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Cometabolic Biodegradation of Halogenated Aliphatic Hydrocarbons by Ammonia-Oxidizing Microorganisms Naturally Associated with Wetland Plant Roots

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Cometabolic Biodegradation of Halogenated Aliphatic Hydrocarbons by Ammonia-Oxidizing Microorganisms Naturally Associated with Wetland Plant Roots Wright State University CORE Scholar Browse all Theses and Dissertations Theses and Dissertations 2015 Cometabolic Biodegradation of Halogenated Aliphatic Hydrocarbons by Ammonia-oxidizing Microorganisms Naturally Associated with Wetland Plant Roots Ke Qin Wright State University Follow this and additional works at: https://corescholar.libraries.wright.edu/etd_all Part of the Environmental Sciences Commons Repository Citation Qin, Ke, "Cometabolic Biodegradation of Halogenated Aliphatic Hydrocarbons by Ammonia-oxidizing Microorganisms Naturally Associated with Wetland Plant Roots" (2015). Browse all Theses and Dissertations. 1430. https://corescholar.libraries.wright.edu/etd_all/1430 This Dissertation is brought to you for free and open access by the Theses and Dissertations at CORE Scholar. It has been accepted for inclusion in Browse all Theses and Dissertations by an authorized administrator of CORE Scholar. For more information, please contact [email protected]. COMETABOLIC BIODEGRADATION OF HALOGENATED ALIPHATIC HYDROCARBONS BY AMMONIA-OXIDIZING MICROORGANISMS NATURALLY ASSOCIATED WITH WETLAND PLANT ROOTS A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy By KE QIN MRes., University of York, 2008 2014 Wright State University i COPYRIGHT BY KE QIN 2014 ii WRIGHT STATE UNIVERSITY GRADUATE SCHOOL JANUARY 12, 2015 I HEREBY RECOMMEND THAT THE DISSERTATION PREPARED UNDER MY SUPERVISION BY Ke Qin ENTITLED Cometabolic Biodegradation of Halogenated Aliphatic Hydrocarbons by Ammonia-Oxidizing Microorganisms Naturally Associated with Wetland Plant Roots BE ACCEPTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF Doctor of Philosophy. ________________________________ Abinash Agrawal, Ph.D. Dissertation Director ________________________________ Donald Cipollini, Ph.D. Director, ES Ph.D. Program ________________________________ Committee on Robert E.W. Fyffe, Ph.D. Final Examination VP for Research and Dean of the Graduate School ________________________________ Abinash Agrawal, Ph.D. ________________________________ Donald F. Cipollini, Ph.D. ________________________________ Hailiang Dong, Ph.D. ________________________________ Mark N. Goltz, Ph.D ________________________________ Michael L. Shelley, Ph.D iii ABSTRACT Qin, Ke. Ph.D. Environmental Sciences Ph.D. Program, Wright State University, 2014. Cometabolic Biodegradation of Halogenated Aliphatic Hydrocarbons by Ammonia- Oxidizing Microorganisms Naturally Associated with Wetland Plant Roots. Bench-scale microcosms with wetland plant roots were investigated to characterize the microbial contributions to contaminant degradation of halogenated aliphatic hydrocarbons (HAHs) with ammonium. The batch system microcosms consisted of a known mass of wetland plant roots in aerobic growth media where the roots provided both an inoculum of root-associated ammonium-oxidizing microorganisms and a microbial habitat. Aqueous growth media, ammonium, and HAHs including trichloroethene (TCE), cis-1,2-dichloroethene (cis-DCE), chloroform (CF), 1,1,2- trichloroethane (1,1,2-TCA), ethylene dibromide (EDB, or 1,2-dibromoethane, 1,2-DBA) and 1,2-dichloroethane (1,2-DCA) were replaced weekly in batch microcosms while retaining roots and root-associated biomass. Molecular biology results indicated that ammonium-oxidizing bacteria (AOB) were enriched from wetland plant roots while analysis of contaminant and oxygen concentrations showed that those microorganisms can degrade HAHs by aerobic cometabolism. Cometabolism of TCE, at 29 and 46 µg/L, was sustainable over the course of 9 weeks, with 20-30 mg/L ammonium-N. However, at 69 µg/L of TCE, ammonium iv oxidation and TCE cometabolism were completely deactivated in two weeks. This indicated that between 46 and 69 µg/L TCE with 30 mg/L ammonium-N there is a threshold [TCE] below which sustainable cometabolism can be maintained with ammonium as the primary substrate. However, cometabolism-induced microbial deactivation of ammonium oxidation and TCE degradation at 69 µg/L TCE did not result in a lower abundance of the amoA gene in the microcosms. In the following experiments with TCE and elevated concentration of 75 mg/L ammonium-N, the deactivation of cometabolism with TCE was observed again when TCE reached from ~50 to ~70 g/L. The cometabolic system was activated again one week after the system was replaced by a TCE-free medium culture. Rate constants did not change significantly during the inactivation cycle if normalized by X. It can be inferred that the drop in ammonium and TCE degradation at certain [TCE] are due to the