YOU ARE DOWNLOADING DOCUMENT

Please tick the box to continue:

Transcript
Page 1: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Development of an attached-growth process for the bioremediation of trichloroethylene- and

1,1,2,2-tetrachloroethane-contaminated groundwater

Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco Doria, Stefano

Lei, Valentina Spaggiari, Nasrin Tavanaie, Florin A. Potra, Roberta Ciavarelli,

Davide Pinelli, Serena Fraraccio, Giulio Zanaroli, Fabio Fava.

DICAM, University of Bologna

Via Terracini 28, Bologna, Italy

Acque sotterranee: protezione dai rischi igienico sanitari e/o tutela delle risorse

RIMINI, 8 NOVEMBRE 2013

Page 2: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Chlorinated aliphatic hydrocarbons (CAHs)

CAH-contaminated waters are usually treated by means of physical–chemical methods

(stripping and adsorption) which simply transfer the contaminants to a different matrix.

On the other hand, several studies have indicated that the biological

degradation/transformation of CAH can occur under aerobic and anaerobic conditions,

offering the possibility to transform several CAHs into non-toxic, easily biodegradable compounds.

Trichloroethylene (TCE) 1,1,2,2-Tetrachloroethane (TeCA)

Chlorinated aliphatic hydrocarbons (CAHs) represent a widespread cause of water contamination. Due to their toxicity and the demonstrated cancerogenicity of some of

them, subsurface contamination by CAH is considered particularly serious and dangerous.

Page 3: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Aerobic cometabolic biodegradation of CAHs

Low-chlorinated CAH (1, 2 or even 3 chlorine atoms) are in general more easily degraded by

means of aerobic (oxidation) processes, which offer the possibility to achieve a complete dechlorination of the parent compounds at high degradation rates. However, most CAH

aerobic degradation processes occur by means of a cometabolic pathway

Thus, it is required to supply a proper growth substrate (an aliphatic or aromatic hydrocarbon), that provides energy and induces the synthesis of enzymes that catalyze CAH

biodegradation.

MONO-OXYGENASE

CAH

CC

ClCl

ClH

O

Epoxide

AlcoholsOrganic acids

O2 H2O

Growth substrate CO2 + H2O + energy

Alvarez-Cohen and Speitel Jr. (2011) Biodegradation 12:105-126.

Suttinun et al. (2013) Rev Environ Sci Biotechnol 12:99-114.

Page 4: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Implementation of CAH aerobic cometabolic

processes

The stimulation of in-situ aerobic cometabolic processes presents significant challenges

related to the risk of a complete consumption of the supplied growth substrate within a

short distance from the injection wells and to the possible clogging of the aquifer porosity.

On the contrary, the on-site implementation of aerobic cometabolic processes presents

quite interesting opportunities, in particular when the need to implement a hydraulic

barrier leads to the choice of a pump and treat remediation approach.

Among the possible bioreactor solutions, packed-bed bioreactors (PBRs) present specific advantages over bioreactors

with suspended cells.

Gandhi et al. (2002) Wat Resour Res 38:11-1–11-18.

Page 5: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Aim of the research

To develop an on-site aerobic cometabolic bioremediation process based on a

packed bed bioreactor (PBRs) for an actual site ground water contaminated by

Trichloroethylene (TCE) and 1,1,2,2-Tetrachloroethane (TeCA) located in Northern Italy.

The site is articulated in a shallow and a confined aquifer contaminated mainly by TCE (0.04-5.8 mg L-1)

1. to obtain and characterize an effective CAH-degrading microbial consortium from the site’s indigenous biomass and select the best growth substrate for the AC process;

Specific aims of this work

2. to select the best carrier for the PBR process;

3. to develop the PBR continuous-flow process with pulsed supply of substrate and

oxygen;

4. to scale-up the PBR continuous-flow process to a 31-L plant.

This research is part of the EU FP7 research program “Microorganism and enzyme

Immobilization: NOvel Techniques and Approaches for Upgraded Remediation of Underground-, wastewater and Soil” (MINOTAURUS).

Page 6: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

1) SELECTION OF SUBSTRATE/MICROBIAL

CONSORTIUM

4 groundwater samples (n. 1 to 4) collected from different zones of the aquifer and a

mixture of the same groundwaters (n. 5) were incubated in the presence of 4 candidate

growth substrates at 30°C for 60 days.

The 20 enriched consortia were

compared in terms of biodegradation capabilities

(normalized net biodegradation

rates) and structure of the microbial community (DGGE).

