Stability and expression of a plasmid-borne TCE degradative pathway in suspended and biofilm cultures by Robert Raymond Sharp III A thesis submitted in partial fulfillment of the requirments for the degree of Doctor of Philosophy in Civil Engineering Montana State University © Copyright by Robert Raymond Sharp III (1995) Abstract: Trichloroethylene (TCE) is a United States Environmental Protection Agency Priority Pollutant. TCE is mutagenic and a suspected carcinogen. TCE is recalcitrant in the environment and has been found to be ubiquitous in soils and ground waters, globally. Use of TCE degradative pathways moderated by enzymes, the expression of which redides on recombinant plasmids, to biologically degrade TCE is a promising new technology to eliminate TCE. One such TCE degradative pathway is the toluene ortho-monooxygenase (TOM) pathway which is borne on the recombinant plasmid pTOM. Research presnted here examines the ability of pTOM pathway to degrade TCE in two different host cells, the original plasmid host Pseudomonas cepacia PR1 and a transconjugant Pseudomonas cepacia 17616. The goal of this research was to determine those mechanisms that cause the loss of the TCE degrading phenotype, associated with pTOM, in both suspended and biofilm cultures. These mechanisms include plasmid instability within the host cell, cell toxicity caused by TCE exposure, and cell injury caused by TCE exposure. In addition this research developed a protocol to determine the severity of these phenotypic losses in any pTOM host organism. With the use of several novel reactor systems, analytical methods, and protocols were developed to determine the suitability of P. cepacia 17616 as a host for plasmid pTOM. Batch studies indicate that P. cepacia 17616 is able to incorporate pTOM and degrade TCE at rates equal to those of other pTOM hosts. Plasmid stability experimental results showed significant segregational plasmid loss in P. cepacia 17616-pTOM in non-selective suspended and biofilm cultures, resulting in almost complete loss of plasmid-bearing cells within 30 days of operation. Continuous culture plasmid loss studies showed that the probability for plasmid loss per cell generation was a function of growth rate and ranged from 0.025/generation at a growth rate of 0.065 hr-1 to 0.035 at a growth rate of 0.17 hr-1. Results indicate there was no significant difference in the probability of plasmid loss between suspended and biofilm culture, suggesting that biofilm growth does not affect plasmid stability the P. cepacia 17616-pTOM system. Use of a novel TCE vapor exposure reactor system showed the TCE exposure can cause serious injury and toxicity to P. cepacia 17616 and can result in catastrophic loss of the TCE degrading phenotype in P. cepacia 17616-pTOM cultures. In addition, the severity of both cell injury and toxicity was found to be a function of TCE exposure time and TCE concentration. Research here indicates that plasmid loss, cell injury, and cell toxicity are significant mechanisms that can result in the detrimental loss of pTOM pathway expression and these mechanisms should be considered in any plasmid mediated catabolism of a toxic waste.  Stability and Expression of a Plasmid-Borne TCE Degradative Pathway In Suspended and Biofilm Cultures by Robert Raymond Sharp III A thesis submitted in partial fulfillment of the requirments for the degree of Doctor of Philosophy in Civil Engineering MONTANA STATE UNIVERSITY Bozeman, Montana July 1995 11 APPROVAL of a thesis submitted by Robert Raymond Sharp III This thesis has been read by each member of the thesis committee and has been found to be satisfactory regarding content, English usage, format, citations, bibliographic style, and consistency, and is ready for submission to the college of Graduate Studies. Dr. James D. Bryers Approved for the Department of Civil Engineering Date Approved for the College of Graduate Studies Dr. Robert Brown (Signature) Date iii STATEMENT OF PERMISSION TO USE In presenting this thesis in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this thesis is allowed only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this thesis should be referred to University Microfilms International, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non-exclusive right to reproduce and distribute my abstract in any format in whole Or in part.” Signature Date Z ACKNOWLEDGMENTS Foremost I would like to thank Dr. Al Cunningham for his guidance, advice, and friendship. I would also like to acknowledge and thank Dr. Malcolm Shields, Dr. Warren Jones, and Dr. James Bryers, who along with Al Cunningham made my Ph.D. experience multidisciplinary, exciting, and unique. A special thanks goes out to Anne Camper and Malcolm Shields who gave me most of my molecular and microbiological knowledge and skills, without which I would not have finished. I would like to thank Raj and Dave who always gave me advice (some good, some bad). I especially thank my lab assistant Jayne Billmeyer, who has become an excellent researcher in her own right. I thank all of the staff at the Center especially John Newman, Susan, Peg, Alma, Brian G. and anyone who ever worked in accounting. Obvious acknowledgment and thanks go out to my family, especially my mother and father for their support and their genes. I would also like to thank Dana Foti for her patience, support, and love. Finally, I would like to thank Dr. William Characklis for giving me the opportunity to pursue my Ph.D. at the Center for Biofilm Engineering where I have learned a great deal and have made life-long friends. I thank the University of West Florida, the National Science Foundation and other sources (James, Al, and Warren) for their financial support of this work which was, for the most part, independent of any Center project. V TABLE OF CONTENTS List of Tables xiii List of Figures xiv Abstract xx CHAPTER I - Goals and Objectives I 1.1 Goal 1 1.2 Thesis Statements 1 1.3 Motivations 2 1.4 Objectives 2; CHAPTER 2 - Introduction 4 2.1 General TCE Biodegradation 4 2.2 Anaerobic TCE Biodegradation 5 2.3 Aerobic TCE Biodegradation 6 2.4 Bulkholderia cepacia G4 7 2.4.1 Plasmids and TCE degradation . 9 2.4.2 Plasmid pTOM31c 9 2.4.3 B. cepacia PR1 -pTOM31c 13 2.5 Biofilms and Biofilm Reactors 13 Page 2.5.1 Biofilm reactors for biodegradation of 14 , organics including TCE 2.6 Loss of TCE Degrading Phenotype - Toxicity, 18 Plasmid Stability, and Cell Injury 2.6.1 Toxicity 19 2.6.2 Plasmid instability 20 Plasmid stability in suspended cultures Plasmid stability in biofilm cultures 2.6.3 Cell injury caused by exposure to toxic and 26 injurious substances 2.7 Plasmid Transfer 28 2.7.1 Pseudomonas cepacia 17616-pTOM31c, a transconjugant 29 of Burkholderia cepacia G4. CHAPTER 3 - Mathematical Models 31 3.1 TCE Degradation and Bacterial Growth Kinetic Models 31 3.1.1 Single substrate saturation kinetics 31 3.1.2 Andrews substrate inhibition growth kinetics 32 3.2 Continuous, Suspended Cell Culture Plasmid Loss Model 33 3.3 Biofilm Culture Plasmid Loss Model . 3 5 vi TABLE OF CONTENTS - Continued Page Vll 3.4 Nomenclature. 38 CHAPTER 4 - Materials and Methods 40 4.1 Bacterial Strains 40 4.2 Plasmids 41 4.3 Media 42 4.3.1 Basal salts medium 42 4.3.2 Rich general growth medium - LBG 44 4.3.3 Non-selective growth medium - HCMM2-sodium acetate 44 4.3.4 pTOM selective medium 47 4.3.5 Growth substrates 47 4.4 Growth and Activity Studies 49 4.4.1 Growth of PR1-pTOM23c on non-selective, 49 non-competitive phthalate-BSM medium 4.4.2 Growth of 17616,17616-pTOM31c, and PR1-pTOM31c on 49 non-selective, non-competitive acetate-HCMM2 medium 4.4.3 Growth characteristics of 17616-pTOM31c on 50 selective medium 4.5 Activity and Expression of pTOM 50 4.5.1 TFMP assays 51 TABLE OF CONTENTS - Continued Page 4.5.2 TCE disappearance assays 53 4.6 Methods for Initial Lab and Field Scale Column 54 Studies Using PR1-pTOM23c 4.6.1 Lab column studies 54 4.6.2 Field scale column studies 56 4.7 Methods for Plasmid Stability and Activity Studies 59 Using P: cepacia 17616-pTOM31c. 4.7.1 Suspended culture plasmid stability and activity studies 59 Batch experiments Continuous culture experiments 4.7.2 Biofilm culture plasmid stability and activity studies 61 4.8 Methods for TCE Exposure Studies Using an Actate Fed-Batch, 64 TCE Vapor Continuous Flow Reactor Studies to Determine Cell Injury, Toxicity, and Plasmid Loss During TCE Exposure 4.8.1 Selective TCE exposure studies 66 4.8.2 Non-selective TCE exposure studies 68 4.9 Analytical Methods and Protocols 68 4.9.1 Protein assay 68 viii TABLE OF CONTENTS - Continued Page ix 4.9.2 Phenol assay 69 4.9.3 Phthalate analysis 69 4.9.4 TCE analysis 69 4.9.5 Acetate analysis 70 4.9.6 pTOM31 selective direct colony transfer methods (PSDCT) 71 4.9.7 Cell enumeration techniques 71 Toxicity fractions Injured cell fractions p(+) and p(-) cell fractions 4.9.8 Biofilm culture suspension (BCS) method 74 4.9.9 pH and temperature 74 CHAPTERS-ResuIts 75 5.1 Results From Initial Lab and Field Studies Using PR1-pTOM23c 75 5.1.1 Initial lab studies 76 Growth of PR1-PTOM230 on phthalate PR1-pTOM23c TCE degradation kinetics Lab column studies using PR1-pT0M23c TABLE OF CONTENTS - Continued Page TABLE OF CONTENTS - Continued 5.1.2 Field studies using PR1 -pTOM23c in a vapor 83 phase bioreactor (VPBR) VPBR start-up Microbial activity of VPBRs TCE degradation in VPBRs 5.2 