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1791. [Article] Grape and Wine Aroma Influenced by Vine Nutrient Status, Vigor and Crop Levels in Oregon Pinot Noir
Wine volatile composition is one of the most important constitutes of wine quality, and it is greatly influenced by number of factors. The studies herein were conducted to evaluate the grape and wine aroma ...Citation Citation
- Title:
- Grape and Wine Aroma Influenced by Vine Nutrient Status, Vigor and Crop Levels in Oregon Pinot Noir
- Author:
- Yuan, Fang
Wine volatile composition is one of the most important constitutes of wine quality, and it is greatly influenced by number of factors. The studies herein were conducted to evaluate the grape and wine aroma affected by different vine nutrient status, vigor and crop levels of Oregon Pinot noir. In the first study, aroma potentials in early and late maturity Pinot noir grapes were investigated in two consecutive vintages. The grape samples were hydrolyzed under acidic conditions and the released odorants were studied by aroma extract dilution analysis (AEDA). Forty-nine main odor-active compounds were detected in the AEDA. The odorants released with high flavor dilution values were 1-hexanal, β-damascenone, guaiacol and vanillin, together with C₆ aldehydes and alcohols, 4-vinylguaiacol, 4-vinylphenol and 1-octen-3-one. The concentrations of aroma-active compounds were further quantitated by SPME-GC-MS and SBSE-GC-MS. Comparing with early harvest grapes, late harvest grapes released more β-damascenone, vanillin, 4-vinylguaiacol and 4-vinylphenol in both years according to both AEDA and quantitation results, suggesting they were important aroma compounds that contribute to the characteristic of mature Pinot noir grapes. The second study evaluated the volatile composition of grape berries subjected to varying levels of nitrogen (N), phosphorous (P) and potassium (K) supply. Pinot noir grapevines were grown in a pot-in-pot system for three years (beginning when they were 4-years-old) and fertigated with either complete nutrition (Control) or with reduced levels of either N, P, or K supply while holding all other nutrients constant. Nitrogen was varied from 7.50 mM total N supply (Control) to 1.13 mM in five discreet treatments, while P and K supply were each varied in four discreet treatments with the lowest rate of 0 mM during fertigation events. Aroma volatiles in berries were determined in each year using SPME-GC-MS technique. Results showed that reducing N supply resulted in lower concentrations of C₆ compounds and total β-damascenone in berries across all three years. Low N supply had little impact on monoterpenes. Reducing N supply resulted in higher bound form α-terpineol in two of the three years. Compared to P and K, N supply showed a more profound influence on the volatile profiles of the grape berries, indicating the important role of N in the biosynthesis of berry volatile and volatile precursors. The volatile composition of resulting wines from the same study was also investigated. Results showed that reducing N supply increased the total phenolic content in wine across all years. N supply has a profound impact on wine volatiles with the greatest effect on yeast-derived esters and higher alcohols. In general, reducing N supply decreased straight-chain esters and many straight chain alcohols, but increased the branched-chain esters, and phenethyl ester and phenethyl alcohol in wine across all years. The effect of N supply on monoterpenes in wine was not consistent from year to year. Low N wines also had reduced levels of volatile sulfur compounds in 2 of 3 years. Compared to the Control, reducing N and K supply resulted in lower quantities of total β-damascenone across all years. Altering P supply did not have reproducible effects on wine volatiles from year to year. The third study investigated the composition of Pinot noir wines produced from vines with varying vegetative vigor levels and two crop levels over three vintages (2011, 2012, and 2013) in the cool climate viticulture region of western Oregon. Wine was produced from grapes grown with two inter-row floor management treatments (tilled or grass) and two crop levels, including full crop and half crop achieved through cluster thinning. Crop thinning treatments didn’t alter the wine volatile composition. Certain wine volatiles were affected by the vineyard floor management treatments. Wines produced from grapes grown in the Grass treatment (lower vigor) had higher levels of grape-derived compounds such as α-terpineol, β-citronellol, vitispirane and TDN compared to wines produced from the Tilled treatment. Wines from Grass treatment also had higher isoamyl alcohol, phenethyl alcohol, isoamyl acetate, ethyl isobutyrate, ethyl isovalerate, and phenethyl acetate but lower levels of linalool, 1-propanol and 1-octanol. A subproject of this study investigated the carotenoid breakdown and C₁₃-norisoprenoids synthesis in Pinot noir grapes from vines of differing vigor and crop levels (yield). Carotenoid degradation and C₁₃-norisoprenoid synthesis during berry development with different vine balance was monitored from véraison to harvest in year 2013. Our data suggests that vineyard floor management had a greater influence than did cluster thinning on carotenoids composition in grapes. Grape berries from Tilled treatment vines have higher carotenoids and C₁₃-norisoprenoids than the grapes from Grass treatment vines. Multivariance analyses also showed some interactions between the vineyard floor treatments and cluster thinning treatments on the composition of carotenoids and C₁₃-norisoprenoids.
