The efficiency of cellulose hydrolysis under straight saccharification and simultaneous saccharification and fermentation (SSF) conditions was evaluated using three lignocellulosic materials (switchgrass, cornstover, and poplar), which had been pretreated with dilute sulfuric acid under conditions which optimized xylose concentrations in the prehydrolysate liquid. Yields of glucose, cellobiose and ethanol obtained from the pretreated feedstocks were measured over 168 hrs. The final theoretical conversions of cellulose from pretreated switchgrass, cornstover, and poplar in straight saccharification were 85-100% (average 94%), 84-100% (average 96%), and 75-100% (average 87%), respectively, while in SSF the conversions were 84-90% (average 87%), 91-96% (average 90%), 72%-82% (average 76%), respectively. The conversion rates of poplar in straight saccharification and SSF were significantly lower than those of switchgrass and cornstover. The effects of reaction parameters such as enzyme activity, cellulose availability, and yeast cell viability on the extent of hydrolysis in straight saccharification and SSF were also studied. Results indicate that the lower glucose or ethanol yields associated with some of the poplar were due to the recalcitrant nature of its cellulose. To compare accurately the efficiencies between straight saccharification and SSF, a direct method for determining the cellulose content of the feedstocks residues resulting from SSF experiments has been developed and evaluated. The method improves on classical cellulose assays by incorporating a yeast lysing enzyme to remove yeast glucans from the feedstocks residue prior to acid hydrolysis and subsequent quantification of cellulose derived glucose. A freeze-drying step was identified as necessary to render the SSF yeast cells susceptible to enzyme lysis. The method was applied to the analysis of the cellulose and yeast-glucan content of SSF residues from the three pretreated feedstocks. Cellulose assays employing the lysing enzyme preparation demonstrated relative errors up to 7.2% when yeast-associated glucan were not removed prior to analysis of SSF residues. Enzymatic lysis of SSF yeast cells may be viewed as a general preparatory procedure to be used prior to the subsequent chemical and physical analysis of SSF residues. / Graduation date: 1996

The novel application of combination, or two stage, photochemical and microbial degradation systems for removal of resin acids from natural river water and single stage photolysis for degradation of naphthenic acids in natural river water was investigated. The organic compounds included in this project comprise naphthenic acid model compounds and mixtures as well as four resin acids. Naphthenic acids are crude oil-derived and accumulate to significant concentrations (>100 mg/L) in tailings pond water at oil sands extraction facilities. Resin acids are pulp and paper mill-derived compounds that tend to persist at low levels in receiving waters. For each compound group, analytical methods utilizing liquid chromatography negative ion electrospray ionization mass spectrometry (LC/ESI/MS) were developed. The main hurdle to developing analytical methods for the naphthenic acids and resin acids are related to their polarity, complexity, and lack of available standards for the various individual components. As well, co-extractives, such as humic and fulvic acids, tend to interfere with the detection of naphthenic acids in aquatic samples (Headley et al., 2002a). Resin acid mixtures are not as complex as the naphthenic acids, although each group of hydrocarbon acids may include several isomeric compounds. The application of photochemical degradation prior to biodegradation was proven to be effective here for rapid degradation of the resin acids. In general, the resin acid precursors were more susceptible to the photolysis than were the naphthenic acids. Through thermal maturation and increased complexity, the naphthenic acids seemingly become more resistant to degradation, as evidenced by their commercial use as anti-microbial agents and the observed resistance to photolysis noted in this research. The results of this research may be significant for the design of staged treatment for reduced microbial shock loading and increased bioavailability (defined here as the ability of microbial organisms to degrade the target contaminants) in both bioremediation systems and receiving waters. Specifically, four selected pulp and paper mill-associated resin acids were exposed to several ultraviolet/visible (UV/vis) spectrum radiation sources in water collected from the River Saale in Germany. Background resin acid concentrations were observed in water collected in 2001 and 2002 from various locations along the well-forested River Saale and a manuscript detailing these results published. Analyses of water samples collected in the pulp and paper milling region of the river (in the state of Thuringia) indicated that resin acids persist through biodegradation treatment systems and for several hundred kilometres downstream. All four resin acids were degraded by facile photochemical and microbial degradation with pseudo-first-order kinetics. Half-life values were in the ranges of 18 to 200 minutes for photolysis applications, 8 to 40 hours for biodegradation applications and 3 to 25 hours for two-stage photochemical-microbial degradation processes, in which photolysis was limited to three hours. From these results, it was shown conclusively that photolysis pre-treatment is a viable and efficient method for reducing both resin acid concentrations and the associated acute toxicity. The naphthenic acids investigated in this study were not effectively degraded via UV/vis radiation, including UV-A/UV-B radiation between 300-400 nm, near-monochromatic UV254-radiation, full spectrum artificial solar radiation and natural sunlight. The photochemical degradation potential of three model naphthenic acid compounds and three naphthenic acid mixtures (one extract from the Athabasca Oil Sands and two commercial mixtures) were examined in Athabasca River water. Photolysis at UV254 was the most successful degradation source in all instances, although most naphthenic acids were not significantly degraded by any of the radiation sources. Therefore, it was determined that photolysis is not likely to contribute significantly to environmental degradation and attenuation in the aquatic ecosystem. The results observed from the various naphthenic acids photodegradation processes, coupled with their low affinity for adsorption to soils, reveal that naphthenic acids are likely to persist in the water column. However, UV/vis radiation is capable of significantly changing the composition of mixtures in the aquatic ecosystem, but not reducing overall naphthenic acid concentrations. This may not be a beneficial as there is the potential for increased toxicity toward the lower molecular weight naphthenic acids.