Results from Suspended Culture Plasmid Stability and 91 Expression Studies Using Pseudomonas cepacia 17616-pTOM31c 5.2.1 Batch suspended culture studies 91 17616 growth on acetate Plasmid loss under non-selective growth conditions TCE degradation and TCE specific activity 5.2.2 Continuous suspended culture studies 96 pTOM specific activity as a function of growth rate Plasmid loss in non-selective, non-competitive continuous culture Plasmid loss in selective continuous culture Plasmid loss in alternative media-fed continuous culture 5.3 Results from Biofilm Culture Plasmid Stability and 108 Expression Studies Using Pseudomonas cepacia 17616-pT0M31c Page 5.3.1 Stability and activity of pTOM31c in non-selective biofilm 108 cultures 5.4 Results From TCE Exposure Studies 117 5.4.1 Selective TCE exposure studies 117 5.4.2 Non-selective TCE exposure studies 119 CHAPTER 6 - Discussion 128 6.1 Failure of PR1-pTOM23cto Establish a Biofilm 130 6.2 pTOM Activity in the Transconjugant 132 P. cepacia 17616-pTOM31c 6.2.1 TCE degradation kinetics 134 6.2.2 Comparisons of pTOM activities in suspended 135 And biofilm 17616-pTOM31c cultures 6.3 Plasmid Loss in P. cepacia 17616-pTOM31c 136 6.3.1 Comparison of plasmid loss in suspended and biofilm 137 cultures of 17616-pTOM31c 6.3.2 Segregational loss of pTOM31c in 17616-pTOM31c cultures 138 6.4 Loss of TCE Degrading Phenotype - Plasmid Loss, 139 xi TABLE OF CONTENTS - Continued Page Toxicity, and Cell Injury , TABLE OF CONTENTS - Continued 6.4.1 Plasmid loss during TCE exposure 139 6.4.2 Toxicity of 17616-pTOM31c incurred during TCE exposure 140 6.4.3 Injury in 17616-pTOM31c caused by TCE exposure 144 6.5 Summary , 145 CHAPTER 7 - Conclusions and Future Work . 1 4 7 7.1 Conclusions 147 7.2 Future Work 149 REFERENCES CITED 151 APPENDICES 164 Appendix A TFMP Assays 165 Appendix B Calculated TCE Henry’s Law Constant 168 Appendix C Calculation for TCE specific activity 169 Appendix D Calculation of acetate specific growth rate 171 Appendix E Protein assay calibration curves 172 Appendix F Phenol assay calibration curve 173 Appendix G HPLC phthalate calibration curve 176 Appendix H TCE calibration curve for G.C.-ECD method 176 Appendix I Acetate calibration curve for I.C. method 177 xii Page Xiii LIST OF TABLES Table I - Applications of Biofilms In Reactor Based and 15 Insitu Bioremediation Processes. Table 2 - Factors Affecting Plasmid Stability and Retention. 24 Table 3 - Basal Salts Mineral Medium (BSM) Formulation. 45 Table 4 - Hydrocarbon Minimal Medium (HCMM2) Formulation. 46 Table 5 - Substrates and Their Uses in the Studies Presented, 48 Table 6 - Plating Methods For Cell Enumerations. 73 Table 7 - Results from Continuous Culture Plasmid Retention 105 and Activity Experiments. Table 8 - Results from Biofilm Plasmid Loss and Activity 116 Experiments. Table 9 - Plasmid-free Cell Concentrations and Fractions 141 of Plasmid Bearing Cells in the Test and Control Non-selective Fed-batch reactors. Page xiv LIST OF FIGURES Figure 1 - Restriction Map of Plasmid pTOM31c. , . 1 1 Figure 2 - Restriction Map of Plasmid pMS64, Derived from pTOM31c. 11 Figure 3 - Genesis of the Constitutive TCE Degrader 12 Burkholderia cepacia PR1 - pTOM31c Via Tn5 Transposon Insertion. Figure 4 - Apparent Losses of TCE Degrading Phenotype. 17 Figure 5 - Methods of Plasmid Transfer. 30 Figure 6 - Detailed Restriction Map of Plasmid pTOM31c. 43 Figure 7 - TFMP Colorimetric Assay Used to Determine the 52 Expression and Specific Activity of the TOM Pathway. Figure 8 - Diagram of Lab Scale Bioreactor Columns. 55 Figure 9 - Diagram of Field Scale Vapor Phase Bidreactor (VPBR) 57 System. Figure 10 - Diagram of Chemostat System Used for Plasmid 62 pTOM31c Stability and Activity Studies. Figure 11 - Diagram of the Annular Reactor System 63 Used for Biofilm Culture Plasmid pTOM31c Activity and Loss Studies. Page XV Figure 12 - Diagram of Fed Batch, TCE Vapor Continuous 65 Flow Reactor Used to Examine the Affects of TCE Exposure. Figure 13 - Monod Growth Kinetics for PR1-pTOM23c on 77 Non-selective Phthalate Medium. Figure 14 - Degradation of TCE by PR1-pTOM23c. 79 Figure 15 - Phthalate Concentrations in the Lab Scale Biofilm 81 Column Reactor Over Time. Figure 16 - Total Heterotrophic Cell Counts and PR1-pTOM23c 82 Cell Counts In the Lab Scale Biofilm Reactor Column. Figure 17 - Total Heterotrophic Cell Counts in the Field 85 Scale Test VPBR Column. Figure 18 - Biofilm Protein Content in the Field Scale . 86 Test VPBR Column. Figure 19 - Total pTOM Activity in the Field Scale Test VPBR Column. 87 Figure 20 - pTOM Specific Activity in the Field Scale Test 88 VPBR Column. Figure 21 - Vapor TCE Concentrations Throughout the Field Scale 90 VPBR Test Column. LIST OF FIGURES - Continued Page xvi LIST OF FIGURES - Continued Figure 22 - Andrews Substrate Inhibition Growth Kinetics For 17616- pTOM31c Growing On Non-selective, Non-competitive Acetate Medium. Figure 23 - TCE Disappearance for pTOM-free and pTOM-bearing 17616 and PR1 Strains. Figure 24 - Monod Kinetics For TCE Mineralization by 17616-pTOM31c. Figure 25 - Linear Relationship Between Growth Rate and pTOM Specific Activity. Figure 26 - Plasmid-bearing Cell Fractions, Total TFMP Specific Activities, and True TFMP Specific Activities Versus Time for an Acetate Fed Continuous 17616-pTOM31c Culture. Figure 27 - Plasmid-bearing Cell Fractions, Total TFMP Specific Activities, and True TFMP Specific Activities Versus Time for an Acetate Fed Continuous 17616-pTOM31c 93 Page 97 98 100 102 103 Culture. Figure 28 - Plasmid-bearing Cell Fractions, Total TFMP Specific 104 Activities, and True TFMP Specific Activities Versus Time for an Acetate Fed Continuous 1761 G-PTOM31c Culture. Figure 29 - Plasmid Loss Factor Versus Growth Rate For 106 17616-pTOM31c Grown Under Non-selective Continuous Culture Conditions. Figure 30 - Biofilm Population Dynamics During the 4.8 mM 110 Acetate Feed, Rotating Annular Reactor Showing Plasmid-bearing, Plasmid-free, and Total Cell Numbers. Figure 31 - Biofilm Population Dynamics During the 8.2 mM 111 Acetate Feed, Rotating Annular Reactor Showing Plasmid-bearing, Plasmid-free, and Total Cell Number. Figure 32 - Plasmid-free Cell Fractions in Effluent and 112 Biofilm Cultures of Both the 4.8 Acetate Feed and 8.2 mM Acetate Feed Rotating Annular Reactors. Figure 33 - Plasmid-bearing Biofilm Cell Fractions and 113 xvii LIST OF FIGURES - Continued Page Biofilm pTOM Specific Activities Found In 4.8 mM Acetate Feed Rotating Annular Reactor. xviii Figure 34 - Plasmid-bearing Biofilm Cell Fractions and 114 Biofilm pTOM Specific Activities Found In 8.2 mM Acetate Feed Rotating Annular Reactor. Figure 35 - Plasmid-selective Test and Control Reactor Total 120 Viable Cell Counts and Fractions of, 17610-PTOM31c Cells Suffering from Toxicity Incurred by TCE Exposure. Figure 36 - Total Viable Cell Counts and Selective Phenol-Km 121 Cell Counts for TCE Test Plasmid Selective Reactor with the Fraction of Injured 17616-pTOM31c Cells. Figure 37 - Ratios of Total Viable Cell Counts and Total 122 Kanamycin Resistant Cell Counts For Both The Test and Control Plasmid-selective Reactors. Figure 38 - Total and True pTOM Specific Activities of the 123 Test and Control Plasmid-selective Cultures. Figure 39 - Total Viable Cell Concentrations for Both the 125 Test and Control Non-selective Reactors Showing Fraction of Toxicity Incurred by Exposure to TCE. Figure 40 - Plasmid-bearing Cell Fractions in the Non-selective 126 Test and Control Fed-batch Reactors. LIST OF FIGURES - Continued Page xix Figure 41 - Total and True pTOM Specific Activities of the 127 Non-selective Test and Control Fed-Batch Reactors. Figure 42 - Biomass and pTOM Specific Activities of B. cepacia 133 PR1 -pTOM31c and P. cepacia 17616-pTOM31c During Batch Growth on 10 mM Acetate-HCIVIIVI2 Medium. Figure 43 - True pTOM Specific Activities Versus Growth Rate for 136 Both Chemostat and Biofilm 17616-pTOM31c Cultures. Figure 44 - Plasmid Loss Factors Versus Growth Rate for Both 138 Chemostat and Biofilm 17616-pTOM31c Cultures. Figure 45 - Percent Toxicity Incurred in Both the Selective and 143 Non-selective Fed-batch Studies at Day 15 (~28 uM TCE) and Day 18 (~280uM TCE). LIST OF FIGURES - Continued Page ABSTRACT Trichloroethylene (TCE) is a United States Environmental Protection Agency Priority Pollutant. TCE is mutagenic and a suspected carcinogen. TCE is recalcitrant in the environment and has been found to be ubiquitous in soils and ground waters, globally. Use of TCE degradative pathways moderated by enzymes, the expression of which redides on recombinant plasmids, to biologically degrade TCE is a promising new technology to eliminate TCE. One such TCE degradative pathway is the toluene ortho-monooxygenase (TOM) pathway which is borne on the recombinant plasmid pTOM. Research presnted here examines the ability of pTOM pathway to degrade TCE in two different host cells, the original plasmid host Pseudomonas cepacia PR1 and a transconjugant Pseudomonas cepacia 17616. The goal of this research was to determine those mechanisms that cause the loss of the TCE degrading phenotype, associated with pTOM, in both suspended and biofilm cultures. These mechanisms include plasmid instability within the host cell, cell toxicity caused by TCE exposure, and cell injury caused by TCE exposure. In addition this research developed a protocol to determine the severity