Jose Julio Ortega-CalvoInstituto de Recursos Naturales y Agrobiologia de S evilla (CSIC)

jjortega@irnase.csic.es

How to influence bioavailability for reducing risks from organic pollutants in

bioremediation

OUTLINE

- BIOAVAILABILITY vs. BIODEGRADABILITY

- ENHANCED PHASE EXCHANGE: BIOSURFACTANTS

- DESORPTION & BIODEGRADATION METHODS

- EFFECTS OF PLANTS:EXUDATES

- COMBINED EFFECTS PLANTS & BIOSURFACTANTS

- CONCLUSIONS

How to influence bioavailability to reduce risks associated to bioremediation of organic pollutants

ES&T (2015) 49, 10255-10264

SETAC Europe setac.org sesss10.setac.org

Bringing different worlds together

Ortega-Calvo et al. ES&T, 2015. 49, 10255-10264

Ortega-Calvo et al. ES&T, 2015. 49, 10255-10264

Why PAHs?

Because…

• ARE UBIQUITOUS & PERSISTENT, RECOGNIZED AS

PRIORITY POLLUTANTS, ARE THE REASON FOR REMEDIATION

• ARE BIODEGRADABLE, SIGNIFICANT POTENTIAL FOR

BIOREMEDIATION

• ARE HYDROPHOBIC, WITH SPECIFIC LIMITATIONS TO

BIODEGRADATION DUE TO LOW BIOAVAILABILITY.

BIODEGRADABLE ≠

BIODEGRADED

-TOXICITY AT POLLUTED SITE

-LOW LEVELS OF NUTRIENT & e- ACCEPTORS

-LOW TOTAL CONCENTRATIONOF SUBSTRATE

-LOW BIOAVAILABILITY:SORPTION, NAPLs

ENHANCED PHASE EXCHANGE

ATTACHMENT

ES&T 2011, 45:1074-1081ES&T 2017, 51:11935–11942

MOBILIZATION

ES&T 2011, 45:3019-3026ES&T 2014, 48:10869-10877

ES&T 2008, 42:1131-1137ES&T 2015, 49:4498-4505ES&T 2016, 50:7633-7640ES&T 2018, 52:10673−10679

Ortega-Calvo et al., 2017. Soil Biological Communities and Ecosystem Resilience, Sustainability in Plant and Crop Protection, M. Lukac et al. (Eds.), DOI: 10.1007/978-3-319-63336-7_19.

BIODEGRADATION

RE (µg/ml)0 50 100 150

Sur

face

tens

ion

(mN

/m)

30

40

50

60

70

80

CMC

Effect of Pseudomonas aeruginosa 19SJ biosurfactants on surface tension: critical micelle concentration

Effect of biosurfactants from Pseudomonas aeruginosa 19SJ on partitioning of pyrene from a NAPL (HMN)

Biosurf. Solids Part. rate CeqCeqsolids

(µg/mL) (mg/mL) (ng/mL/h) (ng/mL) (ng/mg)

0

10

100

0

100

0

0

0

1

1

0,5

1,6

11,6

0,7

27,4

18

85

547

27

650

-

-

-

16

40

M. Garcia-Junco et al., Environ. Sci. Technol. 2003, 37, 2988-2996

Microbial biosurfactants influence on bioavailability of sorbed pyrene: role of desorption kinetics

E. Congiu & J.J. Ortega-Calvo, Environ. Sci. Technol. , 2014, 45:3019-3026

Environmental sample

TENAX

SAMPLESUSPENSION

DESORPTION BIODEGRADATION

SAMPLESUSPENSION

NaOH TRAP

- TENAX EXTRACTION- HPLC ANALYSIS

- MC EXTRACTION- HPLC ANALYSIS (native PAH)- 14CO2 MEASUREMENTS (14C-PAH)

Desorption kinetics using Tenax® extraction

• Sediment or soil suspension

• Liquid medium

• Tenax®

• Biocide (formaldehide)

St / So = Frap exp (-Krap t) + Fslow exp (-Kslow t)

St and So are the sorbed amounts at time t (h) and at the start of the experiment, respectively.

