Feasibility Studies for Encapsulated Cell Bioaugmentation Of

Feasibility Studies for Encapsulated Cell Bioaugmentation of Contaminated Aquifers by Peyman Moslemy Department of Chemical Engineering McGill University, Montreal January 2002 A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements of the degree of Doctor of Philosophy © Peyman Moslemy 2002 National Library Bibliothèque nationale 1+1 of Canada du Canada Acquisitions and Acquisitions et Bibliographie Services services bibliographiques 395 Wellington Street 395, rue Wellington Ottawa ON K1A ON4 Ottawa ON K1 A ON4 canada canada The author bas granted a non L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library ofCanada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies ofthis thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The. author retains ownership ofthe L'auteur conselVe la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts from it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son pemnsslOn. autorisation. 0-612-78740-0 Canada Preface The manuscript-based thesis option was chosen according to the following thesis preparation guideline given by the Faculty of Graduate Studies and Research: As an alternative to the traditional thesis format, the dissertation can consist of a collection ofpapers ofwhich the student is an author or co-author. These papers must have a cohesive, unitary character making them a report of a single program of research. The structure for the manuscript-based thesis must conform to the following: 1. Candidates have the option ofincluding, as part ofthe thesis, the text ofone or more papers submitted, or to be submitted, for publication, or the clearly-duplicated text (not the reprints) ofone or more published papers. These texts must conform to the "Guidelines for Thesis Preparation" with respect to font size, line spacing and margin sizes and must be bound together as an integral part ofthe thesis. (Reprints of published papers can be included in the appendices at the end ofthe thesis.) 2. The thesis must be more than a collection of manuscripts. All components must be integrated into a cohesive unit with a logical progression from one chapter to the next. In order to ensure that the thesis has continuity, connecting texts that provide logical bridges between the different papers are mandatory. 3. The thesis must conform to aU other requirements of the "Guidelines for Thesis Preparation" in addition to the manuscripts. The thesis must include the following: (a) a table ofcontents (b) an abstract in English and French (c) an introduction which clearly states the rational and objectives ofthe research (d) a comprehensive review of the literature (in addition to that covered in the introduction to each paper) (e) a final conclusion and summary 4. As manuscripts for publication are frequently very concise documents, where appropriate, additional material must be provided (e.g., in appendices) in sufficient detail to allow a clear and precise judgement to be made of the importance and originality ofthe research reported in the thesis. 5. In general, when co-authored papers are included in a thesis the candidate must have made a substantial contribution to aU papers included in the thesis. In addition, the candidate is required to make an explicit statement in the thesis as to who contributed to such work and to what extent. This statement should appear in a single section entitled "Contributions ofAuthors" as a preface to the thesis. Preface 11 This thesis is prepared as a result of the experimental studies performed by P. Moslemy to evaluate the feasibility of use of encapsulated cell technology for in situ bioaugmentation of contaminated aquifers. Chapter 1 presents an introduction to environmental pollution with petroleum hydrocarbons and various site bioremediation strategies including in situ bioaugmentation. Chapter 1 alsodescribes the concept of bioencapsulation and the application of encapsulated microbial cells to biological degradation of hazardous compounds. An introduction to gellan gum, a natural polymer used for encapsulation of bacteria in this study, along with its physical and chemical characteristics, methodof gelation, and production of gel microbeads is also covered. In Chapter 2 the objectives of this study are presented. The contents of Chapters 3 to 6 are adopted from the manuscripts submitted or to be submitted to scientific journals for publication. The investigations performed to develop a two-phase dispersion technique for encapsulation of bacteria in gellan gum microbeads are presented in Chapter 3. The influence of various process parameters on size distribution of microbeads, and the repeatability in the microbead formation process and particle size measurement are demonstrated. Chapter 4 presents the experiments performed to evaluate the feasibility of transport of encapsulated cell microbeads through porous soil media. Transport of gellan gum microbeads was studied by pulse injection of a microbead suspension into soil columns packed with different grain size classes of gravel and sand. The effect of grain size distribution on the extent of transport and the variation of microbead dispersion with injection time across the soil matrix are illustrated. The transport of gellan gum microbeads was further investigated by intermittent injection of a suspension of microbeads into sand columns. Uniform dispersion of bacterial carriers across the contaminated area of an aquifer is crucial to the successful formation of a bioactive zone. In Chapter 5 the effect of grain size and sorne operation parameters such as injectant concentration and injection time on distribution patterns of microbeads are presented. Preface 11l The performance of gellan gum-encapsulated bacteria in the biodegradation of gasoline is demonstrated in Chapter 6. The capacity of the encapsulated cells to degrade gasoline under aerobic conditions was evaluated in comparison with free (non encapsulated) cells in liquid suspension and saturated soil microcosms. The influence of initial gasoline concentration and encapsulated cell mass loading on the extent and the rate of biodegradation are also illustrated. Chapter 7 highlights the main conclusions of this experimental study. In Chapter 8 the major contributions to the existing knowledge are emphasized. Chapter 9 will end this text with the author' s recommendations for future work. Contributions of Authors The contents of Chapters 3 to 6 of this thesis are adopted from the manuscripts submitted or to be submitted to scientific journals. The research was conducted by P. Moslemy, under the supervision of Prof. Ronald J. Neufeld and Dr. Serge R. Guiot who are named as co-authors. Dr. Denis Millette provided insight into the design of the soil column experiments in work described in Chapters 4 and 5, and is listed as a co-author. iv Abstract Encapsulated cell bioaugmentation is a novel approach to in situ bioremediation of contaminated aquifers. This study was carried out to develop encapsulated cell microbeads of appropriate size suitable for in situ bioaugmentation, and to evaluate the feasibility of such a remediation strategy based on the performance of entrapped cells in the biodegradation of a common groundwater contaminant and the transport of cell carriers through porous soil media. A two-phase dispersion technique, termed emulsification-internal gelation, was developed to encapsulate a gasoline-degrading bacterial consortium in gellan gum microbeads. The influence of emulsion parameters including stirring rate, disperse phase volume fraction, emulsifier concentration, emulsification time, and cell mass loading on size distribution of microbeads was studied. The microbead diameter was controlled within a narrow range of 10 - 50 ~ by selection of appropriate emulsion conditions. A high degree of repeatability in the microbead formation process and particle size measurement was demonstrated. Transport experiments were conducted in horizontal soil columns (5.2 cm id x 110 cm long) packed with different grain size classes of gravel (2 - 16 mm) and sand (0.125 - 2 mm). The transport of cell-free microbeads through soil was first investigated in pulse injection experiments carried out with gravel- and sand-packed columns. The feasibility of the formation of a bioactive zone consisting of encapsulated cells was then evaluated in intermittent injection experiments performed with cell-free microbeads in columns packed with different grain size classes of sand. A suspension of microbeads in artificial groundwater (AGW) was pulsed into a column for 6 h, followed by injection of bead-free AGW for 42 h. In general, the total amount of microbeads traveling across a given section of the column decreased with the decrease of mean grain size. The results of this study demonstrated the feasibility of transport of gellan gum microbeads through a wide range of soil grains, i.e. medium sand to medium gravel (0.25 - 16 mm), across distances up to 110 cm. Abstract v Intermittent injection experiments were performed to obtain the information on distribution patterns of microbeads across llO-cm

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