of these phenotypic losses in any pTOM host organism. With the use of several novel reactor systems, analytical methods, and protocols were developed to determine the suitability of P. cepacia 17616 as a host for plasmid pTOM. Batch studies indicate that P. cepacia 17616 is able to incorporate pTOM and degrade TCE at rates equal to those of other pTOM hosts. Plasmid stability experimental results showed significant segregational plasmid loss in P. cepacia 17616-pTOM in non selective suspended and biofilm cultures, resulting in almost complete loss of plasmid-bearing cells within 30 days of operation. Continuous culture plasmid loss studies showed that the probability for plasmid loss per cell generation was a function of growth rate and ranged from 0.025/generation at a growth rate of 0.065 hr"1 to 0.035 at a growth rate of 0.17 hr"1. Results indicate there was no significant difference in the probability of plasmid loss between suspended and biofilm culture, suggesting that biofilm growth does not affect plasmid stability the P. cepacia 17616-pTOM system. Use of a novel TCE vapor exposure reactor system showed the TCE exposure can cause serious injury and toxicity to P. cepacia 17616 and can result in catastrophic loss of the TCE degrading phenotype in P. cepacia 17616- pTOM cultures. In addition, the severity of both cell injury and toxicity was found to be a function of TCE exposure time and TCE concentration. Research here indicates that plasmid loss, cell injury, and cell toxicity are significant mechanisms that can result in the detrimental loss of pTOM pathway expression and these mechanisms should be considered in any plasmid mediated catabolism of a toxic waste. I Chapter 1 Goals Aod Objeetiiwes 1.1 Goal The goal of this work is to determine the fate and activity of pTOM31c in the transconjugant host Pseudomonas cepacia 17616 under non-selective and "PTOM31c-SeIective" growth conditions in both suspended and biofilm cultures in order to ascertain the host’s applicability to a TCE biofilm reactor. 1.2 Thesis Statements Hypothetically, the stability and activity of plasmid pT0M31 c in the transconjugant host, Pseudomonas cepacia 17616, may be different in suspended versus biofilm culture. This research project will determine the stability and activity of plasmid pTOM31c in the transconjugant host Pseudomonas cepacia 17616, in both suspended and biofilm cultures. \ Further, this project hypothesizes that TCE toxicity, plasmid stability, and injury caused by TCE exposure will result in the significant loss of the TCE degrading phenotype of B. cepacia 17616-pTOM31c. 2 1.3 Motivations This research was initiated to determine the applicability of PTOM31c to a TCE biofilm reactor, and determine the causes for the apparent loss of the TCE degrading phenotype in both suspended and biofilm cultures. Further, this research was concieved in order to develop an understanding of the extent and severity of phenotypic losses in caused by plasmid/host interactions and the exposure to TCE. Finally, there exists a need to will establish a protocol for determining toxicity, plasmid loss, and injury in pTOM31c bearing microorganisms. 1.4 Objectives ■ Determine growth characteristics for plasmid bearing and plasmid free P. cepacia 17616 cultures under non-selective (acetate) and selective (phenol) growth conditions. ■ Determine the plasmid loss coefficient for pTOM31c in P. cepacia 17616 under non-selective (acetate growth) conditions in suspended and biofilm cultures. ■ Determine the activity of plasmid pTOM31c in suspended and biofilm P. cepacia 17616 cultures when grown under selective and non-selective conditions. 3 ■ Determine the cometabolic TCE degradation kinetics of P. cepacia 17616- pTOM31c cultures and the effect of TCE exposure on the metabolic activity and health of 17616-pTOM31c. 4 Chapter 2 Dinifiredluetioini Contamination of soil and groundwater by organic pollutants has been the focus of much research in recent years. Some of the most notable contaminants in America’s aquifers are volatile organics, a large number of which are chlorinated aliphatic compounds. Trichloroethylene (TCE - CICH=CCI2) is a chlorinated ethene and a member of the chlorinated aliphatic family. Often used as a degreaser, TCE has remained a popular solvent and is used by a great many industries (Westerick et al 1990). TCE is a U.S. Environmental Protection Agency priority pollutant and is one of America’s most ubiquitous and recalcitrant groundwater contaminants (Love and Eilers 1982). 2.1 General TCE Biodegradation Many methods have been developed for remediating TCE laden soil and groundwater. The most common technologies are (1) volatilization into the atmosphere, (2) pump and treat methods using physicochemical processes, and (3) incineration. Many of these technologies involve transfer of the pollutant 5 from one phase or state to another and do not involve actual destruction of the contaminant. This fact, along with the legalities, inefficiency, and cost of these non-biological TCE treatment technologies, have prompted intense research into the biodegradation of TCE (Travis and Doty, 90). 2.2 Anaerobic TCE Biodegradation Although TCE is quite recalcitrant in nature, a number of anaerobic bacteria have been found capable of its degradation. These anaerobes include: the methanotroph Methylosinus trichosporium OB3b (Oldenhuis et al 89), a number of propane-utilizing Mycobacterium (Wackett et al 89), two isoprene-utilizing Alcaligenes denitrificans (Ewers et al 90), the autolithotroph Nitrosomonas europaea (Arciero et al 89) and two consortia of methanogenic bacteria (Fogel et al 86, Vogel and McCarty 85). Each of these anaerobic TCE degraders utilizes different oxygenases to carry out reductive dechlorination of TCE, which often results in the production of the highly recalcitrant and mutagenic vinyl chloride (Vogel and McCarty 85). Also, anaerobic biodegradation of TCE can be as much as ten fold slower and less efficient than aerobic biodegradation processes (Bouwer and McCarty 83, Bouwer et al 81, Freedman and Gossett 89, Kleopfer et al 85). For these reasons, researchers have been searching for aerobic cultures capable of degrading TCE. 6 2.3 Aerobic TCE Biodegradation In recent years, a number of bacterial consortia and isolates capable of aerobic degradation of TCE have been discovered. Some of the more widely known aerobic TCE degraders include: a heterotrophic consortia (Fliermans et a! 88), Pseudomonas putida F1 (Nelson et al 87) (Wackett and Gibson 88), Pseudomonas mendenciha (Winter et al 89), Pseudomonas pickettii PK01 (Kaphammer et al 90), Pseudomonas florescens (Vandenbergh and Kunka 88), Pseudomonas putida B5 (Nelson et al 88), and Burkholderia cepacia GA (Nelson . et al 86). The majority of these aerobic TCE degraders utilize a toluene oxidizing pathway to degrade TCE. Most notable of these toluene oxidizing TCE degraders are P. putida Fl and B. cepacia G4. The biochemistry and TCE degradative pathway of S. putida Fl have been well characterized (Finette et al 84; Gibson et al 82; Subramanian et al 85; Wackett and Householder 89; Wackett and Gibson 88). In addition, the TCE degradative capabilities of G4, and the novel pathway by which it is performed, have received significant research attention (Nelson et al 86,87,88; Folsom and Chapman 91; Folsom et al 90; Shields and Reagin 92; Shields et al 95). Rates of aerobic TCE biodegradation obtained from batch reactor studies have been reported for a number of microorganisms. Monod TCE degradation kinetics for the inducible Burkholderia cepacia GA include a maximum specific activity, Vmax, of 8 nMol TCE/min-mg protein and a half saturation constant, Km, of 4 uM TCE (Folsom et al 90). InitiaIvTCE degradation rates for M. 7 trichosporium 0B3b, B. putida F1, and B. cepacia G4 PR1-pTOM23c were found to be 35 nIVIoI TCE/min-mg protein at 80 uMTCE, 1.8 nIVIoI TCE/min-mg protein at 80 uM TOE, and 1 nIVIoI TCE/min-mg protein at 20 uM TOE, respectively (Tsien et al 89, Wackett and Gibson et al 88, Shields and Reagin 92). These V published TCE degradation rates differ greatly because many of the microorganisms utilize different toluene oxygenase systems. In addition, TCE degradation rates can vary greatly depending on the growth rate of the cells and the carbon/energy source used. It is difficult to determine which microorganisms are most efficient in degrading TCE because all of the physiological and I environmental variables present during the respective studies. 