M1 M2 M3 M4 M5

B1 B2 B3 B4 B5

PE1 PE2 PE3 PE4 PE5

PR1 PR2 PR3 PR4 PR5

Methane (M)

Butane (B)

Pentane (PE)

Propane (PR)

Growndwater samples

1 2 3 4 5

Based on its high k1,TCE (96 L

gprotein-1 d-1), the consortium enriched on butane from

groundwater n. 4 (i.e., B4),

was selected as the best performing one and further

characterized.

Page 7: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

2) SELECTION OF THE BIOFILM CARRIER

4 types of porous biofilm carriers were tested:

Biomax:• Porosity: 60%• Density: 0.66 kg/L• Ceramic

Cerambios:• Porosity: 74%• Density: 0.66 kg/L• Ceramic

Biomech:• Porosity: 64%• Density: 0.68 kg/L• Ceramic

Biopearl:• Porosity: 58%• Density: 0.95 kg/L• Sintered glass

Consortium B4 was subcultured at 30°C and 15°C in 50 mL of sterile groundwater + 60 mL of

carriers, with butane (2 mg/L), oxygen (8 mg/L) and TCE (1 mg/L) pulses. Liquid phase was

periodically replaced with sterile water to remove freely suspended cells.

BIOPEARLBIOMECHBIOMAXCERAMBIOS

After 8 spikes, carriers were washed with sterile

physiological solution. Normalized degradation rates

and attached biomass concentrations were measured and the biofilms characterized.

Page 8: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

0.00

0.04

0.08

0.12

0.16

Biomax Biomech Biopearl Cerambios

30°C 15 °C

0.0

0.2

0.4

0.6

0.8

Biomax Biomech Biopearl Cerambios

30 °C 15 °C

(d-1

)

(gp

rote

in

Lb

iore

acto

r-1)

Biomax was characterized by the highest values of attached cells concentration at both temperatures.

Biomax also exhibited the highest normalized TCE degradation rate at 15°C and at 30°C (along with Biomech).

Frascari et al. (2013) Biodegradation in press DOI: 10.1007 / s10532-013-9664-z.

2) SELECTION OF THE BIOFILM CARRIER (continued)

Biomax was thus selected as the best performing immobilization carrier.

Attached cell concentration TCE normalized degradation rate

Page 9: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Kinetic model: TCE, 30°C

0 2 4 6 8 10

-0,02

0

0,02

0,04

0,06

0,08

0,1

Initial TCE concentration (mg/L)

Initial specific TCE depletion rate (g/g prot./d)

� Model: competitive inhibition

� Butane concentration: 1.5 mg L-1

qmax, TCE, attached cells = 0.021 ± 0.001 g gprotein-1 d-1

Ks, TCE, attached cells = 1.10 ± 0.07 mg L-1

TCE

TCEBcompI

TCEs

TCETCE

CK

CK

Cqq

+

+

⋅=

,,

B

,

max,

1

Ki,comp,B-TCE, attached cells = 0.778 ± 0.23 mg L-1

KINETIC ANALYSIS OF TCE BIODEGRADATION BY BIOMAX-ATTACHED CELLS OF THE SELECTED CONSORTIUM, WITH OR

WITHOUT BUTANE INHIBITION

Page 10: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Goals of the pulsed feed of substrate and oxygen:

� to reduce substrate inhibition on the CAH biodegradation rate

� to extend the bioreactive zone to the entire reactor length, with a homogeneous

biomass distribution

3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED

SUPPLY OF SUBSTRATE AND OXYGEN

Page 11: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

0

5

10

15

20

0 6 12 18 24

Time (hours)

Co

nce

ntr

atio

n (

mg

/L)

Oxygen

Growth substrate

Concentration vs. time at the bioreactor inlet

The overlapping of oxygen and growth substrate , and therefore the uptake of substrate,

occurs in 1 or more narrow bioreactor zones, that shift with groundwater flow and that are

characterized by low oxygen and growth substrate concentrations

� low substrate uptake rate � long bioreactive zone

� substrate inhibition on TCE cometabolism limited to the zones where, at each instant,

substrate is present

0

5

10

15

20

0 0.5 1 1.5 2

Co

nce

ntr

atio

n (

mg

/L)

Concentration vs. space along the bioreactor

Oxygen

Growth

substrate

Length of active aquifer or column height (m)

Supply of alternated pulses of oxygen and growth substrate

3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED

SUPPLY OF SUBSTRATE AND OXYGEN (CONTINUED)

Page 12: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

A 1 L column plant was set-up, filled with the Biomax carrier and inoculated with

consortium B4 (30°C). Passive biomass immobilization was performed in batch for 48 h. The

plant was then fed in continuous mode with alternate pulses of butane (25 mg L-1, pulse length 7.2 h) and oxygen (21 mg L-1, pulse length 16.8 h) and a constant TCE

concentration (1.2 mg L-1) for ̴100 days.

