biological and biologically mediated abiotic transformation of contaminants of emerging concern in...

Biological and Biologically Mediated Abiotic Transformation of Contaminants of Emerging

Concern in Anaerobic Soils

Timothy Strathmann (Colorado School of Mines)Alison Cupples (Michigan State University)

Number 2014-67019-24024

Problem Statement

•Reclaimed wastewater increasingly being considered for crop irrigation

•Valuable resource for improving the sustainability of agricultural production systems

•Concerns due to contaminants of emerging concern (CEC), including pharmaceutically active compounds

Problem Statement •Little known about the dominant biological and abiotic

processes responsible for degradation of CEC in biogeochemically diverse soilsAnaerobic vs. aerobic biodegradationMicrobial community and relevant genesMineral catalysis and other abiotic

mechanisms

Objective

1. Biodegradation of the anticonvulsant drug carbamazepine (CBZ)

2. Abiotic factors influencing CEC in anaerobic soils3. Mineral catalyzed degradation of

organophosphate flame retardants

•Address a critical gap in our ability to predict the fate of important CEC in agroecosystems, identifying transformation processes and microorganisms controlling CEC degradation in biogeochemically diverse soils

•Ongoing activities:

1. Biodegradation of CBZ

• One project has focused on carbamazepine (CBZ) biodegradation

• To determine which phylotypes and functional genes are linked to pharmaceutical biodegradation in agricultural soils

Poor removal efficiency in WWTPsOne of the most commonly detected CECs in soils and biosolidsLong half lives in soilsDetected in plant tissues (roots, leaves, stems)

Removal distribution efficiency of CBZ in WWTPs

Source: Zhang et al, 2008. Carbamazepine and diclofenac: Removal in wastewater treatment plants and occurrence in water bodies. Chemosphere, 73: 1151-61.

MethodsControls (no CBZ) Samples

(w/ CBZ 50, 500, 5000 ng/g)

Nucleic acid extraction

Mothur analysis

QuECHERS extraction

Solid phase extraction

PICRUSt analysis

LC-MS/MS

Experimental Design

Two agricultural soils (soil 1 and soil 2)

Aerobic and anaerobic conditions

Sacrificial sampling days 4 and 14

MiSeq paired end (2 x 250 bp) 16S rRNA gene (V4)

STAMP

Results: CBZ BiodegradationNo significant difference over time in CBZ concentrations for – Soil 2 (aerobic or anaerobic)– Soil 1 (anaerobic)

Decrease in CBZ was only observed in soil 1 under aerobic conditions 50

Aer-obic

500 Aer-obic

5000 Aer-obic

0

30

60

90

120Day 4 Day 14

Similar removal for all concentrations (12.8-14.5%)No removal in abiotic controls

Aver

age

CBZ

Rem

aini

ng (%

)ng/g

Results: Enriched PhylotypesSoil 1: Aerobic Conditions

• These are putative CBZ degraders as appear to be obtaining a growth benefit from CBZ removal– Unclassified Sphingomonadaceae,

Xanthomonadaceae, Sphingomonas and Microvirga

(This level of phylotype enrichment was not observed in soil 2 by day 14)

• Several phylotypes were enriched in the CBZ amended microcosms compared to the controls

Unclass

. Sphingomonadace

aeGp6

Sphingomonas

Microvir

ga

Solirubrobacte

r

Unclass

. Bacte

ria

Unclass

. Betaproteobacte

ria

Unclass

. Xanthomonadace

ae0

0.5

1

1.5

2

Controls (no CBZ) 50 ng/g 500 ng/g 5000 ng/g

NS

NS

NSNS

NS

Soil 1, Day 14

Results: Enriched Phylotypes

p <0.05NS: Not significantly different compared to the controls

Rel

ativ

e A

bund

ance

(%)

An additional 20 phylotypes were enriched at lower levels (<0.05%)

Consistent results from triplicate microcosms

• Mothur was used to create Biom files for PICRUSt from the Illumina MiSeq data

Results: Predicted Metagenomes

• PICRUSt was used to predict the metagenomes using KEGG (Kyoto Encyclopedia of Genes & Genomes) Pathways

