DISTRIBUTION OF METABOLIC CHARACTERISTICS AMONG AEROBIC SOIL BACTERIA AND IMPLICATIONS FOR BIOTRANSFORMATION OF ORGANIC AND METALLIC WASTES by FANGMEI ZHANG Submitted in partial fulfillment of the requirements For the degree of Doctor of Philosophy Dissertation Advisor: Dr. Skubal Department of Civil Engineering CASE WESTERN RESERVE UNIVERSITY January, 2007 UMI Number: 3237870 Copyright 2007 by Zhang, Fangmei All rights reserved. UMI Microform 3237870 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved.
This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P. Box 1346 Ann Arbor, MI 48106-1346 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Fangmei Zhang ______________________________________________________ candidate for the Ph. Karen Skubal (signed)_______________________________________________ (chair of the committee) Dr.
Robert Mullen ________________________________________________ Dr. Aaron Jennings ________________________________________________ Dr. Andrew Swanson ________________________________________________ ________________________________________________ ________________________________________________ 11/29/2006 (date) _______________________ *We also certify that written approval has been obtained for any proprietary material contained therein. Copyright © 2007 by Fangmei Zhang All rights reserved TABLE OF CONTENTS LIST OF TABLES…………………………………………………………….
v LIST OF FIGURES………………………………………………………………. vi ACKNOWLEDGEMENTS……………………………………………………… ix NOMENCLATURE……………………………………………………………… xi ABSTRACT……………………………………………………………. Research hypothesis and significance of the work…………… 3 1. Chromium pollution: sources and chemistry…………………………………………………….
Chromium uptake, transformation, and resistance by soil microorganisms……………………………………………. Microbial partitioning in soil and its implications for pollutant biotransformation……………………………………. 12 Cell partitioning and physiological properties………………. 13 Abundance and activity of planktonic versus attached bacteria 13 Mechanisms of attachment………………………………….
14 Solid surfaces and microbial attachment……………………. 16 Cell surface properties and microbial attachment…………. 16 Cell structures and microbial attachment……………………. 17 Biochemical stimulation of attachment……………………….
Oligotrophy, copiotrophy and bacterial attachment………… 18 CHAPTER 2. Isolation and Physiological Characterization of Microbial Communities and Isolates from Unsaturated Soil……………. Materials and Methods………………………………………… 24 Soil sampling and characterization…………………………… 24 Adhesion-based extraction of soil microorganisms…………. 24 Isolation of dominant microbial populations………………… 25 Characterization of variably-attached microbial communities and isolates……………………………………………….
27 MATH assays………………………………………………… 27 HIC and EIC…………………………………………………. 30 MATH assay for microbial elution fractions F1 and F3……. 31 Gram staining and catalase activity of colony isolates from variably-attached microbial fractions………………………… 33 Cell hydrophobicity and charge of colony isolates from variably-attached microbial fractions……. Growth of Soil Microbial Communities and Isolates on Organic Substrates…….
41 Models of cell growth……. Materials and Methods……. 45 Growth of microbial consortia on yeast extract as a limiting substrate…….……… 45 Growth of microbial isolates on yeast extract as a limiting substrate…….……… 47 Growth of microbial isolates on salicylic acid as a limiting substrate…….……… 49 Growth of isolate F12 on salicylic acid……. 53 Extent of salicylic acid transformation…….
Effect of Chromium on the Growth of Microbial Communities and Isolates Derived from Soil……. Materials and Methods……. 60 Experiments assessing the impact of chromium(VI) on microbial consortia…….… 60 Experiments assessing chromium(VI) impact on microbial isolates grown on yeast extract……. 62 Experiments assessing chromium(VI) impact on isolates grown in simulated comingled waste with salicylic acid…….
62 Impact of Cr(VI) on biomass production by consortia F1 and F3 in yeast extract medium……. 62 ii Impact of Cr(VI) on the growth rate of consortia F1 and F3 in yeast extract medium……. 66 Impact of Cr(VI) on the death kinetics of consortia F1 and F3 in yeast extract medium……. 67 Degrees of inhibition by Cr(VI) to individual microbial isolates in yeast extract medium…….……… 69 Relationships between substrate affinity and Cr(VI) tolerance by microbial isolates in yeast extract medium…….
70 Impact of Cr(VI) on the lag phase of microbial isolates in yeast extract medium……. 72 Chromium tolerance and cell surface properties of microbial isolates……. 75 Inhibition of isolates in simulated comingled waste……. Spectroscopic Studies of Chromium Biotransformation and Uptake by Microbial Communities and Isolates…….
