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TitleAnaerobic transformation of brominated aromatic compounds by Dehalococcoides mccartyi strain ...
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Table of Contents
                            Title Page
Major Theses
1 Introduction
	1.1 Brominated compounds in the environment
		1.1.1 Natural production of organobromine compounds
		1.1.2 Anthropogenic brominated flame retardants
	1.2 Toxicity of brominated flame retardants
	1.3 Microbial reductive dehalogenation
		1.3.1 Organohalide respiration
		1.3.2 Dehalococcoides mccartyi strain CBDB1
		1.3.3 Dehalogenation specificity of reductive dehalogenases
		1.3.4 Progress in microbial reductive debromination
		1.3.5 Growth of D. mccartyi strains with organohalides as electron acceptor
	1.4 Compound-specific stable isotope analysis
	1.5 Objective of this work
2 Materials and Methods
	2.1 Chemicals and gases
	2.2 Cultivation of D. mccartyi strain CBDB1
		2.2.1 Medium preparation
		2.2.2 Different inoculum of cultures
		2.2.3 Brominated benzenes as electron acceptors
		2.2.4 Tetrabromobisphenol A and bromophenol blue as electron acceptors
		2.2.5 Decabromodiphenyl ether and hexabromocyclododecane as electron acceptors
	2.3 Analytical methods
		2.3.1 Cell counting
		2.3.2 Bromide analysis
		2.3.3 Concentration analysis by gas chromatography
		2.3.4 Concentration analysis by liquid chromatography-mass spectrometry (UPLC-MS)
	2.4 RdhA Protein Expression Analysis
		2.4.1 Sample preparation
		2.4.2 nanoLC MS/MS analysis
	2.5 Resting cell activity assay
		2.5.1 Photometric activity assay
		2.5.2 GC-format activity assay
	2.6 Compound-specific stable isotope analysis
3 Results
	3.1 Growth of strain CBDB1 with brominated compounds
		3.1.1 Growth with hexabromobenzene
		3.1.2 Growth with 1,3,5-tribromobenzene
		3.1.3 Growth with bromophenol blue
		3.1.4 Cultivation using tetrabromobisphenol A as electron acceptor
		3.1.5 Cultivation using decabromodiphenyl ether and hexabromocyclododecane as electron acceptor
	3.2 Influencing factors and inhibitory effects in the reductive debromination process catalyzed by strain CBDB1
		3.2.1 Factors influencing cell growth in hexabromobenzene cultures
		3.2.2 Toxicity tests with bromophenol blue
	3.3 Dehalogenation products of brominated organics by strain CBDB1
		3.3.1 Debromination products of 1,3,5 -tribromobenzene
		3.3.2 Debromination products of hexabromobenzene
		3.3.3 Debromination products of bromophenol blue
		3.3.4 Debromination products of tetrabromobisphenol A
	3.4 RdhA Protein Expression
		3.4.1 RdhA protein expression in cultures grown with hexabromobenzene or 1,3,5-tribromobenzene
		3.4.2 RdhA protein expression in cultures incubated with bromophenol blue or tetrabromobisphenol A
	3.5 Resting cell photometric activity assay
	3.6 Effect of dehalogenation of brominated benzenes on the carbon isotope ratio
		3.6.1 Carbon isotope fractionation in enzymatic assays
		3.6.2 Carbon isotope fractionation in live cultures
4 Discussion
	4.1 Growth adaption of D. mccartyi strain CBDB1 to brominated compounds
		4.1.1 Growth adaptation to brominated benzenes
		4.1.2 Growth adaption to brominated compounds with oligocyclic structures
		4.1.3 Inhibitory effects on reductive debromination
	4.2 Dehalogenation Patterns of D. mccartyi strain CBDB1
		4.2.1 Complete removal of bromine in reductive debromination
		4.2.3 Influence of the chemical property of halogenated compounds on reductive dehalogenation
	4.3 Evaluation on resting cell enzymatic activity assay
	4.4 Expression of reductive dehalogenases
	4.5 Potential of applying CSIA to detect reductive debromination
5 Conclusions
Document Text Contents
Page 1