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1792. [Article] Kinetic and molecular effects of sulfate reduction on a dechlorinating culture under chemostat growth conditions
Chlorinated ethenes are common groundwater contaminants that may be treated through in-situ bioremediation. Relationships between the reducing environment, available electron donors and acceptors, reaction ...Citation Citation
- Title:
- Kinetic and molecular effects of sulfate reduction on a dechlorinating culture under chemostat growth conditions
- Author:
- Berggren, Dusty Rose V.
Chlorinated ethenes are common groundwater contaminants that may be treated through in-situ bioremediation. Relationships between the reducing environment, available electron donors and acceptors, reaction kinetics, and microbial community composition must be further understood to successfully engineer remediation schemes in the complex subsurface environment. This thesis work investigated the effect of sulfate reduction on a dehalogenating culture grown under very controlled conditions. Two chemostats containing the Point Mugu (PM) culture were maintained using an influent containing tetrachloroethene (PCE) as an electron acceptor and lactate as a fermenting electron donor. One of these chemostats, PM-5L, was used as a control, while the influent to the PM-2L chemostat was amended with sulfate on an equal electron-equivalent basis to PCE. The effluent composition of these two chemostats was monitored over time, and periodic batch rate tests and molecular analyses were performed with cells harvested from the chemostat to elucidate the changes in performance and microbial composition within the chemostat culture. A numerical model based on Monod kinetics with competitive inhibition was developed to fit data from batch PCE-to-ethene rate tests by simultaneously solving for the k[subscript m]X parameters of each CAH dechlorination step given a standard set of Ks values. Non-linear regression of multi-equilibrium VC Monod test data provided the Monod parameters (k[subscript m]X and K[subscript s]) for VC dechlorination. These parameters were used to quantify changes in dechlorinating performance of each chemostat over time and compare the performance of the two chemostats. The effluent chemical composition of the PM-5L chemostat appeared to be steady after approximately six residence times, with 1120 μM PCE being transformed to 98% ethene and 2% VC, H₂ tensions remaining between 2-3 nM, acetate around 4.3 mM, and biomass around 23 mg protein/L. Batch rate tests during this time showed rapid rates of transformation for all CAHs, agreeing well with chemostat performance. The k[subscript m]X parameters derived from the PCE-to-ethene data and the pseudo-mixed order rate coefficient of VC dechlorination determined through multi-equilibrium VC Monod tests also remained essentially constant over the one-year period of study. Changes in the PM-2L chemostat performance following the initiation of sulfate reduction were observed. Sulfate reduction began almost immediately after its addition to the chemostat, and total sulfide concentrations rose to 100-300 μM. Chemostat performance with respect to CAH and H₂ concentrations was roughly steady over approximately 250 days, with PCE being dechlorinated to 9 μM cis-DCE, 230 μM VC, and 860 μM ethene under H₂ tensions around 4 nM. Total protein levels nearly doubled during this period, increasing from 25 to 47 mg protein/L. Sulfate reduction then rapidly increased to completion, resulting in 620-720 μM dissolved sulfide and a decrease in the H₂ concentration to 2 nM. At this time, the extent of PCE dechlorination also decreased to 280, 760, and 80 μM cis-DCE, VC, and ethene, respectively. Batch rate tests showed a decrease in all chlorinated ethene reduction rates; however, VC dechlorination was the most effected by sulfate reduction, showing a 97% reduction in rate following sulfate addition. Multi-equilibrium VC rate tests were impossible to conduct following the sharp increase in sulfate reduction in the chemostat due to lack of measurable dechlorination over a days' time. A simple chemostat model employed Monod kinetics for the series of CAH reactions to determine the steady-state extent of dechlorination in the chemostat predicted by the best-fit kinetic parameters of each PCE-to-ethene rate test. The extent of dechlorination was well-modeled for the PM-5L chemostat when a H₂ limitation factor of 0.3 was applied to the rate of each CAH. Using a dual Monod kinetic model with H₂ as the electron donor, the limitation factor corresponded with a half-velocity coefficient (K[subscript H]) of 4.6 nM. When this same K[subscript H] was used to model the PM-2L chemostat, a greater extent of dechlorination was predicted than what was observed in the chemostat, possibly