biological and biologically mediated abiotic transformation of contaminants of emerging concern in...
TRANSCRIPT
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
2. Abiotic Factors Affecting CEC Degradation
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)
2. Abiotic Factors Affecting CEC Degradation
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