Bis-GMA-containing resin-composites undergo biodegradation by human saliva derived esterases, yielding Bis-hydroxy-propoxy-phenyl-propane (Bis-HPPP). The hypothesis of this study is that the exposure of dental restorations to saliva-like esterase activities accelerates marginal bacterial microleakage. Resin-composites (Scotchbond, Z250, 3M) bonded to human dentin were incubated in either buffer, or dual-esterase media (pseudocholinesterase/cholesterol-esterase; PCE-CE) with activity levels simulating that of human saliva, for up to 90 days. Incubation solutions were analyzed for resin degradation by-products using high-performance liquid-chromatography. Post-incubation, specimens were suspended in a chemostat-based biofilm fermentor cultivating Streptococcus mutans NG8 for 7 days. Bacterial microleakage was assessed by confocal laser scanning microscopy. Bis-HPPP production, as well as depth and spatial volume of bacterial cell penetration within the interface increased with incubation time. Biodegradation and bacterial microleakage were significantly higher for 30 and 90 day PCE-CE vs. buffer incubated groups (p<0.05). Conclusion: An overall decline in interfacial integrity was observed following exposure to human saliva-like esterases over time.

Bis-GMA-containing resin-composites undergo biodegradation by human saliva derived esterases, yielding Bis-hydroxy-propoxy-phenyl-propane (Bis-HPPP). The hypothesis of this study is that the exposure of dental restorations to saliva-like esterase activities accelerates marginal bacterial microleakage. Resin-composites (Scotchbond, Z250, 3M) bonded to human dentin were incubated in either buffer, or dual-esterase media (pseudocholinesterase/cholesterol-esterase; PCE-CE) with activity levels simulating that of human saliva, for up to 90 days. Incubation solutions were analyzed for resin degradation by-products using high-performance liquid-chromatography. Post-incubation, specimens were suspended in a chemostat-based biofilm fermentor cultivating Streptococcus mutans NG8 for 7 days. Bacterial microleakage was assessed by confocal laser scanning microscopy. Bis-HPPP production, as well as depth and spatial volume of bacterial cell penetration within the interface increased with incubation time. Biodegradation and bacterial microleakage were significantly higher for 30 and 90 day PCE-CE vs. buffer incubated groups (p<0.05). Conclusion: An overall decline in interfacial integrity was observed following exposure to human saliva-like esterases over time.