0%

10%

20%

30%

40%

50%

60%

40 50 60 70 80 90 100 110

TCE removal

Time (d)

TCE REMOVAL VERSUS TIME

- The degree of mineralization to Cl- of the

organic chlorine was equal as an average to 90%

PB

R

n.1

C

arr

ier:

Ce

ram

bio

sTCE

satura

ted

soluti

on

Control

unit

Oxygen

enrichedwater

Butane enrichedwater

3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED SUPPLY OF

SUBSTRATE AND OXYGEN (CONTINUED)

Page 13: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

At the end of operation under continuous flow, carriers were collected from the bottom (inlet,

1), middle (2) and top (outlet, 3) sections of the column, washed in sterile physiological solution, and subjected to the evaluation of:

1

2

3

r/c (1/d)

X (mgprot. /L)

k1 (L/d/mg)

0.15 ± 0.03

28 ± 11 0.06 ± 0.03

0.17 ± 0.06

27 ± 11 0.06 ± 0.03

0.24 ± 0.08

44 ± 18 0.05 ± 0.03

- the TCE

normalized

degradation rate

- the amount

of attached

biomass1 2 3

- the community structure (DGGE

analysis)

- the TCE 1st

order degr.

constant

3) INITIAL DEVELOPMENT OF THE PBR CONTINUOUS-FLOW PROCESS WITH PULSED SUPPLY OF

SUBSTRATE AND OXYGEN (CONTINUED)

Page 14: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

L

reactor

Lmol

L

vD

Lv

dispersiondiffusion

convectionPe

αα≅

⋅+

⋅=

+=

int

int

Pe

cle

t

nu

mb

er

-

+Concentration VS time

INLET profile

butane oxygen

Time (d)

Conc. (m

g L

-

1)

0

10

20

30

40

50

0 1 2 3

Substrate and oxygen

pulsing injection feeding

� The substrate pulses remain

separate, along the reactor

� More space for contaminant

degradation without substrate

inhibition

� Low butane/oxygen

overlapping � low average

substrate rate � possibility to

maintain the entire reactor

length active

� The butane pulses merge

with each other � continuous

butane feed in the 2nd part of

the bioreactor � high rate of

substrate consumption

� Significant substrate

inhibition on contaminant

degradation

convection

dispersion

510=Pe

convection

dispersion

110=Pe

Concentration VS column

heightbutane oxygen

Column height (m)

Co

nc. (m

g L

-1)

0

10

20

30

40

0 0.5 1

butane

oxygen

Column height (m)

Conc. (m

g L

-1)

0

10

20

30

40

0 0.5 1

A crucial aspect for the successful implementation of the pulsed feeding is represented by the

ratio of convection to hydrodynamic dispersion, expressed by the Peclet number:

Longitudinal dispersivity

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT

Page 15: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

� To estimate the longitudinal dispersivity of each tested biomass carrier, the 1-L columns were

exposed to tracer tests consisting of pulses of oxygen, TCE or TeCA

0.0

0.2

0.4

0.6

0.8

1.0

0.00 0.02 0.04 0.06 0.08 0.10

Dimensionless time (t/(Vpores/Q))

Dim

ensio

nle

ss

inle

t concentr

ation

Inlet conditions:

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)

Page 16: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

� The measured outlet concentrations were interpreted with a 1-D model with advection and dispersion:

Column

1

Column

2

Column

3

Column

4

carrierCerambios Biomax Biomech Biopearl

Effective

porosity0.72 0.64 0.65 0.59

Longitudinal

dispersivity (m)0.050 0.054 0.050 0.040

Exper. Oxygen conc.

Exper. TCE conc.Simulation

Typical results: TCE and oxygen pulsed-

injection with Cerambios as the biofilm carrier

2

2

,z

cD

z

cv

t

c iiL

ii

∂⋅+

∂⋅−=

0.0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4

0.0

0.2

0.4

0.6

0.8

1.0

0 1 2 3 4

Time / HRT (-)

Dim

ensio

nle

ss o

utlet

conc.

