Lignin degradation, is quite common in nature and is an important link in the natural carbon cycle. A large variety of microorganisms are know to degrade lignin in nature as well as in contrived fermentation systems. White-rot and soft-rot fungi, as well as Actinomycetes, are apparently the most active lignin degraders in nature. The large, cross-linked, polymeric structure of the lignin macromolecule makes its direct uptake, during the initial stages of its degradation, by microbial cells improbable. Moreover, the fact that the lignin macromolecule is composed of different monomeric units linked by a large variety of non-hydrolyzable intermonomeric bonds precludes hydrolytic cleavage of the biopolymer. Despite the fact that many extracellular and membrane-bound enzymes have been suspected in the initial breakdown of lignin, such activities have not yet been found. A close review of the literature indicates that the initial breakdown of the lignin macromolecule may be nonenzymatic. In addition, the degradation of the lignin polymer appears to follow an exo-degradation mechanism. That is, many lignin degrading microorganisms are apparently incapable of splitting the lignin molecule into intermediate molecular weight polyphenolic moieties which are further degraded; instead, they attack the periphery of the macromolecule. The possible involvement of reduced oxygen species produced by white-rot fungi in the initial breakdown of the lignin macromolecule, during its biodegradation, was investigated. Using Coriolus versicolor as a representative of white-rot fungi, I demonstrated that C. versicolor exports superoxide radical and hydrogen peroxide during lignin degradation, into the lignolytic medium. Results presented in this study indicate that a correlation between the concentration of extracellular superoxide radical in the medium and the extent of lignin degradation may exist. Moreover, I have shown that superoxide radical is produced in the cell membrane, and not the organism's mitochondria. This precludes the possibility that such reduced oxygen species are produced as a result of normal respiration by the organism. An investigation of the effects of aeration and agitation indicated that agitation has a detrimental effect on the extent of lignin degradation. On the other hand, increased oxygen tension in lignolytic cultures appeared to enhance the extent of lignin degradation. Another interesting finding was the fact that conditions leading to the formation of reproductive fruits in the lignolytic microorganism favored the degradation of the lignin fraction in lignocellulosic materials. A comparative study of two different fennentation schemes, designed to degrade lignin in 1ignocellu1osic materials on a large scale, indicated that solid state fermentation of such materials led to greater lignin degradation. Fluidized bed fermentations, on the other hand, appeared to favor the degradation of the carbohydrates rather than the lignin fraction of lignocellulosic materials. Studies of the biodegradation of monomeric lignin model compounds do not shed light on the initial step(s) involved in the breakdown of the lignin polymer. Such studies assume that microbial breakdown of lignin model compounds is similar to microbial breakdown of lignin an assumption that may not be correct. It is true that degradation of monomeric lignin model compounds can conceivably elucidate the mode of degradation of low molecular weight moieties resulting from initial breakdown of the lignin macromolecule. However, the chemical identities of these low molecular weight intermediates are not yet known. The efficacy of studies using aromatic, monomeric lignin model compounds in attempts to identify intracellular pathways for metabolism of lignin depends on the assumption that lignin breakdown products are indeed mononuclear phenolic materials. Careful analysis of soluble and insoluble residual lignin resulting from lignin fermentations is a critical step in assessing the lignolytic ability of microorganisms. Furthermore, such analyses are essential in understanding the steps involved in lignin metabolism by microorganisms. To date the methods for residual lignin analyses are complex, time consuming and error prone. There is an urgent need to develop a quick and simple method for residual lignin analysis that will yield accurate and reproducible results capable of elucidating structural changes in residual, biodegraded lignin. The development of such an analysis technique will undoubtedly lead to a better understanding of the complex problem of lignin biodegration. / Ph. D.

Groundwater contaminants such as chlorinated ethenes, chlorinated ethanes and nitroaromatic explosive compounds (e.g. 2,4,6-Trinitrotoluene (TNT)) degrade in the subsurface primarily by microbially catalyzed reductive transformation reactions. From a regulatory point of view, the capability to simulate the kinetics of these reductive transformation reactions coupled with other attenuation processes in the subsurface (e.g., sorption, advection, and dispersion) is required for site-specific solute transport models. A kinetic model based on Michaelis-Menten type equations (Widdowson 2004) has been successfully validated for the linear reductive dechlorination pathway of chlorinated ethenes, and implemented in solute transport codes such as SEAM3D (Waddill and Widdowson 2000). However, TNT degrades through more complex branched pathways, and kinetic models are lacking in the current literature. This research study was undertaken with the objective of extending the kinetic model developed for the linear reductive pathway of chlorinated ethenes to branched pathways. The proposed extended kinetic model was validated with experimental concentration-time data of TNT and its metabolites from two prior published laboratory studies (Daun et al. 2000; Hwang et al. 2000), both in the presence and absence of sorption. The model-predicted concentrations with time of TNT and its degradation intermediates and end-products correlated well with the experimental data. The model is further compatible with and can be easily incorporated into solute transport codes (e.g., SEAM3D), and used to evaluate the fate and transport of TNT and other similar contaminants in the subsurface. / Master of Science