The effects of natural preconditioning (source wood), wood species, and previous exposure of wood to two types of fungi on the feeding rates and protozoa survival in the Pacific dampwood termite Zootermopsis angusticollis (Isoptera: Termospidae) were examined under laboratory conditions. Termites were exposed to four wood species that had been treated previously with a non-wood decay fungus or a wood decay fungus for various lengths of time, or were untreated. Termite feeding was measured in forced feeding and choice feeding tests, after which two types of gut protozoa (Trichomitopsis spp. and Trichonympha spp.) were counted. Wood species and previous fungal exposure both affected consumption rates and protozoa numbers in Z. angusticollis. The results indicated that pre-conditioning did not affect the amount of wood consumed or protozoa numbers in forced feeding tests, but sometimes affected protozoa numbers in choice feeding tests. Exposure of various conifer woods to an early colonizing white rot fungus, Stereum sanguinolentum, affected how much wood of a particular species was consumed by dampwood termites in both forced and choice feeding tests. Exposure of these woods to a non-wood decay fungus, Trichoderma viride, had little effect on wood consumption but did affect the numbers of one type of gut protozoan, Trichoniitopsis, in choice testing. / Graduation date: 1993

The novel application of combination, or two stage, photochemical and microbial degradation systems for removal of resin acids from natural river water and single stage photolysis for degradation of naphthenic acids in natural river water was investigated. The organic compounds included in this project comprise naphthenic acid model compounds and mixtures as well as four resin acids. Naphthenic acids are crude oil-derived and accumulate to significant concentrations (>100 mg/L) in tailings pond water at oil sands extraction facilities. Resin acids are pulp and paper mill-derived compounds that tend to persist at low levels in receiving waters. For each compound group, analytical methods utilizing liquid chromatography negative ion electrospray ionization mass spectrometry (LC/ESI/MS) were developed. The main hurdle to developing analytical methods for the naphthenic acids and resin acids are related to their polarity, complexity, and lack of available standards for the various individual components. As well, co-extractives, such as humic and fulvic acids, tend to interfere with the detection of naphthenic acids in aquatic samples (Headley et al., 2002a). Resin acid mixtures are not as complex as the naphthenic acids, although each group of hydrocarbon acids may include several isomeric compounds. The application of photochemical degradation prior to biodegradation was proven to be effective here for rapid degradation of the resin acids. In general, the resin acid precursors were more susceptible to the photolysis than were the naphthenic acids. Through thermal maturation and increased complexity, the naphthenic acids seemingly become more resistant to degradation, as evidenced by their commercial use as anti-microbial agents and the observed resistance to photolysis noted in this research. The results of this research may be significant for the design of staged treatment for reduced microbial shock loading and increased bioavailability (defined here as the ability of microbial organisms to degrade the target contaminants) in both bioremediation systems and receiving waters. Specifically, four selected pulp and paper mill-associated resin acids were exposed to several ultraviolet/visible (UV/vis) spectrum radiation sources in water collected from the River Saale in Germany. Background resin acid concentrations were observed in water collected in 2001 and 2002 from various locations along the well-forested River Saale and a manuscript detailing these results published. Analyses of water samples collected in the pulp and paper milling region of the river (in the state of Thuringia) indicated that resin acids persist through biodegradation treatment systems and for several hundred kilometres downstream. All four resin acids were degraded by facile photochemical and microbial degradation with pseudo-first-order kinetics. Half-life values were in the ranges of 18 to 200 minutes for photolysis applications, 8 to 40 hours for biodegradation applications and 3 to 25 hours for two-stage photochemical-microbial degradation processes, in which photolysis was limited to three hours. From these results, it was shown conclusively that photolysis pre-treatment is a viable and efficient method for reducing both resin acid concentrations and the associated acute toxicity. The naphthenic acids investigated in this study were not effectively degraded via UV/vis radiation, including UV-A/UV-B radiation between 300-400 nm, near-monochromatic UV254-radiation, full spectrum artificial solar radiation and natural sunlight. The photochemical degradation potential of three model naphthenic acid compounds and three naphthenic acid mixtures (one extract from the Athabasca Oil Sands and two commercial mixtures) were examined in Athabasca River water. Photolysis at UV254 was the most successful degradation source in all instances, although most naphthenic acids were not significantly degraded by any of the radiation sources. Therefore, it was determined that photolysis is not likely to contribute significantly to environmental degradation and attenuation in the aquatic ecosystem. The results observed from the various naphthenic acids photodegradation processes, coupled with their low affinity for adsorption to soils, reveal that naphthenic acids are likely to persist in the water column. However, UV/vis radiation is capable of significantly changing the composition of mixtures in the aquatic ecosystem, but not reducing overall naphthenic acid concentrations. This may not be a beneficial as there is the potential for increased toxicity toward the lower molecular weight naphthenic acids.