2.4 Burkholderia cepacia;G4 Resent research has been performed on the environmental isolate Burkholderia cepacia G4 to determine the mechanism by which it mineralizes TCE. Results show that B. cepacia G4 degrades TCE via a plasmid-borne cometabolic pathway that must be induced by one of the following: phenol, toluene, m-cresol, o-cresol, or catechol (Folsom et al 90). In addition, a novel pathway involving the toluene degradation pathway has been discovered (Nelson et al 87, Shields et al 95). This pathway involves the sequential hydroxylation of toluene at the ortho- and meta- positions to form 3- methylcatachol (Shields et al 89). Further, research has shown the involvement 8 of a plasmid borne sequence that encodes for toluene ortho-monooxygenase (tom) in the TCE mineralization process (Shields et al 91). Like most aerobic TCE degraders, B. cepacia G4 utilizes a cometabolic pathway to degrade TCE. "Cometabolism" is defined as the metabolic transformation of a substance (TCE) while a second substance serves as the primary energy and/or carbon source (Brock and Madigan 88). This definition of cometabolism is reserved for aerobic microorganisms only, and must involve oxygenase enzymes and the depletion of oxygen during the cometabolic process (Dalton and Stirling 82). Along with a co-metabolite, B. cepacia G4 and most other aerobic TCE degraders also require an inducer to activate the genes responsible for cometabolic TCE degradation. Induction of these cometabolic systems is typically by the primary carbon source/co-substrate, which is usually an aromatic compound that can induce the toluene oxidizing pathway required for TCE degradation. A number of published papers have demonstrated the efficiency of B. cepacia G4 in degrading TCE under various conditions using the appropriate inducers (Folsom and Chapman 91, Folsom et al 90, Ensley and Kurisko 94, Landa et al 94). Results from these studies show that both toluene and phenol can be used effectively as inducers/co-substrates for TCE degradation. However, both toluene and phenol demonstrate competitive inhibition with TCE which can adversely affect the TCE degradation process (Folsom et al 90). The competitive inhibition demonstrated with these inducers/co-substrates, along 9 with the need for induction of the cometabolic TCE pathway by an aromatic compound, has led researchers to derive a TCE constitutive strain of B. cepacia G4. 2.4.1 Plasmids and TCE degradation Plasmids are circular, double stranded, extrachromosomal DNA sequences that are not essential for cell growth and have no extracellular form. Plasmids can be transmitted from one cell to another via replication, transconjugation, transduction, or transformation. Not only are plasmids self- replicating within the cell, but many cells carry multiple copies of their plasmids. Most aerobic TCE degrading microorganisms carry the genes responsible for TCE degradation on a plasmid. In addition, these plasmids usually code for expression of the cometabolic toluene oxidizing pathway proteins that must be induced by an aromatic compound, the primary metabolite. One such plasmid is the large plasmid found in previously mentioned environmental isolate B. cepacia G4. 2.4.2 Plasmid pTOM31c The plasmid PTOM31c (Figures 1 and 2) is a transmissible plasmid that includes gene sequences which encode for all of the proteins needed for constitutive mineralization of TCE via a cometabolic pathway; the newly defined TOM (toluene ortho-monooxygenase) pathway. These proteins include toluene 10 ortho-monoxygenase (TomA) and catechol 2,3 dioxygenase (C230 or TomB) (Shields et al 95). Plasmid pTOM31c also includes a TnS transposon insertion which is a transposable DNA sequence containing a kanamycin resistance marker (Figure 3). Two plasmid maps showing the locations of tomA, tomB (C230), and tomR (tom regulatory region), along with the areas of TnS homology, are shown in Figures 1 and 2. The TOM pathway’s involvement in the aerobic mineralization of TCE has been definitively characterized in the literature (Shields and Reagin 92, Shields et al 95). Plasmid pTOM31c could have a number of important applications in the field of bioremediation. For example, this plasmid could be transferred into indigenous bacterial populations to increase the TCE degradative activity of in- situ bioremediation, or it could be applied to highly effective reactor-based remediation technologies like TCE degrading biofilm reactors. 11 Figure I - Restriction Map of Plasmid pTOM31c. SfrwrI BamHI NJwI SmaI \ I SmaI BamHlV NhnI . NhnI . BamHI * SmaI ^ BamHI I I tom operon 7 RegionofTnS homology TOlVfclC 114 Kb RegionofTnS homology BamHI Figure 2 - Restriction Map of Plasmid pMS64, Derived from pTOM31c Showing position of TomA1 C230, and areas of TnS homology (An EcoRI digest of PR1- pTOM31c plasmid prep cloned into pGEM4Z, Reagin 92). pGBMZ 2.75 kb Nhel Hindlll Bglll BamHI Bgllf Smal Oral Hindlll Sphl BamHI Bglll Pstl Hindlll Pstl Xbal BamHI Smal Kpnl EcoRI Pstl Kpnl Figure 3 - Genesis of the Constitutive TCE Degrader Burkholderia cepacia PR1-pTOM31c Via TnS Transposon Mutagenesis Environm ental Isolate B. Cepacia G4 TnSTransposon Kanamycin (-) Phenol Revertants 52315223 52275220 TCE-Constitutive TCE-ConstitutiveC 2 3 0 pTOM31c Km(-) Tn&pTOM31c Chromosomal DNA Km(-) TnS Chromosomal DNA B. cepacia G4 - RR 123c - (pTO M 23c) B. cepacia G 4 - PR131c - pTOM31 c 13 2.4.3 B. cepacia PR1-pT0M31c B. cepacia PR1-pT0M31c is a transposon mutant of the environmental isolate B. cepacia G4. Like B. cepacia G4, B. cepacia PR1-pTOM31c is capable of aerobic TCE degradation via the cometabolic TOM pathway. 6. cepacia PR1-pTOM31c was obtained by the insertion of a TnS transposon into the large plasmid of 6. cepacia G4, then re-inserting plasmid pTOM31c into B. cepacia G4. The resulting phenol revertants are capable of aerobic TCE degradation via a constitutive cometabolic pathway (refer to Figure 3) (Shields et al 91) without an aromatic inducer and without the possible detrimental effects of competitive inhibition. In addition, the simple TnS insertion resulted in a G4 mutant carrying the plasmid pTOM31c, which includes all of the constitutive TCE degradative pathway and. the kanamycin resistance associated with the TnS transposon (Shields et al 95). 2.5 Biofilms and Biofilm Reactors: Most bacteria found in nature are associated with a surface (Marshall 76) Surface-associated bacteria are physiologically different than suspended bacteria and can be found embedded in an extracellular polysaccaridic (EPS) matrix. Interactions between attached cells and their environment are strongly affected by mass transfer effects, hydrodynamics, and other transport phenomena (Characklis and Marshall 90). Surface-associated bacteria have been designated as biofilms (Costerton et al 87). Biofilms include all surface 14 associated microorganisms in any environment, and are considered a unique and relatively new area of microbiological/bioengineering research. Biofilms can be both detrimental and beneficial. Some examples of detrimental biofilms include biofilms that mediate corrosion of pipes in water and oil distribution systems, biofilms that cause fouling of cooling towers and heat exchangers resulting in inefficiency and increased pressure drop, and biofilms that infect the human body and biomedical prosthetics which are difficult and/or impossible to kill. However, biofilms can also be beneficial. Biofilms that remove pollutants from water and wastewater have been used for hundreds of years. More recently, biofilms have been used in bioremediation technologies to degrade and attenuate xenobiotics found in industrial effluents, groundwaters, and soils. A list of various bioremediation applications of biofilms are shown in Table I . 2.5.1 Biofilm reactors for biodegradation of organics including TCE A number of papers have reported the use of anaerobic and aerobic biofilms to degrade organic compounds. The literature is full of instances, both bench scale and field scale, where biofilm reactors of one type or another have been used to treat simple aromatic pollutants such as phenol, cresol, and the less volatile gasoline components benzene, toluene, ethyl-benzene, and xylene (BTEX). There are only a few examples of biofilm reactors being used successfully to treat TCE laden effluents. Speital and Leonard (92) used a 15 Table 1 - Applications of Biofilms In Reactor Based and Insitu Bioremediation Processes Bioremediation Need Biofilm Process Application 1 - Insitu removal of xenobiotics from soil and groundwater {in- situ) Bacteria associated with soil surfaces are considered a biofilm and may be capable of degrading various xenobiotics (BTEX, TCE, PAHs, etc.). Various techniques can be employed to enhance the degradative capabilities of the indigenous bacteria (addition of oxygen, nutrients, co-substrate). 2 - Reactor based technology for removal of xenobiotics in groundwater. Pump and treat systems where the removed ground water is treated using engineered biofilm reactor's (packed columns, fluidized bed reactors, and rotating disk biofilm reactors). 3 - Removal of metals from industrial waste streams and production effluents. Reactor based biofilm processes with metal binding bacteria colonized on packing surface. 4 - Removal of organics, nitrate, sulfate, and ammonia from waste waters. Trickling filters, activated sludge reactors, and fluidized bed reactors used in community and industrial waste water treatment facilities to maintain effluent water quality. 5 - Treatment of volatile organics in vapor waste streams, soil vapor extraction processes, concentrator effluents, and soil venting technologies. Vapor phase biofilm reactors using engineered biofilms grown on inert packing. Biofilters using surface associated indigenous bacteria present on peat, compost, and other naturally occurring media. 6 - Biobarriers Introduction of large quantities of bacteria down an injection well to develop a cylindrical biofilm "plug" radiating from the well screen. Used to attenuate polluted groundwater flow. 7 - Bioaugmentation Introducing selectively enriched microorganisms into the subsurface to enhance biodegradation capabilities of attached microorganisms. 