suggesting other inhibitory factors of dechlorination were present in the PM-2L chemostat. DNA and RNA analyses of cells periodically harvested from the chemostat were performed by Ian P.G. Marshall at Stanford University. His work revealed shifts in the chemostats' Dehalococcoides population over time. Analysis of the PM-5L culture using the H2ase chip he developed and a clone library of hupL genes showed that the Dehalococcoides population was predominately related to strains BAV-1 or CBDB1/GT and did not undergo a significant shift over time. Clone libraries constructed for cells harvested from the PM-2L chemostat revealed two shifts in the chemostat Dehalococcoides population. A genetically homogenous strain relative of BAV-1 was eliminated following a decline in chemostat H2 tensions from around 27 nM to 2 nM and a corresponding increase in dehalogenation efficiency. In a second shift, a strain 195 relative outcompeted the CBDB1/GT relatives following enhanced sulfate reduction. A general decrease in the Dehalococcoides concentration within the chemostat culture was also suggested by qPCR analysis of Dehalococcoides 16S genes. These molecular results correlated well with the decline in VC reduction rates reported in batch kinetic tests given the characteristic co-metabolic VC reduction of the dominant strain 195 relative and overall lower concentrations of Dehalococcoides. Our work suggests that sulfate reduction in the anaerobic chemostat environment caused a shift in the dechlorinating microbial population to a strain with less efficient VC reduction. This shift was also accompanied by a decline in Dehalococcoides concentration within the culture. Both of these factors contributed to the decline in chlorinated ethene transformation rates observed through batch rate tests. Competition for H₂ was not expected to be the primary cause for the changes observed in the PM-2L chemostat. Long-term batch tests involving the control culture are proposed to elucidate whether sulfide or other factors of sulfate reduction are responsible for this shift, and to confirm the suspected role of H₂ competition between dechlorination and sulfate reduction in the chemostat-grown PM culture.
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1793. [Article] Science and efficacy of mild sodium hydroxide treatments in enzyme-based wheat straw-to-glucose processing
The work described in this dissertation focused on chemistry related to the use of aqueous sodium hydroxide as a treatment in the processing of wheat straw. A major emphasis was the comprehensive evaluation ...Citation Citation
- Title:
- Science and efficacy of mild sodium hydroxide treatments in enzyme-based wheat straw-to-glucose processing
- Author:
- Sophonputtanaphoca, Supaporn
The work described in this dissertation focused on chemistry related to the use of aqueous sodium hydroxide as a treatment in the processing of wheat straw. A major emphasis was the comprehensive evaluation of straw component partitioning due to sodium hydroxide (NaOH) processing. This was evaluated over a range of NaOH concentrations (0‐10%, w/v), all at 50°C, 5 h treatment period, and 3% solid loading. Solid and liquid phases resulting from NaOH treatments were evaluated. Total solids recovered in the NaOH‐treated solid phase ranged from 47.4‐88.0%. Overall carbohydrate recovery in the combined solid and liquid phases was negatively correlated with the alkali concentration of the treatment liquor. The glucan content of the NaOH‐treated solid phase ranged from 37.2‐67.4%. Glucan recovery in the solid phase was relatively high in all cases, the minimum value being ~98%. Increasing amounts of xylan partitioned into the liquid phase as sodium hydroxide concentrations increased – it ranged from 31‐83% of the xylan being recovered in the soluble phase. Carbohydrate analyses of the pretreated liquor revealed that the majority of carbohydrate loss from the solid fraction could be recovered in the liquid phase in form of oligomers and monomers due to alkaline degradation. The interconversion of glucose, fructose, and mannose under the alkaline condition played an important role in the presence of those sugars. Increase in NaOH concentration contributed to increase in amount of cellulose‐derived and hemicellulose‐derived oligomers in the pretreated liquor. All oligomers except fructooligomers in NaOH pretreated liquor were higher than those found in water extraction at 50°C, 5 h. Total carbohydrate recovery from the solid and liquid fractions was as high as 99% for glucose and glucan in 5% NaOH treatment and 80‐95% for xylose and xylan in 1-10% NaOH treatment. The presence of NaOH as extraction reagent dramatically induced lignin and ash removal from the pretreated solid with up to 63% acid insoluble lignin (AIL) and 87% ash extraction. Solid