A laboratory investigation was conducted to determine the effects of biologically produced surfactants (biosurfactants) on petroleum hydrocarbons and their potential for the removal of hydrocarbons from groundwater systems. Bioaurfactanta have been found to be produced by microorganisms during growth on insoluble substrates for the purpose of increasing substrate solubility so as to promote biological degradation. In this study, three types of biosurfactants were produced by microorganisms grown on gasoline and a mixture of glucose with vegetable oil. Solubilization and biodegradation of selected gasoline compounds in the presence of bioeurfactante were measured in both static batch and flow through column systems. Batch experiments were conducted in culture tubes, using only liquid phases. A clean sand was used in the column system to monitor physical and chemical interactions yet minimize adsorption effects. A mixed culture of gasoline degrading microorganisms along with isolated cultures grown on selected compounds were used in the biodegradation studies. The biosurfactants produced and used in this study acted similarly to synthetic surfactants and increased, to various degrees, the solubility of the monitored gasoline compounds. Biosurfactants produced from growth on glucose and vegetable oil were very effective surfactants, markedly increasing solubility of the gasoline compounds, but inhibiting biological degradation of these same compounds. Biosurfactants produced by microorganisms from growth on gasoline were effective surfactants, but they did not inhibit biodegradation of the gasoline compounds. This indicated that the biosurfactants may be substrate or microorganism specific, produced for growth on a particular insoluble substrate by a specific microorganism. Biosurfactants produced from growth on gasoline or an insoluble hydrocarbon could therefore be used to enhance solubility and subsequent biodegradation of that same hydrocarbon. The effectiveness of the biosurfactants during application by injection or recirculation for groundwater remediation would be limited by the adsorption and removal of the biosurfactant to the soil. The surfactant demand (by adsorption) of the soil would have to be met before the effects of the biosurfactants would become apparent. Biosurfactanta added to groundwater could also create an additional oxygen demand in a system already low in oxygen. / Master of Science

Groundwater contamination by man-made chemicals is increasingly being reported in the United States. The potential for detrimental health effects is substantial and has been addressed by the environmental engineering profession. Typically, contaminated groundwater is pumped to the surface and treated in a variety of methods including air stripping, carbon adsorption, and biodegradation. In situ biodegradation is increasingly being considered as an alternative to pump-and-treat technology. The primary goal of this research was to determine the fate of an organic chemical adsorbed to a subsurface soil when exposed to acclimated bacteria. Toluene was chosen as a representative compound because it is a major constituent of groundwater contaminated by gasoline. In addition, toluene is known to be both biodegradable and adsorbable. Sybron Biochemical, Inc. supplied the aerobic bacteria Psgudomonas gutjga known to readily transform toluene. Soil microcosms were established in test-tubes and conditions simulated those of a saturated, aerobic aquifer. Gas chromatography was used to quantify changes in toluene concentration due to adsorption and biodegradation. The addition of an aqueous toluene solution to sterile microcosms resulted in the rapid and extensive adsorption of toluene to the soil. Subsequent analysis revealed the slow adsorption of an additional small fraction of toluene. Biodegradation studies entailed the addition of acclimated bacteria to sterile soil microcosms in which substantial toluene adsorption had occurred. Addition of small doses of hydrogen peroxide effectively maintained aerobic conditions for biodegradation. As a result, E, putjda was able to transform all measurable toluene in the microcosms. Additional desorption studies revealed that a "resistant" component of toluene remained adsorbed to the soil during biodegradation. This component was neither acted upon by bacteria nor readily extractable by methylene chloride. However, slow desorption of toluene was shown to occur at a rate comparable to slow adsorption. To achieve complete removal, groundwater treatment methods must address the rate-controlled desorption of the resistant toluene component. / Master of Science

M.S.

