anaerobic

Low chlorine content

di-, and monochloro-

AerobicAccumulatio

n

of

A good alternative: Series Reactors

Brief example with a chlorinated organic compound

Series Reactors

Anaerobic Reactor

Reactor with second electron acceptor

Tetrechloroethene

TCE, DCE, VC

Further removal of chlorinated aliphatics

Garibay Orijel et al. (2005a) J. Chem. Technol. Biotechnol. In pressCampos-Velarde et al. (1997). Battelle Press.

What’s the problem with series reactors?

2 Reactors

Costs X 2

Better try Simultaneous

Electron Acceptors in One reactor

Salto a la albóndiga

Protection via diffusion barrierin biofilm

CH4

CO2

NO3- + org. matter

N2 +H2O

An

aero

bic

zo

ne

Den

itri

yin

g

zon

e

Liq

ue

d d

if-

fusi

on

lea

yer

Denitrifying microorganisms

Methanogens

Bu

lk l

iqu

id(adapted from López-Navarrete, 2002).

Pollutant

NO3-

Concentration

Carrier

= g COD/g 2nd electron acceptor in the influent

2nd electron acceptor: NO3-

• lambda determines the percentage P of substrate that is channelled into methanogenesis or denitrification in SEA conditions

• it can be demonstrated that P = 50% at = 9 for M-D reactors

Garibay-Orijel et al. (2005b). J. Chem. Technol. Biotechnol.

Objectives

•to evaluate and compare de performance of a fluidized bed bioreactor (FBBR) and complete mix reactors (CM) with suspended biomass, all of them fed with PCE as model chlorinated aliphatic and a low-moderate concentration of degradable organic matter as methanol

• to assess the influence of the biochemical regime (full methanogenic versus M-D), and the effect of in the M-D regime (18 and 9) on performance

Methodology

Nmin

1

2

3

5

7

8

9

6

4

2´

A

Nmin

1

2

3

5

6

7

4

2´

N. max

B Reactor setup: A Anaerobic Fluidized Bed Bioreactor (AFBBR); B Complete Mix Bioreactor (CM). 1A fluidized bed of bioparticles; 2A reservoir and feed of influent with a partial content of methanol; 2´A feed of stock of PCE in methanol; 3A recirculation; 4A liquid trap; 5A effluent reservoir; 6A biogas exit; 7A biogas sampling port; 8A activated carbon trap; 9A biogas measurement by brine meters; 1B suspended biomass; 2B reservoir and feed of influent with a partial content of methanol; 2´B feed of stock of PCE in methanol; 3B effluent reservoir; 4B biogas exit; 5B biogas sampling port; 6B activated carbon trap; 7B biogas sampling port.

Anaerobic fluidized bed reactora Complete Mix Reactorb Complete Mix Reactor2c

Bvd

(gCOD/Ld)

ve

(mg PCE/Ld)

PCEf

(mg/L)

(gCOD/

gN-NO3)

Bvd

(gCOD/Ld)

ve

(mg PCE/Ld)

PCEf

(mg/L)

(gCOD/

gN-NO3)

Bvd

(gCOD/Ld)

ve

(mg PCE/Ld)

PCEf

(mg/L)

g (gCOD/

gN-NO3)

Period 1 1.0 40 40 0 0.066 1.33 20 0 0.066 2.66 0 0

Period 2

1.0 40 40 18 0.066 1.33 20 18 0.066 2.66 40 18

Period 3 1.0 40 40 9 0.066 1.33 20 9 0.066 2.66 40 9

Notes.

1000 mg COD-methanol/L in all periods; PCE: Perchloroethylene.a Anaerobic fluidized bed bioreactor at HRT= 1d, Vop= 2.8L, 35°C; b,c Complete mix reactor at HRT= 15 d, Vop= 2.5L, 35°C.; d Volumetric loading rate of organic matter in gCOD/(L.d), e Volumetric loading rate of PCE in mg PCE/(L.d); f Concentration of PCE in the influent, in mg/L; g Relation of volumetric loading and Nitrogen contend in Nitrate.

Operating conditions of bioreactors in the different periods of the experimental design

Period 1

Period 2

Period 3

40

Operation and monitoring

mesophilic conditions glass column (2.8 L), loaded with 1 L of 1 mm

granular activated carbon, colonized by an anaerobic consortium

methanol (1000mgCOD /L) hydraulic residence time = 1 d (bed basis)

Response variables:– Removal efficiency of organic matter (COD)– Removal efficiency of PCE– Concentration of less substituted chlorinated aliphatics– Specific methanogenic activity, specific denitrifying

activity, specific oxgyen uptake rate

T: transient period with 2vvd

HRT= 1 d, TCPi = 80mg/L, Phei = 20mg/L, CODi =1000 mg/L, mesophilic

15vvd

1 2 3

50

75

100

90 140 190 240 290

D

QO

( %

)

0

10

20

90 140 190 240 290

C

l- (m

g/L

)

0

0.2

0.4

90 140 190 240 290

Ig (L

bio

gas/ L

reacto

r d

)

A

B

C

Dynamic performance of reactors

A: Removal efficiency of COD versus time;

B Biogas productivity;

C Increase of chloride anion.