fractions resulting from NaOH pretreatments (up to 5% NaOH) were tested for their susceptibility to enzymatic saccharification using cellulase and cellulase/xylanase enzyme preparations. The cellulase/xylanase enzyme preparation was found to be more effective at cellulose saccharification than the cellulase enzyme preparation alone. Maximum glucose yield, which corresponded to the 5% NaOH treatment, was 82% over the standard 48 h saccharification period. Extended saccharifications times to 120 h showed that the conversion yield approached 90%. Sequential treatments of the straw (i.e. initial alkali treatment – first enzyme saccharification – second alkali treatment ‐ second enzyme treatment) revealed the NaOH treatment has the potential to render essentially all (~99%) of the straw glucan susceptible to enzyme saccharification. This suggests that the layered molecular arrangement of cellulose, hemicellulose, and lignin in the cell wall impacts biomass recalcitrance and glucan conversion yield. The other major focus of this dissertation research was the characterization of alkali neutralization, which occurs during the aqueous alkali processing of wheat straw. The approach taken was to evaluate the time course of alkali uptake and to determine the underlying nature of alkali uptake. The knowledge generated from this study is useful for understanding the nature of the alkali‐induced chemistry that is at the heart of alkali processing of agricultural byproducts, foods, and forest products. Alkali uptake/acid generation measurements were monitored for wheat straw suspensions at pH 11 and 30°C. The first phase of alkali uptake corresponded to the 30‐second time period over which the pH of the wheat straw suspension was adjusted from its original pH (~6.6) to pH 11. Alkali neutralization during this period was attributed to the instantaneous ionization of solvent accessible Bronstad acids. Following pH adjustment to 11.0, the time course of subsequent alkali uptake was recorded. The time course appeared biphasic. The early phase, which corresponded to the relatively rapid uptake of alkali, was evident during the first 24 hours. The later phase, which was characterized by the relatively slow uptake of alkali, was maintained for the length of the study (up to 96 hours). Alkali uptake during the early phase of the time course appears to be determined by the rate of hydrolysis of readily accessible esters – primarily acetic acid esters (acetyl groups). Alkali uptake during the later phase of the time course appears to be impacted by the rate of alkali penetration into the straw and the rate of production of alkali‐induced acid degradation products. The uptake of alkali in the pH adjustment phase was ~ 120 μEq per gram wheat straw, the uptake of alkali in the early phase of time course was ~ 1,064 μEq per gram wheat straw, and the rate of uptake in the later phase of the time course 6.10 μEq per gram wheat straw per hour. Amount of acetyl groups, ferulic acid, and p-coumaric acid generated during 96-h pretreatment revealed that they are major esters being hydrolyzed under the studied condition. Combined, these ester-derived acids contributed up to ~ 28% of overall alkali uptake. In addition, alkaline degradation products quantified in this study showed additional ~ 28% contribution to the overall alkali uptake.
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I. THE EFFECT OF FEEDING PROGRAMS ON REPRODUCTIVE TRAITS AND SELECTED BLOOD CHEMISTRIES OF CAGED BROILER BREEDER MALES: 1. FEED RESTRICTION LEVELS: The effects of five different feeding levels (136, 125, ...
Citation Citation
- Title:
- The effects of selected feeding programs on the reproductive performance and specific blood plasma chemistries of caged broiler breeder males and dwarf breeder females
- Author:
- Buckner, Robert E.
I. THE EFFECT OF FEEDING PROGRAMS ON REPRODUCTIVE TRAITS AND SELECTED BLOOD CHEMISTRIES OF CAGED BROILER BREEDER MALES: 1. FEED RESTRICTION LEVELS: The effects of five different feeding levels (136, 125, 113, 102, and 91 g/male/day of a 13.1% crude protein (CP), 3167 kcal ME/kg feed) on the reproductive traits of adult broiler breeder males in cages were evaluated from 30 to 60 weeks of age. Individual body weights, semen volume, sperm cells per ejaculate, and fertilizing ability were measured at 30, 40, 50, and 60 weeks of age. To assist in measuring the effect of feed restriction on metabolism during these periods, average hematocrits, plasma cholesterol, total protein, and uric acid levels were analyzed. Significant (P<.05) reductions were observed in average body weights, semen volume, sperm cell numbers per ejaculate, testicular weights, and hematocrits with feed restriction at the 91 g compared to the 136 g feeding level. At 40 weeks of age the percentage of males