activity shut down by ammonia oxidizers. Similar deactivation trends were observed in microcosm amended with cis-DCE, 1,1,2-TCA and EDB when HAHs concentration increased above ~150 g/L. No deactivation was observed in the reactors with CF and 1,2-DCA. A shift of ammonium oxidation production from nitrite of nitrate after HAHs were added was observed in all the HAHs batch systems and those shifts all coincided with a moderate decrease in ammonium and HAHs degradation kinetics. Following sequencing analysis on a cometabolic system with TCE showed that the relative v abundance of nitrite oxidizers (especially Nitrospira) changed significantly after 14 weeks, which suggest that the microbial community changed with the addition of elevated concentration of TCE. The relative abundance of previous dominance genera Nitrosomonas decreased at higher TCE concentrations while the abundance of nitrite oxidizers Nitrospira increased significantly due to the weakened competence for oxygen from Nitrosomonas. This research indicates that microorganisms associated with wetland plant roots can assist in the natural attenuation of HAHs in contaminated aquatic environments, such as urban or treatment wetlands, and wetlands impacted by industrial solvents. vi TABLE OF CONTENTS 1 BACKGROUNDS …………………………………………………………...… 1 1.1 MICROORGANISMS INVOLVED………………………………………….. 4 1.2 AEROBIC AMMONIUM OXIDIZATION…………………………………... 10 1.3 INHIBITION BY HAHS AND RECOVERY FROM THE INHIBITION…… 15 1.4 RESEARCH GOALS………………………………………………………….. 20 1.5 REFERENCES………………………………...……….……………………… 21 2 NATURAL ATTENUATION POTENTIAL OF TRICHOLOROETHENE IN WETLAND PLANT ROOTS: ROLE OF NATIVE AMMONIUM-OXIDIZING MICROORGANISMS……………………………………………………………….... 44 2.1 INTRODUCTION……………………………….……………………………... 44 2.1.1 Ammonium Availability in Wetlands……………………………………... 44 2.1.2 Aerobic Cometabolism of Tricholoethene…………………………………. 46 2.2 MATERIALS AND METHOD………………………………………………… 48 2.2.1 Experimental Set-up………………………………………………………... 48 2.2.1.1 Collection of wetland plants………………………………………………… 48 2.2.1.2 Microbial enrichment with ammonium (in absence of TCE)……………….. 49 2.2.1.3 Cometabolic TCE degradation with ammonium……………………………. 50 2.2.2 DNA Extraction and PCR Amplification…………………………………… 51 2.3 RESULTS AND DISCUSSION……………………………………………….. 51 2.3.1 Ammonium-Oxidizing Microbes in Wetland Plant Roots…………………..51 2.3.1.1 Molecular evidence………………………………………………….……… 51 2.3.1.2 Analytical evidence………………………………………………………… 53 2.3.2 Degradation of TCE and Ammonium……………………………………… 53 vii 2.3.3 Degradation Kinetics and Transformation Yields…………………………. 55 2.3.4 Deactivation of TCE and Ammonium Degradation……………………….. 58 2.4 CONCLUSIONS……………………………………………………………… 59 2.5 REFERENCES………………………………………………………………… 60 3 NATURAL ATTENUATION POTENTIAL OF COMMON CHLORINATED VOLATILE ORGANIC COMPOUNDS IN WETLAND PLANT ROOTS BY NATIVE AMMONIA-OXIDIZING MICROORGANISMS………………………… 70 3.1 INTRODUCTION…………………………………………………………….. 70 3.2 MATERIALS AND METHOD………………………………………………... 72 3.2.1 Experiment Setup………...………………………………………………… 72 3.2.2 Cometabolic Degradation of CAHs with Ammonium…………………….. 72 3. 3 RESULTS AND DISCUSSION……………………………………………… 73 3.3.1 Aerobic TCE Cometabolism: Concentration Effect……………..………… 73 3.3.1.1 Sustained TCE degradation with ammonium……………………………… 74 3.3.1.2 Variations in cell biomass………………………………………………….. 75 3.3.1.3 Variation in degradation kinetics of ammonium and TCE…………………. 76 3.3.1.4 Variation in TCE Transformation Yield……………………………………. 80 3.3.2 Effect of cis-1,2-Dichloroethene (cis-DCE) Concentration…………...…… 82 3.3.2.1 cis-DCE Degradation with ammonium and degradation deactivation..…… 82 3.3.2.2 Variations in biomass……………………………………………………… 84 3.3.2.3 Variation in degradation kinetics of ammonium and cis-DCE……………. 85 3.3.2.4 Variation in cis-DCE transformation yield………………………………... 86 3.3.3 Effect of Chloroform (CF) Concentration………………………………… 87 3.3.3.1 Sustained CF degradation with ammonium……………………………….. 87 viii 3.3.3.2 Variations in Biomass………………..……………………………………. 88 3.3.3.3 Variation in degradation kinetics of ammonium and CF…….…………….. 89 3.3.3.4 Variation in CF transformation yield Ty…………………………………... 90 3.4 CONCLUSIONS…………………………………………………………….. 90 3.5 REFERENCES……………….……………………………………………….. 91 4 NATURAL ATTENUATION POTENTIAL OF EMERGING HALOGENATED ALIPHATIC HYDROCARBONS IN WETLAND PLANT ROOTS BY NATIVE AMMONIA-OXIDIZING MICROORGANISMS.…………………………………. 107 4.1 INTRODUCTION……………...……………………………………………. 107 4.2 MATERIALS AND METHOD……………………………………………… 110 4.2.1 Experiment Setup…………………………………………………………. 110 4.2.2 Cometabolic Degradation of CAHs with Ammonium……………………. 110 4.3 RESULTS AND DISCUSSION…………………………………………….. 111 4.3.1 Aerobic 1.1.2-TCA Cometabolism: Concentration Effect……………….. 111 4.3.1.1 Sustained 1,1,2-TCA degradation with ammonium and degradation deactivation………………………………………………………………………... 111 4.3.1.2 Variations in biomass……………………………………………………… 112 4.3.1.3
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