FBBR

○ CM 1

∆ CM 2

M = 18 = 9

Fluidized bed reactor

Fluidized bed reactor

Complete mix reactors

Complete mix reactors

Complete mix reactors

CO

D (

%)

I g (

L b

iog

as/L

.d)

∆C

l- (m

g/L

)

Time (day)

Average performance of reactors 1/2 Period Parameter 1 2 3 Reactor

PCE (%) a 98.81 1.15 98.67 2.22b

98.62 2.10

98.88 1.12 98.64 3.89 98.10 2.81

98.60 5.95 92.39 2.80 90.01 ( 1.13

AFBBR c CM 1 d CM 2 e

COD (% ) f 94.66 5.21 92.49 2.10b 73.46 2.15

98.85 6.40 96.21 3.33 91.73 3.80

98.87 1.51 96.61 7.35 97.30 8.53

AFBBR CM 1 CM 2

NO3 (% ) g ---- ---- ----

97.88 2.56 99.58 4.34 98.71 3.82

98.91 0.27 98.43 1.31 97.79 0.87

AFBBR CM 1 CM 2

ΔCl- (mg/L) h 11.7 5.3 9.9 3.2b 7.5 5.2

14.53 9.4 9.78 4.2 9.99 3.3

18.7 0.7 16.21 0.8 17.3 0.6

AFBBR

CM 1

CM 2 i 0.30 0.01

0.39 0.01b

0.30 0.01

0.24 0.018 0.28 0.02 0.30 0.02

0.25 0.05 0.28 0.04

0.27 0.05

AFBBR

CM 1

CM 2 CH4 (%) 70.20 7.32

40.30 1.03b

30.37 1.23

60.52 3.65 48.5 4.34 49.5 4.78

65.4 4.45 10.10 3.21

8.50 3.25

AFBBR

CM 1

CM 2 Ig(L/L.d) j 0.41 0.015

0.11 0.01b

0.0140.03

0.39 0.01 0.12 0.01 0.04 0.03

0.280.02 0.022 0.02

0.026 0.02

AFBBR

CM 1

CM 2 VSS (mg/L)k 58.34 2.40

240 3.45b 278 3.65

56.66 ± 3.43 430 5.34 475 5.16

71.34 3.45 420 5.35 410 5.21

AFBBR CM 1 CM 2

NKTbp (mgN/gbpseca)l NKT vss (mgN/L) NKT vss (mgN/L)

11.26 ( 1.23 30.08 ( 0.67b 34.75 ( 0.65

25.45( 2.32 38.72( 3.24 37.23 5.13

32.78 3.24 43.74 2.35 42.45 4.23

AFBBR

CM 1

CM 2 x (mg PCE/g VSS d)m x (mg PCE/g VSS d) x (mg PCE/g VSS d)

17.2 1.2 5.5 0.7 9.6 0.7

7.64 1.3 4.28 0.9 8.91 0.4

5.93 0.3 3.16 0.5 7.81 0.3

AFBBR

CM 1

CM 2 SMAn (mmol CH4/g vss h)