79 The use of XAFS for characterization of metals in environmental samples…. Materials and Methods…….……… 82 Metal uptake experiments…….……… 85 Determination of biosorption kinetics……. 87 Spectral signatures for chromium standards…….……… 87 XAFS determination of chromium reduction by microorganisms……. 89 Normalization of chromium sorption to biomass…….……… 92 Chromium uptake after two hours of exposure- results of the XAFS study…….
96 Chromium sorption versus growth phase in the XAFS study. 97 Chromium sorption and cell surface properties in the XAFS study……. 97 Models of chromium biosorption kinetics……. 103 Chromium biosorption kinetics by microbial isolates in batch systems…….… 104 Equilibrium chromium sorption and cell surface properties….
Summary and Recommendations……. Recommendations for future work…. 119 iv LIST OF TABLES Table 2. Media and resins used in HIC and EIC…….
Biochemical and morphological characterization of five variably-attached microbial isolates eluted from soil……. Relative hydrophobicity and surface charge of five microbial isolates extracted using serial soil elution……. Monod growth parameters for microbial consortia and isolates grown on yeast extract at 19°C……. Kinetic parameters for isolate F12 on two substrates…….
First-order death constants for stationary-phase cultures of consortia F1 and F3 exposed to hexavalent chromium……. Growth rates and lag phases of five microbial isolates in yeast extract medium with hexavalent chromium……. Microbial samples analyzed for chromium uptake and transformation using atomic absorption spectroscopy (AA) and X-ray absorption fine structure (XAFS) spectroscopy……. Chromium standards used to generate Cr(III) and Cr(VI) reference spectra for XAFS analyses…….
Metal uptake in cultures incubated with 2 mM (approximately 100 mg/L) hexavalent or trivalent chromium for two hours……. Chromium biosorption at equilibrium and after 2 and 24 hours of incubation for four microbial isolates……. 109 v LIST OF FIGURES Figure 2. Relative hydrophobicity, based on the MATH assay, of variably- attached microbial fractions eluted from soil and grown to late exponential phase in yeast extract medium……………………… 32 Figure 2.
Relative cell surface hydrophobicity of “loosely-attached” colony isolates F11, F12, and F13, and of “tightly-attached” colony isolates F31 and F32 ……. Relative cell surface charge of “loosely-attached” colony isolates F11, F12, and F13, and of “tightly-attached” colony isolates F31 and F32 ……. Total negative surface charge of cells from “loosely-attached” colony isolates F11, F12, and F13 and from “tightly-attached” colony isolates F31 and F32, as measured by colloid titration…. Two pathways of aerobic salicylate biodegradation.
Growth of two microbial consortia on yeast extract as a limiting carbon source……. Growth of five microbial isolates on yeast extract as a limiting carbon source……. Growth dependence of isolate F11 on salicylic acid concentration……. Growth dependence of isolate F12 on salicylic acid concentration…….
Growth dependence of isolate F13 on salicylic acid concentration……. Growth dependence of isolate F32 on salicylic acid concentration……. Maximum culture density of the five isolates when grown in 40 mg/L salicylic acid……. Monod growth curve of isolate F12 with salicylic acid as the sole carbon source…….
Lineweaver-Burk plot of the growth of isolate F12 on salicylic acid……. Growth data for isolate F12 fit to the modified Monod kinetic model……. Transformation of salicylic acid by isolate F12……. Final biomass concentration versus hexavalent chromium concentration for consortia F1 and F3 grown on 100 mg/L yeast extract and exposed to chromium during the entire two-week incubation period…….
Decrease in biomass production by consortia F1 and F3 with respect to hexavalent chromium concentration……. First-order toxicity model for the impact of 30, 60 and 90 mg/L Cr(VI) on the net biomass production of microbial consortia F1 and F3……. Growth rate of consortia F1 and F3 in the presence of low concentrations of hexavalent chromium……. Normalized growth rate µ versus hexavalent chromium concentration (0 - 15 mg/L) for microbial consortia F1 and F3 in sucrose medium…….
Decrease in viable cell counts X for stationary-phase cultures of consortia F1 and F3 that had been grown in 200 mg/L yeast extract medium and were subsequently exposed to 0, 5 or 25 mg/L Cr6+. Growth rates of five microbial isolates in 100 mg/L yeast extract medium with hexavalent chromium at 0, 5, 20, 50, 100, and 200 mg/L……. Growth rates of five microbial isolates in 500 mg/L yeast extract medium with hexavalent chromium at 0, 5, 20, 50, 100, and 200 mg/L……. Relationships between the Monod affinity constant and chromium tolerance (expressed as µ/µo) for microbial isolates F11, F12, F13, F31, and F32…….