• Statistical analysis of data was performed with STAMP

CBZ amended samples were compared to the controls (no CBZ)

Soil 1: 5000 ng/g CBZ Compared to Controls (p<0.001)

Significantly different pathways

Mean proportions (%) Difference in mean proportions (%)

CBZ amended > controls CBZ amended < controls

Similar trends at 50 ng/g and at 500 ng/g

Soil 2: Treatment vs. Controls50 ng/g

500 ng/g

5000 ng/g

Results: Predicted MetagenomesConsidering all concentrations together (soil 1, day 14)• 8 pathways contained more genes in the CBZ

amended samples compared to the controls

(p<0.05)

Control 50 ng/g 500 ng/g 5000 ng/g

Num

ber o

f seq

uenc

es

Xenobiotic degradation: Aminobenzoate degradation

p=5.99 e-6

Aminobenzoate Degradation Pathway (KEGG)

• CBZ is recalcitrant under O2 depleted conditions• Under aerobic conditions, CBZ removal is also limited

Conclusions on CBZ Biodegradation

• PICRUSt has the potential to be a powerful approach for determining the capacity of soils to biodegrade CECs

• Several phylotypes were linked to CBZ removal– Unclassified Sphingomonadaceae, Xanthomonadaceae

Sphingomonas and Microvirga

2. Abiotic Factors Affecting CEC Degradation • To screen reactivity of representative CEC with

abiotic constituents that are abundant in anaerobic environments

Reduced SulfurSpecies

Redox-activeorganic matter

Ferrous Iron

Fe oxide, Fe sulfide minerals

2. Abiotic Factors Affecting CEC Degradation

• Antibiotics• Anticonvulsants• Antiinflammatories• Antihypertensive• Flame retardants• Herbicide

• LC-MS/MS used to simultaneously screen degradation of 14 structures representative of CEC detected in domestic wastewater

Adsorption to FeS mineral

Amine-containing structures-atenolol, trimethoprim, Ciprofloxacin, amitriptylene

Atenolol

Reduction by Fe(II)ads, FeS

Structures with N-O or C-Cl bonds-Carbadox, Sulfamethoxazole,TDCPP, TCPP, TCEP, TBPP


pH 7, 25C, 30 g/L contaminant

Reaction with thiol

Aromatic C-Cl bonds-atrazine

Reaction with Fe oxide mineral

Fluoroquinolone (ciprofloxacin), phosphate esters (TDCPP, TCPP, TCEP)


TDCPP

pH 7, 25C, 30 g/L contaminant

3. Fe oxide catalyzed degradation of organophosphate flame retardants

• Phaseout of brominated flame retardants leading to increased use of organophosphate substitutes

• Halogenated structures impart high recalcitrance to aerobic biodegradation

• Goethite (-FeOOH) catalyzes hydrolytic decomposition

Metal Oxide

25C, 200 g/L TDCPP, 1 g/L FeOOH(s)

Time (d)

Ln(C

/C0)

pH 9

pH 8pH 7

pH 6

Control(pH 7)

FeOOH(s)

Fe(II) +FeOOH(s)

3. Fe oxide catalyzed degradation of organophosphate flame retardants

• Mineral-catalyzed mechanism consistent for range of organophosphate flame retardants

• Reactivity related to acidity of the leaving group• Highly chlorinated/brominated analogues enhanced by

Fe(II) addition (abiotic reduction?)

pH 7, 25C, 1 g/L FeOOH(s), 56 mg/L Fe(II)

Ongoing Work

• Screening fate of a the wider range of CEC structures under variable anaerobic (nitrate reducing, sulfate reducing, and methanogenic) conditions

• Identification of the phylotypes and genes associated biodegradation processes observed

• Apply high resolution mass spectrometry to identify transformation products

• Structure-reactivity analyses

Acknowledgements

• Center for Environmental Risk Assessment (CERA) at CSM – mass spectrometry

• Research Technology Support Facility at MSU– Genomics and mass spectrometry services

• Dr. Chris Higgins (CSM), Dr. Hui Li (MSU)• Paul Merrifield (soil collection)

Number 2014-67019-24024