16 anaerobic biofilm reactor to treat TCE. Fennell et al (93) used a methonotrophic attached film expanded bed reactor to degrade TCE at a maximum rate of 0.8 mg TCE/g VSS-day. There are a few other accounts of anaerobic biofilm reactors used to treat TCE (Strandberg et al 1989). However, most of these processes involve slow growing and low yield anaerobic cultures that do not readily produce a biofilm. In addition, many of the anaerobic TCE biodegradation rates are extremely slow and would be impractical for long term use in reactor based bioremediation applications. Researchers have been trying to apply aerobic TCE degraders to biofilm reactors with limited success and little literature on aerobic TCE degrading biofilm reactors can be found. A few attempts have been made to utilize biofilm reactors to treat either liquid and vapor TCE wastes using pure cultures of TCE degrading .microorganisms. Some of these attempts have had limited success during short term operation. However, when these systems were used for long term treatment of TCE waste, many lost their ability to degrade TCE at a significant rate. This apparent loss of TCE degrading ability was noted by decreasing efficiency, lack of biomass accumulation, and failure to degrade TCE. There are a number of explanations for such failures. Figure 4 enumerates phenomena to which the apparent loss of a plasmid borne TCE degrading phenotype of a particular microbial population can be attributed. These phenomena include toxicity, plasmid instability, and cell injury. In addition, mono- and engineered mixed culture systems operated under field or Figure 4 - Apparent Losses of Plasmid Borne TCE Degrading Phenotype PHENOTYPE LOSS MECHANISMS Toxicity Cell Lysis TCE Toxicity Intermediate Toxicity Segregational Recombination Injury Induced Injury Deactivation Inhibition Catabolic Repression 18 non-sterile conditions can suffer from competition and predation by invading microorganisms. Other TCE degrading microorganisms may be washed out of the biofilm system due to both their inability to attach to the. reactor packing and their inability to produce a significant biofilm. The key to developing an efficient, reliable TCE degrading biofilm reactor is finding a suitable host to harbor and express the desired TCE degrading plasmid phenotype. Suitable host characteristics include: ability to produce copious amounts of biofilm, resistance to TCE related injury and toxicity, and ability to retain and express plasmid during long term operation and other favorable plasmid-host interactions. 2.6 Loss of TCE Degrading Phenotype - Toxicity, Plasmid Stability, and Cell Injury. Loss of a TCE degrading phenotype in TCE degrading biofilm processes has been noted by many researchers. Many times, the performance of TCE degrading biofilms in long term application is not as effective as observed in short term experiments. The lack of efficiency can be attributed to scale-up factors and poor reactor design. However, many times it is a decline in the health and activity of the TCE degrading biofilm cells that is affecting reactor performance. When TCE degrading cells are grown in a biofilm and exposed to TCE for a prolonged period of time, a number of physiological changes can 19 occur that result in the loss of the desired TCE degrading phenotype. Such physiological changes include toxicity, plasmid instability, and injury. 2.6.1 Toxicity Death or permanent inactivation of a given microbial population can occur when the population is exposed to a toxic substance either in a sufficiently high concentration or for a long enough period of time to fatally affect the function of the cells. Toxicity can involve severe damage to the cell wall and cell membrane which in turn can cause cell lysis and loss of the cell's structure and function. Cell lysis is usually caused by anti-microbial agents, strong solvents, endogenous proteolytic enzymes, strong acids, or highly reactive substrates dissolving the cell wall. Toxicity can also involve permanent damage to a desired phenotype or set of genes within the cell’s chromosomal or accessory DNA. This form of toxicity may be caused by the toxin itself or the production of harmful intermediates or by-products. Toxicity of this nature is very common in TCE degrading microorganisms. Intermediate and product toxicity among TCE degrading methane oxidizing cultures has been noted by Alvarez-Cohen and McCarty(91 a and 91b) and Janssen et al (87). This type of toxicity results in decreased TCE degradation along with decreased methane conversion, thus leading one to believe that the whole pathway for methane oxidation (and TCE degradation) has been damaged (Ely et al 95). Other accounts of intermediate toxicity among phenol 20 and methane oxidizing TCE and DCE degraders (Bielefeld et al 95) and toluene oxidizing biofilm cultures ( Arcageli et al 95) have been presented in the literature. Toxicity is an obvious concern when designing TCE biofilm reactors. Unlike cells in continuous flow stir tank reactors or batch stir tank reactors, biofilm cells are continuously exposed to a relatively steady, low level concentration of TCE. Some environmental and nutrient conditions may decrease intermediate and product toxicity effects; but ultimately, the degree of TCE related toxicity will be a function of the TCE concentration, toxigenicity to the cell, and the pathway utilized to degrade TCE. 2.6.2 Plasmid Instability Plasmids may place a metabolic burden upon their host cell because of the energy needed for their maintenance and replication. This burden may or may not be significant depending on the plasmid/host relationship and the total amount of accessory DNA the cell is maintaining. Depending on the growth conditions, plasmids may increase the fitness of the cell, even if those growth conditions do not select for the plasmid (Bouma and Lenski 88, Zund and Lebek 80). The interactions between a given plasmid and its host can not be generalized. Each plasmid-host relationship has unique characteristics and 21 differs from one plasmid type to another in the same cell. Plasmid-host relationships can be strongly influenced by growth and environment conditions. Microorganisms carrying plasmids are susceptible to plasmid instability which can lead to the loss of a desired plasmid-borne phenotype, or complete loss or inactivation of a desired plasmid-borne genotype. Plasmid instability can occur in two ways: (1) segregations! instability and (2) structural instability. Segregations! instability is the consequence of random and irregular separation of a plasmid between daughter cells during cell division, and leads to new generations of daughter cells that do not contain the plasmid. The plasmid free daughter cells produced by segregational instability will have lost all of the genotypes/phenotypes carried on that plasmid, including all selection markers, anti-microbial resistant sequences, and any amended DNA sequences such as transposons and other insertion sequences. Structural instability of a plasmid involves the actual change or recombination (deletion, insertion, and rearrangement) of a single gene or several genes in the plasmid. Structural instability can result in a portion of the plasmid DNA being incorporated into the chromosomal DNA. In addition, structural instability may involve the loss of a certain plasmid borne phenotype, but not the loss of other phenotypes carried by the plasmid. For instance, structural instability of plasmid pTOM31c may result in the loss of the TCE degrading phenotype, but not the loss of the plasmid borne kanamycin resistance. Since most TCE degrading microorganisms carry their 22 TCE degradative ability on plasmids, plasmid stability will be a major factor affecting the performance of TCE bioreactors. Plasmid stability in suspended culture Due to increased environmental concerns and the promise of genetic manipulation in biotechnology related industries, much research on the stability of plasmids in suspended cell cultures has been performed (Sherrat 82, Ensley 86, Ollis 82, Noack et al 82). Such research has focused on determining the factors that govern plasmid stability and expression in well controlled suspended pure culture systems (Grand! et al 81, Kumar et al 91, and Seo and Bailey 85). In addition, a number of kinetic models have been developed to quantify and predict plasmid loss in suspended culture (Summers 91). Researchers have found that plasmid maintenance can reduce the overall growth rate of cells in continuous culture (Uhlin and Nordstroml 978, Peretti and Bailey 87). While Chao et al (83), and Bouma and Lenski (88) note instances where plasmid maintenance did not result in a growth rate disadvantage relative to the plasmid bearing cells. Results indicate that plasmid maintenance can actually enhanced cell health even under non-selective conditions. Researchers have measured significant segregational plasmid loss under both non-selective (Grand! et al 81, Kadam et al 87) and highly selective (Peretti et al 89, Wood and Peretti 91, Roth et al 80) continuous growth conditions. Noack et al (82) have shown structural instability of plasmid pBR325 in E. coli 23 cultures where certain antibiotic resistances are lost at a slower rate than other plasmid borne antibiotic resistances. Other researchers have found that plasmid loss can be decreased or eliminated by using either a selective growth