Anaerobic transformation of brominated aromatic
compounds by Dehalococcoides mccartyi strain CBDB1

vorgelegt von

Master of Engineering

Chao Yang
geb. in Henan. China

von der Fakultät III – Prozesswissenschaften

der Technischen Universität Berlin

zur Erlangung des akademischen Grades

Doktor der Naturwissenschaften

- Dr.-rer. nat. -

genehmigte Dissertation


Vorsitzender: Prof. Dr. Stephan Pflugmacher Lima
Gutachter: Prof. Dr. Peter Neubauer
Gutachter: Prof. Dr. Lorenz Adrian
Gutachter: PD Dr. Ute Lechner

Tag der wissenschaftlichen Aussprache: 28. August 2017

Berlin 2017

Page 2

Chao Yang

Declaration for the dissertation with the tittle:

“Anaerobic transformation of brominated aromatic compounds by Dehalococcoides

mccartyi strain CBDB1”

This dissertation was carried out at The Helmholtz Centre for Environmental Research-UFZ,

Leipzig, Germany between October, 2011 and September, 2015 under the supervision of PD Dr.

Lorenz Adrian and Prof. Dr. Peter Neubauer. I herewith declare that the results of this

dissertation were my own research and I also certify that I wrote all sentences in this dissertation

by my own construction.

Signature Date

Page 51


Figure 3.9: Cultures of strain CBDB1 incubated with decabromodiphenyl ether (deca-BDE) or

hexabromocyclododecane (HBCD) as the electron acceptor. Left: the measured bromide concentration in

cultures incubated with either deca-BDE (●) or HBCD (○). Right: the cell density of strain CBDB1 in

cultures with one of the two compounds after inoculation (black bars) and after 65 days of incubation

(gray bars). Shown are means of triplicate cultures± SD.

For a new set up of cultures of strain CBDB1 with either decabromodiphenyl ether or

hexabromocyclododecane as electron acceptor, decabromodiphenyl ether or

hexabromocyclododecane were supplied as crystalline form similar to the approach for

hexabromobenzene cultures described previously. The inoculum of strain CBDB1 was from

well-grown hexabromobenzene debrominating cultures and the starting cell densities in both

cultures were around 5 × 106 cells mL-1. For theses cultures, no increase in bromide

concentration was detected in both cultures after 90 days of incubation. There was also no cell

growth during the whole incubation, indicating strain CBDB1 might not be able to use either

decabromodiphenyl ether or hexabromocyclododecane for reductive dehalogenation. No

debromination activity for both compounds by strain CBDB1 was confirmed after repeating all

experiments three times.

Page 52


3.2 Influencing factors and inhibitory effects in the reductive debromination

process catalyzed by strain CBDB1

3.2.1 Factors influencing cell growth in hexabromobenzene cultures

(1) Cultures using different inocula

In this test, two different parent cultures were used for a new transfer with hexabromobenzene as

an electron acceptor to test the importance of adaptation to an electron acceptor. One parent

culture was previous grown with hexabromobenzene, and the other was previous grown with

hexachlorobenzene as electron acceptor. New transfer cultures were set up in triplicates with

crystalline hexabromobenzene using the same cultivation approach described previously. The

starting cell density in both cultures was 2.0 × 106 cells mL-1. After 46 days of incubation, the

cell density of the cultures inoculated from hexabromobenzene cultures reached 9.6 × 106 cells

mL-1 while the cell density of the cultures inoculated from hexachlorobenzene cultures was still

around 2.7 × 106 cells mL-1 (Figure 3.10).

Figure 3.10: Cell growth of D. mccartyi strain CBDB1 with hexabromobenzene as electron acceptor.

Two different inocula were used: (○) inoculated from cultures of strain CBDB1 pre-grown with

hexabromobenzene, (●) inoculated from cultures of strain CBDB1 pre-grown with hexachlorobenzene.

Shown are means of triplicate cultures± SD.

The growth rate of hexachlorobenzene-inoculated cultures was slower than the rate of

hexabromobenzene inoculated cultures, and this difference became larger with the cultivation

continued. The cell density of hexabromobenzene-inoculated cultures reached 6.8 × 107 cells

mL-1 on day 78 and finally went up to 2.3 × 108 cells mL-1 after 126 days of incubation. On the

other hand, the cell density of hexachlorobenzene inoculated cultures slowly reached 5.0 × 107

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