producing semen was significantly reduced for the 102 and 91 g compared to the 136 g feeding level. Average plasma cholesterol levels were significantly increased at the 91 g compared to the 136 g feeding level, but no significant effect (P>.05) was observed in plasma total protein and uric acid levels. Correlation coefficients were negative for plasma cholesterol to body weights, sperm numbers per ejaculate, and testicular weights but positive for body weights to hematocrits and testicular weights. II. THE EFFECT OF FEEDING PROGRAMS ON REPRODUCTIVE TRAITS AND SELECTED BLOOD CHEMISTRIES OF CAGED BROILER BREEDER MALES: 2. FREE CHOICE, LOW ENERGY RATIONS OF 9, 7, AND 5 PERCENT CRUDE PROTEIN: Three isocaloric feeds consisting of 9, 7, and 5% CP each containing 2315 kcal ME/kg were fed free choice to individually caged broiler breeder males from 22 to 65 weeks of age. To obtain these protein and energy levels, sand was incorporated into the rations at levels ranging from 26.1 to 33.6% of the feed. The average daily protein intake per male was 18.7, 14.7, and 10.7 g for the 9, 7, and 5% CP, respectively, with an average caloric intake of 480 kcal ME. Individual body weights, semen characteristics, testicular weights, hematocrits, plasma cholesterol, total protein, uric acid and triglyceride levels were evaluated during each 4-week period from 24 to 44 weeks and also at 50 and 65 weeks of age. Average body weights from 24 to 32 weeks of age were significantly lower for the males fed 5% CP then those fed 9% CP rations. Semen volumes and sperm cell numbers per ejaculate were significantly reduced only at 24 weeks of age for males fed 5% CP feeds. At 65 weeks of age there were no significant differences among treatments means for body weight, sperm cells numbers per ejaculate, nor testicular weights. Plasma cholesterol levels of males fed the 5% CP diet were significantly higher at 28 and 32 weeks while levels of plasma total protein were decreased at 28 weeks of age. Plasma uric acid levels were consistently and significantly decreased as protein in take was reduced. The results suggest that within the parameters of this study broiler breeder males can be successfully maintained on low energy (2315 kcal/ kg) feeds of 7% CP from 22 to 40 weeks and then 5% CP from 40 to 65 weeks of age. III. INFLUENCES OF 16 AND 17.5 PERCENT DIETARY PROTEIN AND SUPPLEMENTAL METHIONINE ON THE REPRODUCTIVE TRAITS AND SELECTED PLASMA CHEMISTRIES OF CAGED DWARF (dw) BROILER BREEDER FEMALES: A total of 240 individually caged dwarf (dw) broiler breeder females were fed one of six isocaloric treatment diets (2740 kcal/kg) containing 16 or 17.5% CP to which d,l-methionine (MET) was supple mented at 0, .08, or .15% from 22 to 50 weeks of age. The resulting dietary MET levels ranged from .256 to .424% and total sulfur amino acids (TSAA) ranged from .527 to .718% of the rations. Treatment means were compared for body weights, mortality, total egg produc tion, egg weights, and egg fertility and hatchability resulting from artificial insemination with .05 ml broiler breeder semen. Blood plasma was analyzed at 32, 40, and 50 weeks of age for cholesterol, uric acid, total protein, and albumin as possible monitors of dietary protein utilization and of the physiological effects of protein and methionine levels on reproductive traits. There were no significant differences observed among dietary treatment means for body weights, mortality, total egg production, fertility, nor hatchability from 24 to 50 weeks of age. Significant differences were not observed among the treatment means for plasma cholesterol, total protein, and albumin at 32, 40, or 50 weeks of age. Between the two CP levels, plasma uric acid was significantly increased for the females on 17.5% compared to those on 16% CP rations. A consistent although nonsignificant trend of decreased uric acid levels with supplemental methionine was observed only in the females fed 16% CP diets and not in those on 17.5% CP. Under the conditions of this experiment, protein requirements for caged dwarf broiler breeder females from 22 to 50 weeks of age appear to be satisfied with daily dietary intakes of 20.3 g CP, 325 mg MET, and 670 mg TSAA.
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1795. [Article] Addressing wastewater epidemiology limitations with the use of dynamic population surrogates, complementary urinalyses and in-situ experiments
Wastewater epidemiology is an emerging discipline that requires collaborative research involving analytical chemists, drug epidemiologists, and wastewater engineers. Wastewater epidemiology involves the ...Citation Citation
- Title:
- Addressing wastewater epidemiology limitations with the use of dynamic population surrogates, complementary urinalyses and in-situ experiments
- Author:
- Brewer, Alex J.