<p>Dibenzothiophene (DBT) is a constituent of creosote and petroleum waste contamination, it is a model compound for more complex thiophenes, and its degradation by mixed microbial communities has received little attention. The chemical characteristics, environmental fate and ecotoxicology of DBT degradation products are not well understood. This research investigated DBT degradation in an enrichment culture derived from creosote-contaminated estuarian sediment using a suite of assays to monitor bacterial populations, bacterial growth, degradation products, DBT loss, and toxicity. Ultraviolet (UV) irradiation was evaluated as a sequential treatment following biodegradation. Additionally, to advance SYBR-Green qPCR methodology for characterizing mixed microbial communities, an alternative approach for evaluating qPCR data using a sigmoidal model to fit the amplification curve was compared to the conventional approach in artificial mixed communities. The overall objective of this research was to gain a comprehensive understanding of the degradation of a model heterocyclic PAH, DBT, by a mixed microbial community, particularly within the context of remediation goals.</p><p>DBT biodegradation was evaluated in laboratory scale cultures with and without pH control. The microbial community was monitored with 10 primer sets using SYBR-Green quantitative polymerase chain reaction (qPCR). Twenty-seven degradation products were identified by gas chromatography and mass spectrometry (GC/MS). The diversity of these products indicated that multiple pathways functioned in the community. DBT degradation appeared inhibited under acidic conditions. Toxicity to bioluminescent bacteria <italic>Vibrio fischeri</italic> more than doubled in the first few days of degradation, was never reduced below initial levels, and was attributed in part to one or more degradation products. UV treatment following biodegradation was explored using a monochromatic (254 nm) low-pressure UV lamp. While DBT was not extensively photooxidized, several biodegradation products were susceptible to UV treatment. At higher doses, UV treatment following DBT biodegradation exacerbated cardiac defects in <italic>Fundulus heteroclitus</italic> embryos, but slightly reduced toxicity to <italic>V. fischeri</italic>.</p><p>This research provides a uniquely comprehensive view of the DBT degradation process, identifying bacterial populations previously unassociated with PAH biodegradation, as well as potentially hazardous products that may form during biodegradation. Additionally, this research contributes to development of unconventional remediation strategies combining microbial degradation with subsequent UV treatment.</p> / Dissertation