Chromium tolerance (µ/µo) versus the lag phase observed before the onset of growth of microbial isolates F11, F12, F13, F31, and F32……. Chromium tolerance (µ/µo) versus negative cell surface charge of microbial isolates as determined by colloid titration……. Chromium tolerance (µ/µo) versus negative cell surface charge of microbial isolates as determined by electrostatic interaction chromatography. Graphical representation of the experimental analysis of chromium adsorption and transformation by microbial consortia and isolates.
Chromium fluorescence XAFS spectra (fluorescence intensity in arbitrary units versus incident x-ray photon energy) for a number of trivalent and hexavalent chromium standards. Cr fluorescence XAFS spectra (fluorescence intensity in arbitrary units versus incident x-ray photon energy) for chromium sorbed to cells from consortia F1 and F3 and isolates F12, F13, F31, and F32……. Chromium fluorescence XAFS spectra (fluorescence intensity in arbitrary units versus incident x-ray photon energy) for trivalent chromium standard, hexavalent chromium standard, and microbial samples containing cell-sorbed chromium…………. Relationships between protein mass and dry cell mass for the microbial consortia and isolates tested…….
A comparison of chromium sorbed to cell biomass versus that remaining in solution following two hours of incubation with 100 mg/L chromium……. A comparison of sorbed and aqueous chromium following two hours of incubation with 100 mg/L hexavalent or trivalent Cr and consortia F1 and F3 and isolates F12, F13, F31, and F32 in exponential and stationary phase………………………………. Chromium partitioning between cells and supernatant for microbial consortia and isolates……. Chromium biosorption with respect to growth phase for four bacterial isolates and two consortia following two hours of exposure to 100 mg/L trivalent or hexavalent chromium at pH 7.
Cr(III) and Cr(VI) were initially present as Cr(NO3)3 and K2Cr2O7, respectively. Chromium biosorption by microbial isolates F12, F13, F31, and F32 with respect to cell surface charge and relative hydrophobicity……. Chromium sorption by microbial isolate F11 represented by a pseudo-second order model……. Chromium sorption by microbial isolate F12 represented by a pseudo-second order model…….
Chromium sorption by microbial isolate F13 represented by a pseudo-second order model……. Chromium sorption by microbial isolate F31 represented by a pseudo-second order model……. Chromium sorption by microbial isolate F32 represented by a pseudo-second order model……. pseudo-second order chromium sorption model parameters for the five variably-attached microbial isolates F11, F12, F13, F31, and F32…….
The pseudo-second order equilibrium chromium sorption constant qe plotted against microbial isolates’ negative surface charge……. 110 viii ACKNOWLEDGEMENTS I would like to acknowledge many people for helping me during my doctoral work. Without their support and encouragement, my dissertation would not be completed. Firstly, I would like to give special thanks to my dissertation committee, Dr.
Karen Skubal, Dr. Aaron Jennings, Dr. Robert Mullen, and Dr. Andrew Swanson for their input, valuable guidance, and suggestions.
I would especially like to thank my advisor, Dr. Karen Skubal, for her continuous support and encouragement in multiple ways in the pursuit of this degree. I am grateful for her guidance, understanding, patience, and mostly her friendship during my graduate studies at CWRU. She has always been there to listen to me, comfort me, help me, and advise me as much as she could not only as an advisor but also like a friend and a sister.
I have learnt much from her for both my professional and personal growth. Aaron Jennings’s constructive suggestions have always motivated me when I have talked to him. I greatly value his suggestions, his patience, and encouragement. I have also always enjoyed his anecdotal lectures, his sense of humor, and his broad range of conversational topics.
I owe a special note of gratitude to Dr. Robert Mullen, the chairman of Department of Civil Engineering at CWRU. I really appreciated his efforts in finding financial support for my last semester. I would like to acknowledge Dr.
Laura Skubal for conducting XAFS analysis in Argonne National Lab. This is a very important and supportive experiment in my dissertation. ix The support of the Civil Engineering Department personnel and other friends has been tremendous. My deepest thanks go to Dr.
Jun Ma, who graduated from Civil Engineering earlier, and his wife. As my best friends, they have given me much comfort, help, and encouragement these years.