0.060 0.02 0.067 0.03 0.058 0.04

0.067 0.03 0.065 0.03 0.033 0.01

0.075 0.043 0.010 0.004 0.022 0.012

AFBBR

CM 1

CM 2 SDAo (gNO3

- /gNKT h) 0.17 0.09 0.27 0.12 0.20 0.10

0.17 0.05 0.25 0.07 0.20 0.02

0.29 0.02 0.17 0.04 0.15 0.08

AFBBR

CM 1

CM 2

Average performance of reactors 2/2Metabolites and dechlorination efficiencies

Period

Parameter

1

2

3

Reactor

Metabolites in the effluent PCE ( mol/L) a 2.85 4.1

3.2 2.9 3.3 0.7

3.14 2.1 3.3 1.2 4.6 2.1

3.3 1.2 18.4 2.1 23.6 2.4

AFBBR c CM 1 d CM 2 e

TCE ( mol/L) f 62.7 8.2 29.6 16.0b 134.8 12.8

12.3 1.3 22.91 12.2 49.12 5.4

11.5 3.2 10.8 2.1 30.4 5.9

AFBBR c

CM 1 d

CM 2 e

DCE (mol/L) g 4.1 1.5 2.6 1.2b

0.5 0.4

undetectable 8.99 4.5

41.92 2.0

18.1 2.8 25.1 1.4 35.0 3.5

AFBBR c

CM 1 d

CM 2 e

VC (mol/L) h 127.6 4.3 138.5 8.5b

85.7 0.6

125.9 9.7 138.7 3.2

154.0 10.9

74.6 10.1 79.58 9.2 160.4 9.4

AFBBR c CM 1 d CM 2 e

Metabolites by stripping PCE (mol/gAC) 1.1E-2 1.1E-2

1.7E-4 5.0E-4b

9.1E-4 7.2E-4

1.1E-2 7.4E-3 1.7E-3 1.0E-4 9.0E-4 1.1E-4

1.1E-3 1.2E-2 1.7E-3 1.4E-3 9.0E-4 4.5E-4

AFBBR c

CM 1 d

CM 2 e

TCE (mol/ gAC ) 3.6E-2 6.3E-3 1.6E-4 1.2E-4b 3.1E-4 1.1E-4

2.7E-2 1.4E-2 1.0E-4 0.5E-4 2.3E-4 1.3E-4

1.2E-2 0.4E-2 1.1E-4 0.3E-4 2.3E-4 1.3E-4

AFBBR c

CM 1 d

CM 2 e

DCE (mol/ gAC ) 3.5E-2 3.2E-3 undetectableb

1.0E-3 1.2E-3

1.7E-2 1.1E-2 undetectable

5.12E-4 3.2E-4

3.2E-3 2.5E-3 undetectable

5.1E-3 3.2E-3

AFBBR c

CM 1 d

CM 2 e

VC (mol/ gAC) 1.1E-1 1.2E-2 2.7E-4 1.4E-4b

5.1E-2 1.2E-4

2.8E-2 1.3E-2 4.2E-3 2.1E-3 1.3E-2 0.9E-2

6.3E-3 2.1E-3 4.1E-3 2.3E-3 1.2E-2 1.0E-2

AFBBR c

CM 1 d

CM 2 e

Adsorption of metabolites on the bioparticles

PCE (mol/ gbp) 1.7E-1 2.1E-2 1.8E-1 3.03E-2 4.4E-1 1.63E-3

TCE (mol/ gbp) 1.4E-3 0.3E-3 undetectable 5.7E-1 1.63E-3

DCE (mol/ gbp) 7.1E-1 1.1E-2 3.6E-2 1.15E-2 5.5E-1 3.19E-2

VC (mol/ gbp)

7.3E-1 6.2E-2 9.6E-1 1.42E-1 1.2 1.42E-1

AFBBR e

Declorination Efficiency CL b(%) j 64.19 2.19

48.94 1.98 47.49 4.21

81.86 3.21 50.33 5.54 57.78 3.53

83.39 3.21 57.56 2.43 56.63 4.31

AFBBR c

CM 1 d

CM 2 e

Poggi’s discrete divergence index

n’A = only green

n’B = only yellow

nA = green plus white

nB = yellow plus white

Microbial community

A

Microbial community

B

Zárate-Segura et al. (2005). Battelle

Poggi’s discrete divergence indexand dynamic divergence coefficient

Poggi = (n’A + n’B)/(nA + nB)

wherewhere

n’n’AA = number of bands in A that are not in B = number of bands in A that are not in B

n’n’BB = number of bands in B that are not in A = number of bands in B that are not in A

nnAA = total number of bands in A = total number of bands in A

nnBB = total number of bands in B = total number of bands in B

complete similarity 0 complete similarity 0 PoggiPoggi 1 complete divergence 1 complete divergence

Poggi = d(Poggi)/dt; dynamic divergence coefficient

.

Zárate-Segura et al. (2005). Battelle

Lanes 1 and 2, methanogenic period with no PCE; Lanes 3 to 5, methanogenic period with 20 mg/L PCE; Lanes 6 to 8 methanogenic period with 40 mg/L PCE. All lanes contain 5 g of 16S rDNA PCR product. Poliacrylamide gel at 8% (Acrylamide/N-N´methylenbisacrylamide 37.5:1) Buffer TBE 1X, Urea -Formamide 10-50% (8M of Urea and 40%v/v formamide equivalent to 100% denaturing agents); 30 V, 13-15 mA, 8 h, 60°C.