Microbial populations from subsurface soil collected from a hydrocarbon contaminated site and a pristine site with no history of contamination had the ability to degrade pentachlorophenol (PCP) in anaerobic enrichment cultures. Increasing concentrations of PCP in nitrate, sulfate and yeast extract-mineral salts media were used to acclimate the cultures. Nitrate enrichments, previously incubated in an anaerobic phenol-mineral salts medium, showed 23% degradation in medium containing 40 μg ml⁻¹ PCP during a 32 d incubation period. Cultures not adapted to phenol degradation did not degrade PCP at concentrations over 20 μg ml⁻¹. Enrichment cultures grown in the anaerobic yeast extract-mineral salts medium did not degrade PCP at concentrations over 20 μg ml⁻¹ and phenol adaptation did not enhance PCP degradation. The sulfate reducing enrichment containing 1 μg ml⁻¹ PCP showed 71.3% degradation after 32 d incubation. No degradation occurred at or above 5 μg ml⁻¹ PCP. PCP intermediates, 2,4,6-trichlorophenol (TCP) and 3,4,5 TCP were found in the spent culture of the nitrate reducing enrichment. In the spent culture of the sulfate reducing enrichment, 3,4,5 TCP and 2,3,4,5-tetrachlorophenol were found. Attempts to obtain a pure culture of an anaerobic PCP degrading bacterium were unsuccessful. / Master of Science

Cellulose to lignin ratio is one of the widely used indicators of degree of landfill stabilization. This ratio shows the amount of carbohydrate or cellulose consumed by anaerobes compared to relatively inert lignin. However, the method of lignin measurement contains an intrinsic error. Plastics are contained in the landfill samples and these are characterized as lignaceous materials due to their acid-insolubility. Lignin is typically measured as the organic residue that is acid insoluble but is combustible upon ignition. Additionally lignin may degrade under anaerobic, high temperature conditions associated with wet conditions in sediments and bioreactor landfills. In this study, it has been found that the typical measure of lignin, a gravimetric measure, also includes plastics, leading to erroneous measures of both lignin and the cellulose/lignin (C/L) ratio. Typically, 100% of the plastic will be measured as lignin. Since plastic amounts to approximately 10% of landfill contents, lignin measurements will be 10% greater than actual amounts. Laboratory reactors were set up with known amounts of paper and plastic. The degradation of the cellulose and lignin in paper was measured and compared to plastics, which was collected by hand and weighed. Ratios of cellulose to plastics and lignin to plastics were obtained. It was found, based on the cellulose to plastic ratio and lignin to plastic ration that lignin degrades under anaerobic conditions although at a much slower rate than cellulose. These findings indicate that the cellulose to lignin ratio cannot be used as the sole indicator of stabilization in the landfills. The inclusion of the biochemical methane potential test data along with C/L is thought to provide a better indication of landfill stabilization. / Master of Science

Beech (<u>Fagus grand1folia</u> Ehrh.> bark contains appreciable quantities of Z- (cis) con1feryl and Z-s1napyl alcohols and not the corresponding E- (trans) alcohols. Previous rad1otracer experiments suggested that the Z-coniferyl alcohol in beech bark is formed by isomerization of E-coniferyl alcohol which proceeds either directly or through the corresponding aldehydes. In the work conducted in this thesis, is has been found that crude cinnamy1 alcohol dehydrogenase isolated from beech bark shows a strong substrate preference for E-coniferyl alcohol (as opposed to Z-coniferyl alcohol) thereby suggesting that the E to Z isomerization described occurs directly at the alcohol level. Administration of (2-¹⁴C) ferul1c acid to feland wheat (<u>Triticum aest1yum</u> L.) over extended durations (21 days) and subsequent isolation of the lignin from the root tissue as its acetal derivative demonstrated the incorporation of the labelled feru1ic acid into the lignin component of the tissue. Through sim1lar administrations of (1-¹³C, 2-¹³C and 3-¹³C) ferul1c acid and subsequent analysis of the root tissues by solid state ¹³C nuclear magnetic resonance (NMR) spectroscopy, it was possible to determine the bonding patterns of lignin in situ. The lignin component of each ¹³C feru1ic acid enriched root tissue was then isolated as its acetal derivative and analyzed by solution state ¹³C NMR. Through comparison it was shown that the enhanced resonances observed in the solution state ¹³C NMR spectra of the ¹³C ferulic acid enriched acetal lignins corresponded to the enhanced resonances in the respective spectra of the intact root tissues. This indicated that minimal changes to the lignin bonding patterns occurred during the isolation procedure. The dominant presence of ¹³C NMR resonances corresponding to hydroxycinnamic acid functionalities in the solid and solution state NMR spectra demonstrates the important role of hydroxyc1nnamic acids in wheat root lignin. However, no evidence of the formation of dimers such as 4,4'-dihydroxytruxillic acid was noted. Thus, such structures do not represent an important bonding pattern in wheat root lignin. / Master of Science