Frap and Fslow are the rapidly and slowly desorbing fractions.

Krap and Kslow (h-1) are the rate constants of rapid and slow desorption.

M. Bueno-Montes et al., Environ. Sci. Technol. 2011, 45:3019-3026

The study of the complete desorption kinetics is very important in determining the magnitude of the different desorbing fractions present in this soil

DESORPTION OF PAHs WITH TENAX IN GREENHOUSE SOIL

St / S0 = Frap * exp (-Krap * t) + Fslow * exp (-Kslow * t)

2. DESORPTION OF PAHs

EXTRACTING F rap as T20 (ISO 16751)

1. COMPLETE DESORPTION KINETICS OF PAHs

Benzo(a)pyrene

Phenanthrene

T20 = 99.9 % Frap

T20 = 97.3 % Frap

With this test we can assess quickly bioavailability through Frap in a high number of samples during the

greenhouse experiment

BIOACCESIBILITY ASSAY

250 ml Erlenmeyer flask

B. HPLC analysis

40 g Sample

Teflon-lined stopper

Lateral body

A. 14CO2 production

Syringe sampling

Alkali trap40 g sample + radiolabelled PAH

Main body

Teflon-lined stoppers

Biometer flask

14CO2

Bioaccessibility of phenanthrene in creosote-polluted soil

Phen

Time (days)

0 10 20 30 40 50

Phe

nant

hren

e (m

g kg

-1)

0

200

400

600

800

1000

1200

1400

1600

% 1

4 C m

iner

aliz

ed

0

10

20

30

40

50

60

70

J.L. Niqui-Arroyo et al., IUPAC Ser. Biophys. Proc. Env. Syst. Vol 3 (2011)

14CO2

12C-Phen

R. Posada-Baquero et al., Environ. Geochem. Health 2008, 30, 159-163

River sediment(Stokholm, industrial pollution)

Forest soil(Cádiz, background pollution)

Native PAH conc. (mg/kg)

Initial Fslow Control +Brij35

Phen 42 37 43 18

Pyr 48 41 38 8

Benzo(a)pyr 22 19 22 10

EFFECT OF SURFACTANTS ON BIODEGRADATION OF PAHs SOILREM SOIL-

AUTOCTHONOUS POP.

M. Bueno-Montes et al., Environ. Sci. Technol. 2011, 45:3019-3026

Phen Pyr

EFECT OF PLANTS ON BIOAVAILABILITY OF PAHs

Breakthrough curves of Pseudomonas putida G7 transportedthrough sand columns: exudates from Helianthus annuus vs. other DOM sources

C. Jimenez-Sanchez et al. Environ. Sci. Technol. , 2015, 49:4498-4505

High TOC (100 mg/L) Low TOC (10 mg/L)

Motility pattern mediated by taxis affects bacterial dispersal and biodegradation rate in porous media

C. Jimenez-Sanchez et al. 2018, 52:10673−10679

M. C. Tejeda-Agredano et al., Soil Biol. Biochem. 2013, 57:830-840

GREENHOUSE BIOAVAILABILITY EXPERIMENT

1. BIODEGRADATION

2. BIOAVAILABILITY

Chemical method :Desorption extraction.

Kinetics and single-point extraction at 20 h

(ISO 16751)

MEASURE THE TOTAL PAHs CONCENTRATION WITH SOXHLET EXTRACTION AND HPLC ANALYSIS TO STUDY THE EFFECT OF PLANTING ( SUNFLOWERS) AND BIOSURFACTANTS ADDITION ( RHAMNOLIPIDS AT 7 mg/g)

1.A

SEPARATE BIODEGRADATION EXPERIMENT UNDER LABORATORY CONDITIONS USING AN EXCESS OF NUTRIENTS, RADIORESPIROMETRY DETERMINATIONS WITH 14C-PYRENE AND ANALYSIS OF RESIDUAL CONCENTRATIONS OF NATIVE PAHs