substrates or antibiotics that are selective for the plasmid (Tiedji et al 89). Dyhuizen and Hartl (83) review the effects of continuous culture growth on plasmid stability and expression and suggest that continuous culture may either enhance or deter plasmid stability depending on the plasmid/host system and the growth factors involved. Dwilvedi et al (82) found plasmid stability was increased in continuous E. co//cultures, while stability significantly decreased when the cultures were grown in batch. Additionally, certain methods for increasing plasmid stability require continuous culture dynamics (Primrose et al 84, Roth and Noack 82). It is obvious that the plasmid-host relationship is unique for any given plasmid-host system; however, a number of environmental and physiological factors have been found to influence plasmid stability that may have some general implications. A list of these factors are.presented in Table 2. Kumar et al (91) present a review of strategies for improving plasmid stability. Many of these strategies include cellular/molecular techniques used either to control plasmid partitioning during cell division (Austin 81, Summers and Sherrat 84) or to kill plasmid free cells after segregation (Lauffenberger 87, Gerdes 88, and Rosteck and Hershberger 83). Other plasmid stabilizing strategies include bioprocess control strategies to separate plasmid free cells from the culture or to 24 Table 2 - Factors Affecting Plasmid Stability and Retention Environmental and Physiological Factors References Growth R ate - Increases plasmid loss with Stewart and Carlson 86 increased growth rate. Taxis Du Poet 87 Seo and Bailey 86 P lasm id co p y nu m b er - decreased plasmid Jones et al 80 loss rate with increased plasmid copy number. Plasmid copy numbers can range from 1 to 700. Sayadi et al 89 Uhlin and Nordstrom 79 Carbon to n itrogen ratios - Increased nitrogen growth conditions can increase plasmid stability. Huang et al 94 Sayaldi et al 89 Selection - Selection of plasmid using a selective carbon source or antibiotic resistance markers can increase plasmid retention in a given population. Lauffenberger 87 Tiedje et al 89 Wood et al 90 N utrient L im itations - Nitrogen, phosphorous, Godwin and Slater 79 potassium, magnesium, and carbon limitations may either increase or decrease plasmid Jones and Melling 84 Jones et al 80 stability. Noack et al 82 Im m obilization a n d A ttachm en t - plasmid bearing populations that are immobilized or in a biofilm culture may display either increased Huang et al 93 Kumar and Schugerl 90 Inloes et al 83 or decreased plasmid stability. Dykhuizen and Hartl 83 Exposure to in jurious o r toxic substances - injury and toxicity may lead to increased plasmid loss. Ridgway 94 ■ 25 inhibit the growth of plasmid free cells (Stephens and Lyberatos 92, Siegel and Ryu 85, and Henry et al 90). Plasmid stability in bio films When compared to suspended cultures, biofilm and immobilized cells may experience increased plasmid stability for two reasons: 1) the proximity of immobilized and biofilm cells to one another may improve intra-cellular transfer mechanisms and 2) the mass transfer limitations in biofilm and immobilized cell systems. Because immobilized and biofilm cells exist in close proximity to one another, the likelihood of plasmid transfer by conjugation could be increased. Thus the net rate of plasmid loss may be decreased when the number of plasmid transfers from plasmid bearing cells to plasmid free cells is included. Due to mass transfer limitations, there is the tendency for growth rate gradients to develop within immobilized and biofilm cultures systems. Since plasmid loss has been shown to be a function of growth rate, the cells experiencing a slower growth rate due to nutrient and carbon source.limitations may also experience decreased plasmid loss. Inloes et al (82) have reported increased plasmid stability in E. co//cultures immobilized in a non-selective hollow fiber system. Taxis du Poet et al (86) saw increased stability of plasmid pTG201 in k-carrageenan encapsulated E. coli cultures. These results are attributed to mass transfer effects resulting in slower growth rates, the possibility of increased plasmid transfer, and the reduction of 26 structural instabilities. Similar results using different plasmid/host systems and different immobilization techniques have been reported by Nash et al (87), Sayadi et al (89), and Kumar and Schrugel (90). In biofilm cultures, a number of researchers have found significant plasmid transfer that may result in a net decrease in plasmid loss ( Levin et al 79, Stewart and Carson 86, Saye et al 87). In contrast to these reports, Huang et al (93) found decreased stability of plasmid pMJR1750 in E. coli DHSa when grown in a biofilm. This decrease was attributed to copy number differences between suspended and biofilm cultures and the energy needed for production of the biofilm extracellular matrix polymers that might compete for plasmid maintenance/replication energy. As with suspended plasmid bearing cultures, biofilm plasmid bearing cultures have their own unique plasmid/host characteristics. In order to utilize certain recombinant plasmids in industrial microbial systems, specific plasmid/host interactions must be well understood irrespective of whether the system involves suspended or attached cells. 2.6.3 Cell injury caused by exposure to toxic or injurious substances The word injury is generally used to explain the loss of a given phenotype under certain environmental conditions. Injury differs from toxicity in that injury implies that the lost phenotype can be regained under non-selective general growth conditions. The concept of injury in microorganisms arose in the water 27 treatment industry where coliforms and other indicator microorganisms were found to be "inactivated" but not killed by traditional chlorine disinfection (Camper and McFeters 79, LeChevaIiier and McFeters 85, Waters et al 89). The key element to injury that mak^s it such an intriguing phenomena is that the cells can recover to their full pre-injury capacity. A number of challenges including: chlorine and other antimicrobial agents, specific metabolites, oxidative conditions, DNA damaging agents, and hydrocarbon exposure have been shown to cause microbial injury (Ridgeway et al 94, Arcangeli et al 95). Injury can be determined in a number of ways, the simplest of which are growth-related methods. Injured cells are non-culturable on selective media, yet they are recoverable on general growth media. By comparing the differential microbial counts on selective and non-sdlective plates, one can get an idea of the extent of injury experienced in a given microbial population (McFeters et al 86). Injury, however, is a poorly understood phenomena and to date there are no mathematical models explaining its affect on engineered microbial systems. Nevertheless, along with toxicity and plasmid instability, injury may be an important element governing the performance of a TCE biofilm reactor. 28 2.7 Plasmid Transfer The growth of biotechnology and environmental-related industries has prompted the need to exploit certain recombinant organisms possessing desired phenotypes/genotypes in a variety of microbial systems. Since the persistence and expression of a desired recombinant plasmid is uniquely dependent upon the health, maintenance, and interactions of the plasmid-host system, techniques have been developed to increase the retention and expression of a plasmid in a given microbial system. One method consists of transferring the plasmid to a more suitable host. Plasmid transfer can be done three ways: transconjugation, transduction, and transformation. Figure 5 illustrates the three plasmid transfer methods. The method most commonly used to transfer a plasmid from one microbial strain to another is transconjugation, often called " mating". In this method, a plasmid-bearing cell conjugates with a plasmid-free cell via a pilus, resulting in a new plasmid bearing cell. The new plasmid-bearing cell is called a transconjugant. Not all bacteria are capable of conjugation and not all plasmids can be transferred by this method. In order for a given plasmid to be transferred by conjugation, the donor cell must contain the proper transfer gene (tran) and the plasmid must be mobile (mobgenes). In addition, the recipient cell must be able to incorporate, maintain, and express the plasmid in order for the transconjugation to be successful. 29 2.7.1 P seudom onas cepacia 17616-pTOM31c - a transconjugant of B urkholderia cepacia PRI-PTOM31c. Over two dozen transconjugants of Burkholderia cepacia PR1-pTOM31c have been obtained. One of these transconjugants is Pseudomonas cepacia 17616- PTOM31c, which is an environmental isolate capable of producing copious amounts of biofilm (Murgel et al 91 ). The DNA content of Pseudomonas cepacia 17616 has been well characterized by Cheng and Lessie (94), and it was chosen I , as a host for plasmid pTOM31c for both its ability to produce a biofilm and its apparent health and competitiveness in open systems. 