DGGE profiles of major bacterial communities present in fluidized bed bioreactor

∆Poggi Jaccard index of similarity

Index Reactor FBBRa CM1a CM2a FBBRa CM1a CM2a FBBR 0.05

0.71

0.44

0.73

0.17

0.39

CM1 ------

0.33

0.67

------

0.50

0.20

CM2 ------ ------ 0.67 ------

------

0.20

Variation of bacterial communities in methanogenic FBBR and CM reactors in a period of operation with no PCE in the influent:

Diagonal: comparison between start and end of the period for a given bioreactor

Other cells: comparison between bioreactors

∆Poggi Jaccard index of

similarity Index Reactor FBBRa CM1a CM2a FBBRa CM1a CM2a FBBR 0.05 0.65

0.65

0.91

0.22

0.22

CM1 ------

0.00

0.86

------

1.00

0.08

CM2 ------ ------ 0.50 ------

------

0.32

Variation of bacterial communities in methanogenic FBBR and CM reactors in a period of operation with 40 mg PCE/L for

FBBR and CM2 and 20 mg PCE/L in CM1:Diagonal: comparison between start and end of the period for a given bioreactor Other cells: comparison between bioreactors

∆Poggi Jaccard index of

similarity Index Reactor FBBRa CM1a CM2a FBBRa CM1a CM2a FBBR 0.10

------

------

0.82

------

------

CM1 ------

0.63

------

------

0.22

------

CM2 ------ ------ 0.60 ------

------

0.25

Variation of bacterial communities in methanogenic FBBR and CM reactors between periods with no PCE and with PCE

in the feed:Diagonal: comparison between periods for a given bioreactor Other cells: empty

System Poggi (1/day) Reference

Methanogenic fluidized bed bio-reactor, 1000 mg/L COD-MeOH

0.0003 This work

Methanogenic complete mix bio-reactor, 1000 mg/L COD-MeOH

0.0022 This work

Methanogenic complete mix bio-reactor, 1000 mg/L COD-MeOH

0.0045 This work

Sequencing batch reactor treating piggery wastewater

0.260 1

Membrane-coupled bioreactor dedi-cated to biological nutrient removal

0.084 2

UASB reactor treating a penta-chlorophenol-contaminated wastewater

0.010 3

. .

Ref.: 1. Juteau, P., Tremblay, D., Villemur, R., Bisaillon, J.-G. and Beaudet R. (2004). Analysis of the bacterial community inhabiting an aerobic thermophilic sequencing batch reactor (AT-SBR) treating swine waste. Appl. Microbiol. Biotechnol. 66, 115-122.

2. Ghosh, S. and LaPara, T.M. (2004). Removal of carbonaceous and nitrogenous pollutants from a synthetic wastewater using a membrane-coupled bioreactor. J. Industrial Microbiol. Biotechnol. 31, 353 –361.

3. Tartakovsky, B., Manuel, M.F., Beaumier, D., Greer, C.W. and Guiot, S.R. (2001). Enhanced selection of an anaerobic pentachlorophenol-degrading consortium. Biotechnol. Bioeng. 73, 476-483.

Dynamic divergence indices of bacterial communities of several bioreactors

Conclusions

- In Period 1, methanogenic regime, AFBBR showed the best performance of the three reactors with higher values of both organic matter removal and PCE, in spite that volumetric loadings on AFBBR were 15-fold higher and some process stress would have been expected.

- During Period 2, simultaneous M-D regime at =18 gCOD/gN-NO3

-, an improvement in performance of CM2 was observed. The other two reactors displayed similar performances than the corresponding in Period 1.

-In Period 3, simultaneous M-D regime at = 9 gCOD/gN-NO3

-, and concerning the two reactors fed with the highest PCE concentration 40 mg/L (FBBR and CM2), the FBBR outperformed CM2 in almost all the performance parameters and its metabolite profile was better than both of CM2 and CM1 (lower TCE, DCE, and VC in the effluent).

This pattern, along with highest dechlorination efficiency of FBBR strongly suggests that FBBR under M-D conditions may provide a more integral treatment to wastewaters polluted with significant concentrations of PCE.

– The bacterial communities in the CMs were richer than those of FBBR during three operations periods

– Generally, bacterial profiles in each reactor varied with time in a given period of operation. There was a relative higher stability of consortium in the FBBR as displayed by the lowest dynamic divergence coefficients values

– The above described pattern was accompanied by a better biochemical peformance of FBBR (stable methanogenesis, high removal of PCE and lower concentrations of intermediate chlorinated aliphatics)

– Despite relative variation of bacterial consortia with time, bioreactors showed steady state biochemical performance

– PCE had a negative impact on the richness of CMs consortia whereas this impact was less noticeable in the FBBR.

Questions and [email protected]

Despedida Farewell

Me miro al espejo I look at myself in the mirrory sólo veo but I can only seela desnuda pared a mis espaldas. the bare wall behind me.Me estremezco y me pregunto: I shiver and mutter:¿quién soñará nuestros sueños? Who will dream our dreams?¿quién peleará nuestras batallas? Who will fight our battles?¿quién llorará nuestras derrotas? Who will cry for our defeats?¿quién ganará nuestras victorias? Who will win our victories?