SAMPLESUSPENSION

NaOH TRAP

1.B

D20 = T20/Ctotal

C D

RESULTS IN DIFFERENT ASSAYS

A B

NO SIGNIFICANT EFFECTS A SIGNIFICANT EFFECTAFTER THE ADDITION OF THAMNOLIPIS

A SIGNIFICANT INCREASE IN THE RAPIDLY DESORBABLE FRACTION

WAS OBSERVED AFTER RHAMNOLIPIDS ADDITION FOR

MINERALIZABLE AND COMETABOLIZABLE PAHs

Study of biodegradation experiment under laboratory conditions

BIODEGRADATION EXPERIMENT USING AN EXCESS OF NUTRIENTS, RADIORESPIROMETRY, SHAKING, DETERMINATIONS WITH 14C-PYRENE AND ANALYSIS OF RESIDUAL CONCENTRATIONS OF NATIVE PAHs

THE EFFECT OF BIOSURFACTANT WAS OBSERVED ONLY IN THE PRESENCE OF PLANTS DUE TO ROOT COMPONENTS OR EXUDATES COULD PROMOTE THE BIOSURFACT ACTION

With this experiment we can say that the concentration was lower under laboratory conditions

PAH Soil Soil+ rhamnolipid

Planted soil Planted soil + rhamnolipid

Pyrene D20a 0.04 0.13 0.10 0.20

Min. rate (µg/kg/h)b 1.5 ± 0.1ad 1.7 ± 0.2ab 3.9 ± 0.9bc 5.3 ± 0.01cMin. extent (%)b 40 ± 6 38 ± 8 48 ± 6 40 ± 7C0 (mg/kg)b 4.0 ± 0.2Ae 6.3 ± 0.3A 5.0 ± 1.0A 13.8 ± 4.4 ACf (mg/kg)b 1.0 ± 0.1Aa 0.5 ± 0.1Bab 0.6 ± 0.2Bab 0.4 ± 0.01BbC210d (mg/kg)c 2.8 ± 0.8A 2.4 ± 0.2AB 3.7 ± 0.4A 2.7 ± 0.2AB

Benzo(a)pyrene D20a 0.10 0.10 0.18 0.45

C0 (mg/kg)b 1.9 ± 0.5A 1.6 ± 0.01A 1.8 ± 0.15A 2.5 ± 0.5ACf (mg/kg)b 0.8 ± 0.1Aa 0.6 ± 0.04Bab 0.7 ± 0.05Aab 0.5 ± 0.01BbC210d (mg/kg)c 1.6 ± 0.2A 1.8 ± 0.01A 1.5 ± 0.5A 1.6 ± 0.04AB

LIFE15 ENV/IT/000396

RESULTS IN DIFFERENT ASSAYS

PARTNER

OUR ROLE: BIOAVAILABILITY ASSESSMENT

THE OBJECTIVE OF THIS PROJECT IS TO OPTIMIZE A BIOREMEDIATION METHOD WHERE THE TRANSFORMATION MADE BY CONSORTIA OF FUNGI AND BACTERIA IS FINALIZED BY THE FINAL STEP OF REVEGETATION IN A SOIL HIGHLY CONTAMINATED BY PAHS, BTEX AND ALKANES

BIOAVAILABE CONCENRATION FOR MINERALIZABLE AND COMETABOLIZABLE PAHs IN LIFE PROJECT

No significant effect was observed in

cometabolizable PAHs

A

B C

CONCLUSIONS

Tenax extraction during 20 hours has resulted a reliable and robust method to determine bioavailability in a wide set of operational conditions ranging from a different time scale to dissimilar treatments (planting, biosurfactant application, etc.).

We used different bioremediation approaches, operating on the bioavailability of PAHs, as a proof-of-concept for the use of desorption extraction methods to estimate bioavailability.

Magdalena Grifoll (Universidad de Barcelona)Joop Harmsen (Wageningen Environmental Research (Alterra)

Projects from Spanish Ministry of Economy, Industry and Competitiveness (CGL2013-44554-R and CGL2016-77497-R), Andalusian Government (RNM 2337) and European Commission (LIFE15 ENV/IT/000396)

Acknowledgements

Thank youfor your attention