Biodegradation of Di-(2ethylhexyl)

Phthalate (DEHP) by Rhodococcus

Rita Chen, Audrey Pottschmidt, John Schlichting
Overview
● Properties of DEHP
● Exposure & Health Hazards
● Microbial Taxa
● Proposed Pathways
Free of phthalates!...What does this mean to consumers?
What is DEHP?
● Phthalic acid esters (PAEs) - group of chemicals commonly used as
plasticizers
● DEHP is the most common and widely manufactured phthalate
● Very cheap
● Can be found in:
○ Tablecloths
○ Floor files
○ Shower curtains
○ Toys
○ Personal care products
○ Disposable medical plastic applications
○ And more!
What is DEHP?
● Chemical formula: C24H38O4
● Organic
● Oily colorless liquid with almost no odor
● Solubility: minimal solubility in water, soluble in oil
● Melting point: -47 deg C wikipedia.org

● Boiling point: 386 deg C

● Very slow natural photodegradation
○ Half life: 2000 years
Overview
● Properties of DEHP
● Exposure & Health Hazards
● Microbial Taxa
● Proposed Pathways
Exposure & Impact
Exposure
● Transferred from plastic food packaging, containers, or processing
● Medical procedures via transfusions, dialysis, other disposable equipment
● Present throughout the environment in low levels
● Can transfer from mother to baby Question:
If exposed to DEHP, which pathway
Regulation would be most preferred?
● Listed on Proposition 65 A) Inhalation
● USEPA RfD: 20 µg/kg body weight/day B) Ingestion
C) Transfusion/Medical procedure
● EU CSTEE TDI: 37 µg/kg body weight/day
● 2003 study: median intake 13.8 µg/kg body weight/day
○ 95th percentile: 52.1 µg/kg body weight/day
Exposure & Health Hazards
Health Impacts and Hazards

● Acute
○ Large oral doses (5-10g) can lead to gastrointestinal distress
● Chronic (noncancer)
○ No established information
● Reproductive
○ Newborns with detectable DEHP in blood had shorter gestation periods (teratogens)
● Cancer risk
○ EPA: probable carcinogenic
○ Laboratory rodents exhibited oral dose-related increases in liver tumors
Which of the following items have DEHP/Phthalates in them?

Answer: They all do!

Overview
● Properties of DEHP
● Exposure & Health Hazards
● Microbial Taxa
● Proposed Pathways
 Degradation of DEHP by Rhodococcus pyridinivorans XB
● Facultative anaerobe
● Cell morphology: gram positive, non-flagellated,
short rod
● Colony structure: opaque, salmon pink, rounded
● Optimal conditions for degradation:
○ pH = 7.08
○ Temperature = 30.4°C
● Isolated from activated sludge in China

Zhao et al. 2018

R. pyridinivorans XB degrades
98% of DEHP within 48 hours
on MSM without lag phase.

● Uses phthalic acid as sole carbon Question:

and energy source. Why does EPS production give r.
● Produces extracellular polymeric XB a competitive
pyridinivorans
substances (EPS) as responsein
advantage toresponse to DEHP stress?
DEHP stress.
● Able to degrade other PAE
compounds and their potential
metabolites as well.

Zhao et al. 2018

 Rhodococcus ruber YC-YT1 completely degrades
DEHP after 72 hours.
● Cell morphology: gram positive, short rod
● Isolated from marine plastic off the coast of China
● Performs best in slightly alkaline solutions
○ pH = 6-10
○ Temperature = ~30°C
○ 10 hour lag phase
○ Max salinity = 120 g NaCl/L

Yang et al. 2018

 R. ruber YC-YT1 also capable of degrading other
PAEs and their intermediates.

Yang et al. 2018

 Further research: using community of microbes rather
than individual isolated strains to degrade PAEs.
● Consortium enriched from activated sludge found to degrade 93.64% of 1000 mg/L
DEHP after 48 hours.
● Optimal conditions:
○ pH = 6
○ Temperature = 30°C
○ Highly halotolerant

Li et al. 2018
Overview
● Properties of DEHP
● Exposure & Health Hazards
● Microbial Taxa
● Proposed Pathways
DEHP Bioaccumulation and Prevalence

Phthalates have been observed in

natural waters, river sediments,
lakes, groundwater, wastewater,
and sludge.