Wastewater epidemiology is an emerging discipline that requires collaborative research involving analytical chemists, drug epidemiologists, and wastewater engineers. Wastewater epidemiology involves the sampling and quantitative analysis of raw wastewaters from communities for illicit drugs and their metabolites. Mass loads (mass per day) and per capita (mg per day per person) are then calculated from concentrations and indicate the approximate quantity of illicit drugs used and excreted by the community. Limitations to wastewater epidemiology include that the population served by wastewater treatment plants within a day and between days is not well known. In addition, biodegradation of illicit drugs during transit in sewers may affect the concentration and mass flows that reach wastewater treatment plants. This thesis describes a series of studies conducted by an international collaboration between scientists and engineers from the United States and Switzerland to answer these two limitations. The experimental approaches for these studies used included high-frequency wastewater sampling strategies, the use of creatinine as a human urinary biomarker, as well as the use of unique locations as test sites including an open community, a prison in the state of Oregon, and a 5 km section of sewer in Zürich Switzerland. In Chapter 2, the diurnal study on the mass flows of illicit drugs or metabolites was formed over four days in a municipality with a population of approximately 55,000 people. The diurnal trends in illicit substances vary by substance. The high (g/day) mass flows of caffeine, methamphetamine, and creatinine indicate that lower-frequency sampling (approximately one sample per h) may representatively capture the use and excretion of these substances. However, lower and episodic mass flows of cocaine and its primary human metabolite, benzoylecgonine, indicate that higher-frequency is needed to accurately assess the use of the cocaine within the municipality. Normalization of illicit substances to creatinine gave between-day trends in illicit and legal substances that differed from non-normalized trends. Resident use of cocaine and methamphetamine were indicated by normalized mass flows that increased during early morning hours while commuters are largely absent from the community. Chapter 3 describes a series of experiments conducted at an Oregon state prison. The prison setting provided a unique opportunity to study a nearly-fixed population of individuals and their corresponding mass flows of illicit substances, the number of doses per person consumed, as well as an opportunity to quantify the level of agreement between numbers of individuals and the measured mass flows of creatinine. Methamphetamine use was more prevalent than cocaine/benzoylecgonine in the prison over the one month study in which single daily (24 h) composite samples of wastewater were collected. The hypothesis that the mass flows of methamphetamine and cocaine would be lower on days on which random urinalysis testing (RUA) is typically conducted by the prison (Monday-Thursday) was rejected. While the mass flows (mg/d) of methamphetamine were less than those for a nearby open community, the number of estimated doses per person was higher for the prison population. A higher number of positive RUA results were obtained for methamphetamine while none were positive for cocaine, which is consistent with the data obtained from wastewater. The hourly (diurnal) trend in methamphetamine mass loads indicated continual methamphetamine use/excretion inside the prison while cocaine and benzoylecgonine were detected in five hourly composite samples. Use of methamphetamine and cocaine by inmates could not be unambiguously distinguished from that of non-inmates (employees and visitors). The observed diurnal trends in creatinine mass loads were similar to those of an open community and are indicative of the general pattern of human wakefulness/activity. Predicted creatinine mass loads based on the total prison (inmates + non-inmates) were in good agreement with the measured mass loads, which indicates the potential use of creatinine as a quantitative population indicator. Additional research on the biodegradability of creatinine is needed because the prison setting was deliberately selected to minimize the potential for creatinine biodegradation. Chapter 4 addresses the data gap that exists on illicit drug transformation during in situ transit in sewers. The rates of in situ biodegradation have not yet been determined for conditions that are relevant to sewers, which include low to variable oxygen concentrations, the presence of a biofilm, and temperatures ≤ 20 °C. For this reason, two tracer tests were conducted in a 5 km stretch of sewer located near Zürich, Switzerland. The stable-isotope forms (deuterated) of cocaine and benzoylecgonine were injected into flowing wastewater and three locations up to 5 km downstream were sampled over time. Breakthrough curves were constructed from measurements of cocaine-d3 and benzoylecgonine-d3 concentration with time. The area under the curve (mass) was determined by integrating concentration over time. Benzoylecgonine-d3 was present in the injectate that should have only contained cocaine-d3; because the benzoylecgonine-d3 formation prior to injection is not known. The injected mass of cocaine-d3 did not decline over the 5 km distance. The observed mass of cocaine-d3 at 5 km was 10% greater than at 500 m, which indicates that the transformation of cocaine was not significant over the 1.5 h experiment. At 5 km downgradient, the apparent mass of benzoylecgonine-d3 had increased by 35% over that observed at 500 m. However, the apparent increase in benzoylecgonine-d3 mass was not accompanied by a corresponding loss of cocaine-d3. While uncertainty is apparent in the increase of both cocaine-d3 and benzoylecgonine-d3, the ratio of cocaine-d3/benzoylecgonine-d3 is subject only to analytical error because any errors associated with sampling and the integration of masses cancel out. The ratio of cocaine-d3/benzoylecgonine-d3 declined from 2.98 in the injectate to 1.66 at Location 3, which indicated a greater increase in benzoylecgonine-d3 relative to cocaine over the 5 km distance. Due to the benzoylecgonine-d3 that was present in the injectate, any biodegradation of cocaine-c3 to form benzoylecgonine-d3 could not be unambiguously distinguished. During the second tracer test in which benzoylecgonine-d3 was injected, the mass of benzoylecgonine-d3 did not significantly decline, which suggests that the apparent loss of benzoylecgonine-d3 during the cocaine-d3 test cannot be attributed to in-situ biodegradation. Overall, while uncertainty exists about the integrated masses for cocaine-d3 and benzoylecgonine-d3, the 5 km distance was too short in order to observe a significant loss of cocaine-d3 and formation of benzoylecgonine-d3. Recommendations for future research include conducting analysis on the injectate solution to ensure that only cocaine-d3 is introduced so that any formation of benzoylecgonine-d3 is readily apparent and quantifiable. In addition, the tracer tests should be repeated in a longer section of sewer to increase the residence time beyond 1.5 hr and degradation products of benzoylecgonine-d3 should be monitored including ecgonine and ecgonine methyl ester.