30 Figure 5 - Methods of Plasmid Transfer Transconjugation Transduction T ransformation Q Plasmid DNA Chromosomal DNA Virus Naked DNA 31 Ghapfieir 3 MathematieaD Models ( 3.1 TCE Degradation and Bacterial Growth Kinetic Models 3.1.1 Single substrate saturation kinetics Michaelis-Menten kinetics are used to describe the saturation kinetics of enzyme-catalyzed reactions. Enzyme saturation kinetics can be used to model cometabolic TCE degradation because TCE degradation is driven by the enzymes produced by the TOM pathway (Folsom et al 90, Landa et al 94, Barrio-Lage et al 87, Shamat and Maier 80). Equation 3.1 is the Michaelis- Menten enzyme saturation kinetic expression, where V is the specific activty of the enzyme system ([TCE]/min. - protein), Vmax is the maximum specific activity of the enzyme system, and Km is the Michaelis constant ([TCE]). The amount of enzyme produced in a cell is a function of growth rate, thus, since Michaelis- Menten kinetics are a function of the total amount of enzyme present, they are only valid in describing whole cell TCE degradation kinetics at a single growth rate of the cells. The Michaelis-Menten kinetic expression can be used to describe the TCE degradation kinetics of cells at a single growth rate. Monod kinetics are the whole cell growth rate analog of Michaelis-Menten specifc activity kinetics. Monod saturation kinetics have been historically used to model microbial growth that follows the same form as Michaelis-Menten enzyme saturation kinetics (Monod 49). Data for the Monod kinetic model can be obtained from either a series of batch growth studies, each initiated at a different substrate concentration or a series of continuous culture experiments, each run at a different dilution rate. The parameters used in Monod kinetics include the maximum growth or utilization rate (Mmax) and the half-saturation constant (Ks). Using methods presented in Appendix A, the specific growth rate for a given system can be determined and plotted against concentration to yield a typical Monod kinetics curve. The curve can be fit using the single substrate Monod kinetics expression presented in Equation 3.2. ( K + S ) (3-2) 3.1.2 Andrews substrate inhibition growth kinetics The Andrews substrate inhibition growth kinetics model is a modified version of the Monod kinetic model (Andrews 68). Andrews kinetics account for 33 inhibitory effects that may occur at high substrate concentrations. Using the same methodology presented in Appendix A, a series of initial growth rates can be plotted against initial substrate concentration to give a standard growth rate (p) versus substrate concentration curve. This curve can be fit using Equation 3.3 to determine the following Andrews kinetic parameters: maximum growth rate (Mmax), half-saturation Constant(Ks), and the Andrews substrate inhibition constant (Kj). From these parameters, the growth rate limit for a given set of Andrews kinetic parameters occurs at a substrate concentration equal to the square root of the product of half-saturation constant and the Andrews inhibition constant (KiltKs)172. M . Chemostat For Inoculatio Sampling Ports Phthalate HCMM2 Medium Packing Support Screen Medium Effluent/Recycle TC E/Air Influent 56 The reactor was operated in a batch fed mode with 2 liters of phthalate-HCMM2 medium which was drained and replaced every 2 days. During the two day period the medium was recycled through the column with a residence time of approximately 1 hour. The system was inoculated with 1 liter of PR1-pTOM23c cells at an A600 of I under heavy kanamycin selection. The system was not sterile because the oyster shell could not be sufficiently sterilized. Periodically the batch feed solution was amended with 40 gamma kanamycin to select for PR1-pTOM23 cells. Daily analysis of the system included: measurement of phthalate concentration, total heterotrophic cell counts in bulk fluid, selective cell counts in bulk fluid, biofilm protein measurements, and a TCE disappearance assay on biofilm/oyster shell samples. 4.6.2 Field scale column studies A field study was conducted to determine the ability of PR1-pTOM23c to be utilized in a large scale TCE vapor phase bioreactor (VPBR) packed with crushed oyster shell using both selective/inhibitory and non-selective/non- inhibitory carbon sources for start-up and operation. The reactor system was set up at a TCE concentrator plant at the Hanscom Air force Base outside Boston, Massachusetts. A diagram of the 110 liter, counter current VPBRs used in the field is shown in Figure 9. Two VPBRs were used in this study, one cell- free control reactor (uninoculated) and one test reactor. TCE vapor to be treated 57 58 was the effluent from a TCE concentrator. TCE influent was fed to the VPBRs from the bottom of the each reactor along with toluene vapor that was used as a selective growth substrate. Nutrient media and liquid growth media were introduced at the top of the columns at a rate of approximately 0.5 liters/hr. Start-up of the test VPBR involved a large volume inoculation of PR1- PTOM23c cells harvested from a continuous culture. Over 5 liters of high cell density culture were added to the system at the top of the column for inoculation. The system was also fed toluene vapor as a selective growth source to enhance start-up and PR1-pTOM23c biofilm formation. A number of different feed scenarios involving toluene vapor, phenol-BSM medium, and phthalate-BSM took place during the operation of the system. TCE vapor was introduced to the columns after 27 days of reactor start-up and inoculation. When TCE was introduced, 20 mM phthalate was fed to the reactor systems in place of the inhibitory toluene vapor. Oyster shell packing samples from both the test and control reactors were taken every, day at each of the three sample ports. The oyster shell samples were analyzed for the following: total heterotrophic cell numbers using LBG, protein content using the protein assay, pTOM activity using the TFMP biofilm assay, and PR1-pTOM23c cell number using phenol-Km-HCMM2 selective plates. Samples of the effluent liquid were also analyzed for total cell counts, protein content, PR1'-pTOM23c cell counts, and carbon source concentration. TCE vapor 59 was analyzed at the inlet, outlet, and all three of the sampling ports in each column. 4.7 Methods for Plasmid Stability and Activity Studies Using P. cepacia 17616-pTOM31c Stability and activity of PTOM31c in the transconjugant host P. cepacia 17616 was determined in both suspended and biofilm cultures under non- selective growth conditions. 4.7.1 Suspended culture plasmid stability and activity studies Batch experiments Experiments using PR1-pT0M31c and the transconjugant 17616-pTOM310 growing on non-selective acetate-HCMM2 media were carried out to compare pTOM activities, acetate growth characteristics, and pTOM31c plasmid stability between the two strains during batch growth. A series of growth studies at acetate concentrations ranging from 2-20 mM were performed. Acetate growth characteristics were determined by monitoring acetate and biomass concentrations over the duration of each complete batch growth curve (Appendix D). Plasmid pTOM31c activities were determined periodically during each batch experiment using the TFMP suspended culture assay and the TCE disappearance assay. 60 Total, plasmid-free, and plasmid-bearing cell concentrations were determined periodically throughout each batch growth study to monitor the loss of.plasmid PTOM31c in the batch 1761 G-PTOM31c cultures. Total cell numbers were determined by dilution plating on" LBG agar plates. Plasmid-free cell count to total cell count ratios and plasmid-bearing cell count to total cell counts ratios were determined using the pTOM31c selective direct-colony transfer (PSDCT) method. Continuous culture experiments Acetate fed chemostat studies were carried out to determine the stability and activity of pTOM31c in host 17616 during continuous culture. The chemostat system used is shown in Figure 10. Chemostats were run at a number of different dilution rates ranging from 0.06/hr to 0.19/hr. Steady-state values for pTOM specific activity were determined every day for each dilution rate, using the suspended culture TFMP assay to determine if pTOM activity was a function of growth rate. Steady-state plasmid-bearing, plasmid-free, and total cell counts were determined daily for each dilution rate, using the PSDCT method. Cell counts were used to monitor plasmid loss during each continuous growth experiment to determine if plasmid loss was a function of growth rate. Continuous culture plasmid loss was modeled using the Ollis model presented in Chapter 3. 61 A set of pTOM selective, phenol fed chemostat studies were performed to determine if phenol could be used to either stabilize the pTOM31c plasmid in 17616 or select for pTOM31c bearing cells in continuous culture. Phenol concentrations were measured using a colorimetric phenol assay. Biomass measurements were made using the protein assay. Plasmid loss was determined using the PSDCT method. All continuous culture experiments were inoculated with a pure culture of 17616-pTOM31c cells harvested from a highly selective starter culture. 4.7.2 Biofilm culture plasmid stability and activity studies Pseudomonas cepacia 17616-pTOM31c biofilm cultures were grown on non- selective acetate-HCMM2 media to determine the stability and activity of PTOM31c in biofilm cultures. Biofilm cultures were grown using a rotating annular reactor shown in Figure 11. Annular reactors were initially colonized under batch operation with high kanamycin selection (80 gamma or ug/ml) to insure the initial biofilm was made up of all plasmid-bearing cells. No cells were added to the system after initial inoculation. Biofilm reactors were operated at two different influent acetate concentrations, 4 mM and 10 nhM acetate-HCMM2. The annular reactors were run at a dilution rate of at least 1.0/h (at least 5 times greater than the maximum growth rate of 17616) to insure no replication of detached biofilm cells occurred in the bulk fluid phase. 