Phthalates are not chemically

bonded to plastic polymers. They
can readily migrate into the
environment.

This chemical leaching is

prolonged with microplastics.
DEHP Biodegradability and Industrial Synthesis
General esterification process.
DEHP is recalcitrant due to the
steric effect of its ester chains.

Common industrial synthesis of DEHP

DEHP
2-ethylhexanol
phthalic anhydride

They
Whyinhibit
wouldhydrolysis
longer esterby This process occurs in two steps: General structure
of phthalates
hindering hydrolytic
chains cause DEHPenzymes
to be
recalcitrant?
from binding. 1) Rapid esterification of first carboxylic group in
absence of catalyst
DEHP has long ester chains, 2) Slow esterification of second carboxylic group
therefore it’s less susceptible to that is catalyst facilitated
biodegradation
Enzymes used in Phthalate Degradation
Common microbial PAE
enzymes:

● Oxygenase
● Dioxygenase
● Dehydrogenase
● Decarboxylase
● Lipase
● Esterase
Lipase removal ratio for DEHP found as 1 mg Porcine pancreas lipase isolated
DEHP / 1 ml enzyme (Kurane et al. 1980) from slaughterhouse wastewater
decreased a 100 mg/L DEHP sample
Commercial lipase found to remove 85% of a 7
by 93% (Saito et al. 2010)
mg/L DEHP sample (Gavala et al. 2004)
Overview DEHP Degradation Pathway
In aerobic and anaerobic
conditions, microbial and enzymatic
degradation pathways generally
occur in 2 main steps:
1) Primary degradation,
PDE to PME then to PA
2) Ultimate degradation,
PDE = Phthalate diester
PA to CO2 or CH4 PME = Phthalate monoester
PA = Phthalic acid
PCA = Protocatechuate
Common degradation pathways:
• De-esterification Question:
• Transesterification Which degradation pathway is
most commonly used when
• β-oxidation
converting DEHP into PA?
• Demethylation (hint: opposite general DEHP synthesis
r. ruber YC-YT1

Using HPLC-MS analysis, three metabolites MEHP, PA, and BA were observed.
This pathway utilizes selective hydrolysis of the 2 ester bonds in the primary
degradation.

Ultimate degradation occurs with decarboxylation forming benzoic acid that will be
used for cell growth in the benzoate metabolic pathway.
Primary degradation Ultimate degradation

Yang et al. 2018


r. pyridinivorans XB
Primary degradation
Using HPLC-MS analysis, four intermediates
were observed in primary degradation; DBP,
MEHP, 2-Ethylhexanol and PA. PA can be
converted to PCA via 3,4 - dihydroxyphthalate
pathway with dioxygenase.
PCA is converted
to small organic
acids by
3,4-dioxygenase
and finally
degraded by
Ultimate degradation
tricarboxylic
acid cycle.
Zhao et al. 2018
References
1. Li, F., Liu, Y., Wang, D., Zhang, C., Yang, Z., Lu, S., & Wang, Y. (2018). Biodegradation of di-(2-ethylhexyl) phthalate by a

halotolerant consortium LF. Plos One, 13(10). doi: 10.1371/journal.pone.0204324

2. Yang, T., Ren, L., Jia, Y., Fan, S., Wang, J., Wang, J., … Yan, Y. (2018). Biodegradation of Di-(2-ethylhexyl) Phthalate by

Rhodococcus ruber YC-YT1 in Contaminated Water and Soil. International Journal of Environmental Research and Public

Health, 15(5), 964. doi: 10.3390/ijerph15050964

3. Zhao, H.-M., Hu, R.-W., Chen, X.-X., Chen, X.-B., Lü, H., Li, Y.-W., Wong, M.-H. (2018). Biodegradation pathway of

di-(2-ethylhexyl) phthalate by a novel Rhodococcus pyridinivorans XB and its bioaugmentation for remediation of DEHP

contaminated soil. Science of The Total Environment, 640-641, 1121–1131. doi: 10.1016/j.scitotenv.2018.05.334

See the in-class handout or paper for the remaining references.

Questions,
comments, or
concerns?