(-)

Dim

ensio

nle

ss o

utlet

conc. (-

)

Time / HRT (-)

vD LiL ⋅≈ α,

with

αL = longitudinal dispersivity

A = section of the column

Φe = effective porosity

OxygenTCE

eA

Qv

φ⋅≈

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)

Page 17: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

� With a longitudinal dispersivity of 0.054 m for the selected carrier, a reasonable compromise to avoid an excessive reactor length was found in Pe = 300 total

reactor length = 16 m

� This led to the design of a modular plant, obtained by connecting in series 14 glass columns of 1.2 m of length, each

� The diameter was set to about 0.05 m, to avoid an excessively low value of the ratio of column diameter / carrier diameter

� The reactor total volume resulted therefore equal to 31 L

� To ensure a residence time of 4 days, the groundwater flowrate has to be = 0.2 L/h

� The interstitial velocity results equal to 4 m/d

Reactor design:L

reactorLPe

α≅

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)

Page 18: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

Flow sheet of UNIBO’s pilot plant:

1 2 14

Temperature-controlled bath(T = 10-30 °C)

Sampling ports

TCE

saturate

d

solution

Control unit

Oxygen

enrichedwater

Butane enrichedwater

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)

Page 19: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

UNIBO’s pilot plant – preliminary pictures:

Top view

Bottom view

Page 20: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

0 300

0,84

1,68

2,52

3,36

4,2

5,04

5,88

6,72

7,56

8,4

9,24

-0,2

0

0,2

0,4

0,6

0,8

1

1,2

time (d)

rTC

E/C

TC

E (

d-1

)

Substrate/Oxygen pulsing schedule applied

TCE conversion attained in the 31-L plant: significantly higher than that attained in the 1-L column; TCE normalized degradation rates (r/C): comparable in the two systems

Goal: optimize the pulsed feeding strategy in order to attain higher TCE biodegradation yields.

SUCCESSFUL SCALE-UP OF THE PROCESS

OPERATING CONDITIONS: - T = 30 °C- HRT = 3.7 - 5.5 days

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)

Page 21: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

0 0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

10

5

10

15

20

25

30

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

dimensionless reactor length

Oxygen, Butane [mg/L]

0 0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

10

5

10

15

20

25

30

0

2

4

6

8

10

12

dimensionless reactor length

Oxygen, Butane [mg/L]

THIRD PHASE:• t cycle = 2 days,• Butane pulsed feed = 19% of

the cycle duration, • TCE inlet conc. = 1.37 mg L-1• rTCE/CTCE,in = 0.11 ± 0.02 d-

1• TCE conv. = 49 ± 12%

NINTH PHASE:• t cycle = 4 .66 days, • Butane pulsed feed = 14.3% of

the cycle duration, • TCE inlet conc. = 0.5 mg L-1• rTCE/CTCE,in = 0.19 ± 0.06 d-1• TCE conv. = 76 ± 9%

4) SCALE-UP OF THE PBR CONTINUOUS-FLOW PROCESS TO A 31-L PLANT (CONTINUED)

Page 22: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

CONCLUSIONS

• Preliminary investigation in a 1 L Biomax packed column plant evidenced that the alternated pulse feeding of butane and oxygen leads to an homogenous growth and activity of B4 consortium on Biomax.

Further work is in progress: o to identify biomass carriers characterized by lower longitudinal dispersivities and to

optimize the pulsed substrate/oxygen pulsed feed, in order to attain outlet TCE concentrations as close as possible to the limits for the remediation of CAH-polluted

aquifers;o to test the PBR process at the temperature of the site (15 ° C).

• Butane was selected among 4 tested substrates, and a suspended-cell consortium capable to degrade TCE was developed from the site’s indigenous biomass (B4).

• The attachment of the selected consortium to 4 porous biofilm carriers led to the selection of a ceramic porous carrier (Biomax).

• A 31-L packed-bed reactor, consisting of 14 columns connected in series, was designed, set-up, inoculated and operated for 170 days with satisfactory results; a substrate pulsed feed was set up to avoid excessive inhibition between growth substrate and TCE.

• The high longitudinal dispersivity of the selected carrier determines a significant widening of the butane pulses while they travel along the columns � despite the progressive optimization of the butane/oxygen pulsed feed, substrate inhibition on the TCE biodegradation rate results not negligible.

Page 23: Development of an attached-growth process for the ...newweb.riminifiera.it/upload_ist/AllegatiProgrammaEventi/Rosato... · Antonella Rosato, Dario Frascari, Giacomo Bucchi, Francesco

ACKNOWLEDGEMENTS

This research was funded by the EU through the FP7 program

Project MINOTAURUS (GA n. 265946)

Microorganism and enzyme Immobilization: NOvel Techniques and Approaches

for Upgraded Remediation of Underground-, wastewater and Soil

Thank you for your attention!


Related Documents