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Because many coral-reef fishes are observable in situ, are amenable to transplantation, have small home ranges and short generation times, they provide a excellent system to investigate many topics within ...
Citation Citation
- Title:
- Coral-reef fishes : insights into larval dispersal and invasion ecology
- Author:
- Pusack, Timothy J.
Because many coral-reef fishes are observable in situ, are amenable to transplantation, have small home ranges and short generation times, they provide a excellent system to investigate many topics within general ecology, fisheries biology, and conservation biology. The primary goal of this dissertation was to use the coral-reef fishes system to investigate two pressing sets of issues that face marine ecologists and managers of living marine resources. The first topic is the spatial and temporal patterns of larval dispersal and reproductive success in a marine metapopulation (Chapter 2). Because miniscule larvae are difficult to track in the vast pelagic environment, little is known about the patterns of larval dispersal. Yet, the more that is understood about the spatial and temporal variability in larval dispersal, the easier it will be to identify sites that are self-sustaining and exporting larvae to unprotect sites, a common goal of marine reserves. Incorporating this information into siting of marine reserves will improve their effectiveness. The second topic is fundamental in the ecology of biological invasions: species specific interactions between an invasive predator and native species. Specifically, I investigated the ability of an invasive predator to disrupt natural population regulation of a native prey species (Chapter 3), and the ability of a native predator to provide biotic resistance against the invasive species (Chapter 4). Because management of the lionfish invasion is needed throughout the Caribbean and Atlantic waters, it is necessary to understand not only how lionfish can change the native system, but also potential ways to moderate the negative effects. To address the first topic, we collected a total of 3,278 genetic tissue samples from bicolor damselfish (Stegastes partitus) over a four year period from reefs near four islands that encompass Exuma Sound, Bahamas (Chapter 2). Using a Bayesian parentage analysis, eight parent-offspring pairs were detected, which directly documented both connectivity between and self-recruitment on an ecological time scale. Remarkably, some larvae returned to the exact same reef where they were spawned, while others traveled to sites greater than 100 km distance. The only study island without a detected parent-offspring pair, Lee Stocking Island, was also the island that showed the most restrictive gene flow on evolutionary time scales. Additionally, variability was documented in the spatial and temporal signatures of sweepstakes reproduction and Wahlund effects. The variation we observed may be influenced by seasonal mesoscale gyres present in Exuma Sound, which play a prominent role in shaping local oceanographic patterns. Understanding how to identify pathways of larval dispersal is important to designing networks of marine reserves, because a common goal of reserves is to protect populations that are self-seeding and can export larvae. Thus, this research not only demonstrates that temporal variability is a prominent characteristic of larval dispersal, but provides an example of how to identify these populations. To address the second topic, two studies using both lab observations and manipulative field experiments were conducted to study the interaction between invasive Indo-Pacific red lionfish (Pterois volitans) and two different native species. In the first study (Chapter 3) three different data sets were used to document the effect of lionfish predation on their top prey species, the bridled goby (Coryphopterus glaucofraenum). The first data set was extracted from three previous studies to compare the change in abundance of bridled goby between patch reefs with lionfish and patch reefs that were predator-free. The second data set came from laboratory feeding trials to test for the presence of a size refuge for bridled goby caused by lionfish gape limitation. The third data set came from a manipulative field experiment using 22 nearshore reefs where the per capita mortality of bridled gobies was compared among four orthogonal predator treatments: (1) predator-free control, (2) a single native predator only -- the graysby grouper (Cephalopholis cruentatus) -- representing the pre-invasion system, (3) a single lionfish only, and (4) one native grouper and one lionfish, representing the invaded system. The combined results from these three data sets demonstrated that lionfish can consistently consume a significant amount and an extremely high proportion of bridled goby on small patch reefs. While small lionfish cannot eat bridled goby larger than 0.42 times their body size, large lionfish eat virtually the entire size range of bridled goby. These findings indicate that lionfish have the potential to extirpate local goby populations. In the second experiment, lionfish were exposed to different abundances of a native grouper, the Nassau grouper (Epinephelus striatus), on 28 nearshore patch reefs in the Bahamas. Lionfish