62 Figure 10 - Diagram of Chemostat System Used for Plasmid PTOM31c Stability and Activity Studies. Media Influent Air Effluent Air In 0.2 um filter Humidifier Stir BarReactor Effluent 63 Figure 11 - Diagram of the Annular Reactor System Used for Biofilm Culture Plasmid pTOM31c Activity and Loss Studies. Dilution water Influent Port w Removable Slide (12) (Biofilm Substratum) Rotating Inner Cylinder Acetate/ Mineral Solution/ Buffer Cork Outer Drum Bulk Liquid I Phase Recirculation j Tube (4) Outlet 64 Protein content and acetate concentrations of effluent and biofilm samples were determined periodically throughout the biofilm experiments. Plasmid­ bearing, plasmid-free, and total cell counts in both effluent and biofilm samples from each annular reactor were determined every two days using the PSDCT method. Samples for the PSDCT method were harvested from the annular reactor slides and effluent, and prepared using the biofilm culture suspension method (BCS). Cell counts were used to determine the growth and plasmid loss characteristics of 1761 G-PTOM31c in biofilm culture. Plasmid pTOM31c specific activities of the biofilm cultures were determined using the suspended culture TFMP assay on a 5ml aliquot of the BCS sample. Results were modeled using the biofilm plasmid loss model presented in Chapter 3. 4.8 Methods for TCE Exposure Studies Using an Acetate Fed-Batch, TCE Vapor Continuous Flow Reactor to Determine Cell Injury, Toxicity and Plasmid Loss During TCE Exposure. An acetate fed batch reactor with continuous TCE vapor flow was designed to examine the effect of TCE exposure on the toxicity, plasmid-loss, and injury of 17616-pTOM31c cell cultures. The system used is shown in Figure 12. A fed batch system was used to mimic a biofilm system, in that the cells were continuously exposed to a pseudo steady-state TCE concentration (unlike a batch reactor), while they did not have a finite residence time (like in a continuous culture). Cells in the fed batch reactor system had a residence time 65 Figure 12 - Diagram of Fed-Batch, TCE Vapor Continuous Flow Reactor Used to Examine the Affects of TCE Exposure. Compressed Air Dilution TCE Vapor Out TCE Vapor Iny Humidifier 25 ml Pulse Acetate And Sampling HCMM2 Medium Mini-inert valve 150 ml Liquid Culture Compressed Air 700 uM TCE in air 66 of 6 days. Two different experiments, with the appropriate controls, were run using the fed batch system. 4.8.1 Selective TCE exposure studies The selective fed batch reactor system was operated by taking a 25 ml sample from the 150 ml liquid volume every 24 hours. The 25 ml sample was then replaced by 25 mis of 24 mM acetate-HCMM2 medium amended with 120 gamma kanamycin, resulting in a 6 hour residence time for the system. This volume extraction resulted in a concentration of 4 mM acetate/20 gamma kanamycin at the end of each batch feed. The kanamycin was used to select for 17616-pTOM31cby killing plasmid-free cells generated by segregational plasmid- loss. It was assumed that the 4 mM acetate was completely utilized within the 24 hour time period between batch feeds. In addition, the kanamycin was assumed to be utilized and degenerated to a substantially lower level between batch feeds. It was also presumed, with the combination of a 6 day residence time (dilution) and the utilization and degeneration of the kanamycin, that the concentration of kanamycin would not exceed the lethal limit for 17616-pTOM31c, which was found to be approximately 280 gamma. If the maximum kanamycin concentration were ever reached, it would be noted by a severe decline in both total and selective cell counts. A test and a control reactor were run. Each reactor was inoculated with the same concentration of 17616-pTOM31c cells harvested from a pure culture (high 67 i selection) 17616-pTOM31c batch culture. After 8 hours of unincumbered growth, 70 uM TCE (in air) vapor was introduced to the test reactor at a flow rate of 50 mls/min. After 15 days of 70 uM TCE exposure , the TCE concentration was raised to 700 uM for three days to determine the effect of concentration. No TCE was introduced to the control reactor. Daily analysis of the reactors included: protein contents, acetate concentrations, pTOM specific activities, total cell counts, TnS selective plate counts, and pTOM selective plate counts. Total cell counts were determined by plating on LBG media. TnS selective cell counts were determined by plating on LBG-Kanamycin agar. The pTOM selective cell counts were determined by dilution plating on phenol-kanamycin-HCMM2 low carbon agar plates. Comparison of the control and test reactor total cell counts demonstrates the extent of toxicity incurred by the 17616-pTOM31c cells by prolonged exposure to TCE. Differences between the test reactor’s total counts and selective counts indicates the number 17616-pTOM31c cells injured as a result of TCE exposure. Finally, the occurrence of significant numbers of TFMP-negative LBG-kanamycin plates would symbolize that the plasmid pTOM31c may have been recombined as a result of structural instability and would insinuate that the kanamycin resistance marker either had been incorporated into the cells’ chromosomal DNA or remained active while the TOM pathway had been deemed inactive. 4.8.2 Non-selective TCE exposure studies A non-selective fed batch reactor was run under the same conditions as the v. selective reactors, except kanamycin was not provided in either the test or control reactors. Analysis of the non-selective test and control reactors included: protein content, pTOM specific activity, and pTOM31c selective direct colony transfers to determine if TCE exposure resulted in increased plasmid loss. Results from this experiment also give information on the toxicity and degree of selection that TCE has on 17616-pTOM31c cells. 4.9 Analytical Methods and Protocols 4.9.1 Protein assay - Protein content was determined using the enhanced BA Protein assay (Pierce Co.) This protein assay was used in both the suspended and biofilm culture TFMP assays. Suspended protein determinations were usually made using the appropriate A600 versus protein calibration curve. The calibration curves were determined for each specific microbial strain and medium (Appendix E). Direct protein measurements (no Calibration curve) were determined for all biofilm packing and suspended phenol growth samples. All absorbance measurements were taken using a Milton Roy Spectronic 601 photospectrometer. 68 69 4.9.2 Phenol assay Phenol concentrations were determined using a colorimetric phenol assay which involved the addition and thorough mixing of 50ul of 2 N NH4OH and 25 ul of 2% 4-aminoantipyrene (Aldrich Chemical Co., Inc., Milwaukee Wise.) to a 1ml phenol sample. After mixing, 25 ul of 8% K3Fe(CN)6 (Sigma Chemical Co., ST. Louis, Mo.) was added, and the contents were then mixed again and centrifuged (14,000 x g) for 2 minutes. A500 of the centrifuged sample supernatant was measured. Phenol concentrations were calculated by reference to a standard curve (Appendix F). 4.9.3 Phthalate analysis Phthalate was analyzed using a Hewlitt Packard-1050 High Pressure Liquid Chromatograph equipped with variable wavelength and multiple wavelength detectors using a HP, OD Hypersil, Sum, 100x2.1 mm analytical column and a HP OD Hypersil, Sum, 20 X 2.1 mm guard column. A calibration curve for the IC phthalate analysis is shown in Appendix G. 4.9.4 TCE analysis Vapor and liquid phase TCE samples were analyzed using a Schimadzu GC9A gas chromatograph equipped with an electron capture detector (Shimadzu Scientific Instruments, Inc., Columbia, MD) usipg a 30 m, 0.53mm ID Vocal mega-bore column (Supelco, Inc., Bellefonte, PA.). The TCE method was 70 isothermal (110° Celsius oven temp., 200° C detector and injector temp.) with a carrier gas flow of 5 ml/min and a make-up gas flow of 50 ml/min. Ultra-high purity nitrogen was used for both the carrier and make-up gases. Since the method was isothermal using nitrogen, there were two linear calibration curves, one for high TCE concentrations (~10 nM to 10uM, detector range set at 2, current set at I ) and one for low TCE concentrations (~10 uM to 1000 uM, detector range set at 1, current set at 2). Vapor TCE samples were injected directly. Liquid TCE samples were extracted using an equal volume of pentane, and a 2 ul sample of the pentane TCE extraction was analyzed. Liquid TCE standards were made with high grade TCE (Sigma Chemical Co.) in HPLC grade methanol or pentane. Vapor TCE was supplied from a 700 uM TCE-ultra zero air balance compressed gas bottle (Air Liquide, La Porte, Texas). A representative calibration curve for the G.C. TCE analysis is presented in Appendix H. 4.9.5 Acetate analysis Acetate was analyzed using a Dionex ion chomatograph (Model AI-450; Dionex Co., San Francisco) with a pulse electrochemical detector (Model DX300) using a IonpacASI 0 column(4mm). Calibration information is presented in Appendix I. 71 4.9.