By shearing activated sludge using a high shear rotor stator device, bioavailable proteinaceous material can be produced. Operation at elevated temperatures, serves to increase the amount of material that is rendered soluble (<0.45 um) and biodegradable. The storage of sludge under anoxic condition prior to shearing does not appear to enhance solublization of solids, though deflocculation and deterioration of dewaterablility was observed. Anaerobic digestibility appears to be enhanced by the addition of a high shear as shown by increases in gas production and volatile solids destruction. The dewatering properties of activated sludge, measured by capillary suction time, deteriorated with the addition of sheared solids, but reaeration resulted in near complete recovery. The role of iron and iron chemistry plays a critical role in the activated sludge. Iron apparently selectively removes protein, in particular material ranging in the 1.5 um to 30K size range. The addition of ferric iron was found to increase SVI and decrease zone-settling velocity, when added to reactors with mechanically disintegrated sludges. Similar trends were not observed in reactors dosed with ferrous iron. Preliminary results suggest that the ferric/ferrous redox chemistry may serve to enhance floc structure, as observed by increased settling velocity and shear resistance for sludges dosed with ferrous sulfate. / Master of Science

Thesis advisor: Babak Momeni / Thesis advisor: Charles Hoffman / Aflatoxins (AFs) are secondary fungal metabolites that contaminate common food crops and are harmful to humans and animals. The ability to remove AFs from feed commodities will improve health standards and counter the economic drain inflicted by AF contamination. Strategies to mitigate AF contamination fall into three categories: physical, chemical, and biological. In this thesis, I explore the identification of degraders and degradation mechanisms, as well as their enhancement, within the context of chemical and biological strategies. Known chemical strategies have used strong acids and bases to remove contaminating AF, but these methods often lead to ecological waste issues downstream. Chapter 3 investigates the application of weaker acidic and alkaline conditions to remove two types of AFs, AFB1 and AFG2. I find that a weakly alkaline environment is sufficient to degrade AF, providing an alternative solution for chemical decontamination. Biodetoxification is a promising solution to AF contamination because of its low cost and few undesired environmental side-effects. Microbes possess a rich potential for removing toxins and pollutants from the environment. Despite the fairly wide availability of this potential, identifying suitable candidates and improving them remain challenging. In Chapter 2, I explore the use of computational tools to discover strains and enzymes that detoxify harmful toxins. Of focus is the detoxification of mycotoxins by biological enzymes. Existing computational tools can be used to address questions in the discovery of new detoxification potential, the investigation the cellular processes that contribute to detoxification, and the improvement of detoxification potential in discovered enzymes. I showcase open bioremediation questions where computational researchers can contribute and highlight relevant existing and emerging computational tools that could benefit bioremediation researchers. In Chapter 4, I screen several environmental isolates for their AF detoxification ability, using AFG2. I used different carbon sources (glucose and starch) as isolation and culturing media to examine the effect of the environment on degradation ability. Overall, I find that starch medium expedites the screening process and generally improves the performance of isolates, making this a promising method for identifying new degraders and enhancing their performance. Chapter 5 highlights the characterization of degradation by two promising Rhodococcus species, R. erythropolis and R. pyridinivorans. While previous work has identified their degradation ability, further investigation into degradation mechanisms has been understudied. Here, I explore the characterization of degradation mechanisms toward enzyme identification. Finally, the appendix starts to broach the question of enhancing degradation of known degrading enzymes, the example here is laccase from the fungus Trametes versicolor. Using molecular dynamic and quantum mechanics simulations to identify mutations of interest in increasing the affinity of laccase toward AF, I create five mutants to test their degradation against the performance of wildtype. These mutants show a range of improvements against AF and showcase the efficacy of this approach to enhancement. Together, this body of work highlights the importance of understanding AF degradation for the creation of new strategies of AF mitigation. My thesis provides a framework for developing AF decontamination strategies, from identifying degraders and unlocking their mechanisms to enhancing their performance. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.