This document summarizes research on the biological and abiotic transformation of contaminants of emerging concern (CECs) in anaerobic soils. The research has three main objectives: 1) study the biodegradation of the anticonvulsant drug carbamazepine (CBZ) in soils, 2) examine abiotic factors influencing CEC degradation, and 3) investigate mineral-catalyzed degradation of organophosphate flame retardants. For CBZ, results show removal only under aerobic conditions in one soil, and enrichment of phylotypes like Sphingomonadaceae linked to degradation. Screening of CEC reactivity with abiotic soil components found reactions with reduced sulfur, iron, and

1. 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

2. 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

3. Problem Statement
•Little known about the dominant biological and abiotic
processes responsible for degradation of CEC in
biogeochemically diverse soils
1.0
0.8
0.6
0.4
0.2
0.0
-0.2
-0.4
terminal
e- acceptor
stable
reduction
product
EH
0(w)
(Volt)
O2 H2O
NO3
- N2
MnO2(s) Mn2+
Fe(OH)3(s) Fe2+
SO4
2- HS-
HCO3
- CH4
morefavorablelessfavorable
Anaerobic vs. aerobic biodegradation
Microbial community and relevant genes
Mineral catalysis and other abiotic
mechanisms

4. Objective
1. Biodegradation of the anticonvulsant drug
carbamazepine (CBZ)
2. Abiotic factors influencing CEC in anaerobic soils
3. 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:

5. 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 WWTPs
One of the most commonly detected CECs in soils and
biosolids
Long half lives in soils
Detected 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.

6. Methods
Controls (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

7. Results: CBZ Biodegradation
No 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 0
30
60
90
120
50 Aerobic 500 Aerobic 5000 Aerobic
Day 4 Day 14
Similar removal for all concentrations (12.8-14.5%)
No removal in abiotic controls
AverageCBZRemaining(%)
ng/g

8. Results: Enriched Phylotypes
Soil 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

9. 0
0.5
1
1.5
2
Controls (no CBZ) 50 ng/g 500 ng/g 5000 ng/g
NS
NS
NS
NS
NS
Soil 1, Day 14
Results: Enriched Phylotypes
p <0.05
NS: Not significantly
different compared to the
controls
RelativeAbundance(%)
An additional 20 phylotypes were
enriched at lower levels (<0.05%)
Consistent results from
triplicate microcosms

10. • 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)

11. 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

12. Soil 2: Treatment vs. Controls50 ng/g
500 ng/g
5000 ng/g

13. Results: Predicted Metagenomes
Considering 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
Numberofsequences
Xenobiotic degradation:
Aminobenzoate degradation
p=5.99 e-6
Aminobenzoate Degradation Pathway (KEGG)

14. • 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

15. 2. Abiotic Factors Affecting CEC Degradation
• To screen reactivity of representative CEC with
abiotic constituents that are abundant in
anaerobic environments
“biological” “abiotic”
Reduced Sulfur
Species
Redox-active
organic matter
Ferrous Iron
Fe oxide,
Fe sulfide
minerals

16. 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

17. 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
2. Abiotic Factors Affecting CEC Degradation
pH 7, 25C, 30 g/L contaminant

18. Reaction with thiol
Aromatic C-Cl bonds
-atrazine
Reaction with Fe oxide mineral
Fluoroquinolone
(ciprofloxacin), phosphate
esters (TDCPP, TCPP, TCEP)
2. Abiotic Factors Affecting CEC Degradation
TDCPP
pH 7, 25C, 30 g/L contaminant

19. 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
25C, 200 g/L TDCPP,
1 g/L FeOOH(s)
Time (d)
Ln(C/C0)
pH 9
pH 8
pH 7
pH 6
Control
(pH 7)
FeOOH(s)
Fe(II) +
FeOOH(s)

20. 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, 25C, 1 g/L FeOOH(s), 56 mg/L Fe(II)

21. 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

22. 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