Natural and synthetic estrogens are some of the most potent hormones detected in the environment. Agriculture fields often release higher concentrations of natural estrogens to the environment, but wastewater treatment plants (WWTPs) commonly release higher concentrations of synthetic estrogens. Estrogens can disrupt endocrine functions in wildlife and humans. Less attention has been paid to the fate and occurrence of estrogens in agricultural operations than WWTPs. Their fate is influenced by major mechanisms such as sorption and biodegradation. Sorption typically accounts for less than 10 percent of estrogen removal in WWTPs. However, biodegradation is a primary method for estrogen loss at high ammonia concentration in the agricultural and municipal operation. Less attention has been paid to the biodegradation kinetics of estrogens in the field application. Therefore, this dissertation focused on the occurrence of estrogens in agricultural fields and their biodegradation by a mixed culture and a pure culture. The estrogens in turkey litter amended fields might be biodegraded to some degree by turkey litter borne bacteria. The estrogen biodegradation by a mixed culture showed different mechanisms for each estrogen. E1 and E2 were easily degraded as a carbon source of the mixed culture. E3 and EE2 were favorable for cometabolic degradation by AOB. EE2 was not readily biodegraded by the mixed culture due to a steric hindrance of enzyme expression and EE2 metabolism in the ethynylgroup of EE2. The cometabolic kinetics of individual estrogen was evaluated by using a pure culture. The cometabolism of estrogen was demonstrated by a reductant model. This model appropriately estimated the cometabolic kinetics of individual estrogens. In addition, the effect of antibiotics on the hormone degradation was investigated in Sequencing Batch Reactors (SBRs). No significant difference was detected for the removal efficiency of target compounds in the SBRs in presence or absence of antibiotics (oxytetracycline and chlortetracycline) during long sludge retention time (SRT). However, the effluent organic matter (EfOM) was less decomposed with the presence of antibiotics, especially causing less degradation of the humic-like substances in EfOM. The results indicated the flux of antibiotics to WWTPs did not affect hormone degradation, but reduced the decomposition of humic-like substance. Finally, the findings from the research provide insight into how biodegradation influences estrogen removal in agricultural fields and municipal WWTPs. The models developed in this research yielded valuable predictive values for engineered systems.

The cyanide-degrading bacterium Klebsiella oxytoca SYSU-011 was isolated from the waste water of a metal-plating plant. In this study, we found out that K. oxytoca was capable of utilizing tetracyanonickelate {K2[Ni(CN)4]}(TCN) as its sole nitrogen source. This organism could degrade TCN both aerobically (D.O.¡×100¢H) and anaerobically (D.O.¡×0¢H).The addition of ammonia (5 mM) in the growth medium would inhibit TCN-degrading. The TCN-degrading by-product, a greenish precipitate, was found in the spent medium and was identified as nickel cyanide [Ni(CN)2] by FT-IR spectroscopic studies. Ammonia was demonstrated as a product of the TCN-degrading process by K. oxytoca resting cells. The addition of glucose could greatly enhance the TCN-degradation. Nitrogenase was found to be the cyanide degrading enzyme in this organism. The activity of nitrogenase was inhibited by ammonia but could be induced by the addition of TCN or KCN.

The purpose of this study is to isolate and identify the crude oil-degrading bacteria from oil polluted soil. Their physiological characteristics and oil-degrading capability were also studied. Eight polluted soil samples were taken from the Kaohsiung Refingery Factory of the Chinese Petroleum Corporation (CPC). The microbiota of the Kaohsiung refinery soil sample P37-2 (#6) could degrade crude oil from 2000 ppm to 572 ppm in 10 days. Bacteria in polluted soil samples were selected and isolated by minimal medium with 2000 ppm crude oil as the sole carbon source. Biochemical test, PCR-DGGE, and 16S DNA sequencing were used to identify and characterize the bacteria isolates. Three strains were identified as Pseudomonas aeruginosa (NSYSU-1-1), Acinetobacter sp. (NSYSU-4-1), and Pseudomonas sp. (NSYSU-7-1). These three strains and microbiota #6 were tested for their capability of degrading the total petroleum hydrocarbons (TPH). We found that microbiota #6 performed better than the other three bacterial strains in degrading the crude oil. In this study, we also found temperature was not the major factor of influcing the biodegradation; however, high oxygen concentration and providing nitrogen soure couled improve the biodegradation rate. Although both NSYSU-1-1 and NSYSU-7-1 are Pseudomonas strains, they performed different on degrading the oil. All strains tested could degrade the crude oil to a concentration below 1000 ppm to meet the government emission standard. The bacterial strains and techniques developed in this study provide a choice for future bioremediation of crude oil pollution.