persistence and growth was monitored over 10 weeks, as well as the abundance of small, < 5 cm total length (TL), native reef fishes. Nassau grouper did not affect lionfish persistence or growth. Yet, reefs with a lionfish and many Nassau grouper saw a significant increase in the abundance of small reef fishes compared to reefs with only lionfish or lionfish with few grouper. Thus, it appears that high densities of Nassau grouper provide some biotic resistance on Bahamian patch reefs by potentially interfering with lionfish predation on native fishes, perhaps by interrupting stalking behavior. This dissertation highlights the flexibility of the coral-reef fishes system for investigation of broadly different topics in basic and applied ecology. Coral-reef fishes are a tractable system for both large and small scale studies, as well as laboratory observations and manipulative field experiments. The findings of this dissertation advance the understanding of metapopulation dynamics and have implications for fisheries management and marine reserve design. This research also documents further evidence of the negative effects of lionfish, yet provides some promising findings that may indicate some native biotic resistance to this devastating of marine invasions.
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1797. [Article] Population Genetics of American Pika (Ochotona princeps) : Investigating Gene Flow and Genetic Diversity Across Multiple, Complex Landscapes
American pikas (Ochotona princeps) are considered an indicator species of climate change. Adaptations for cold climates and active winters make pikas particularly sensitive to increasing temperatures. ...Citation Citation
- Title:
- Population Genetics of American Pika (Ochotona princeps) : Investigating Gene Flow and Genetic Diversity Across Multiple, Complex Landscapes
- Author:
- Castillo, Jessica A.
American pikas (Ochotona princeps) are considered an indicator species of climate change. Adaptations for cold climates and active winters make pikas particularly sensitive to increasing temperatures. This, combined with evidence that multiple historically occupied populations have been extirpated within the past century, contributed to American pikas becoming a focal species for climate change research. This dissertation is primarily the product of a collaboration among the National Parks Service and multiple academic institutions whose principal goal was to assess the vulnerability of the American pika within eight national parks representing a variety of habitat types and environmental conditions. While much of the previous climate change-related research on American pikas has focused on how climate affects occupancy, this work sought to establish how landscape characteristics and climate may interact and influence dispersal, population connectivity, and ultimately population vulnerability or resilience. I addressed these questions using population and landscape genetics approaches within nine national parks, two national wildlife refuges, and a national forest. American pikas represent one of the best described mammalian metapopulation systems. They are restricted to rocky habitats, such as talus and lava, which are frequently patchily distributed on the landscape, both at the local and regional scales. Functional connectivity, the combination of habitat configuration and an organism’s ability to move through the landscape, is essential for maintaining robust genetic populations. This is particularly true within metapopulations which, by definition, rely on dispersal of individuals among habitat patches for overall long-term stability. In Chapters 1 and 2, I used a landscape genetics approach to identify features that either promote or inhibit dispersal of American pikas. There has been significant debate over the past few years about the best statistical approaches within landscape genetics and the appropriateness of certain methods in particular. In Chapter 1, I used Crater Lake National Park as a test case to evaluate the effectiveness of Mantel and partial Mantel tests in a causal modeling framework to correctly identify underlying landscape variables (e.g., topography, aspect, water barriers) and their effect on gene flow. I demonstrated, through simulations, that this approach was able to correctly identify the landscape variables, but not precisely the magnitude of their effect on gene flow. I concluded that while results need to be interpreted with caution, this method was effective for identifying which variables are important in shaping functional connectivity. In Chapter 2, I applied the causal modeling approach from Chapter 1 to seven additional study sites. I identified a general trend that south-west facing aspects pose greater resistance to dispersal than north and east-facing aspects, suggesting that exposure to high temperatures may limit dispersal in American pikas. I also found that amount and configuration of habitat (i.e., rocky substrate) influenced the degree to which other landscape variables impact dispersal. I then applied the models of landscape resistance to investigate habitat patch connectivity within each site using a graph theoretic approach. This allowed me to assess overall patch connectivity, as well as identify specific patches and areas within study sites that are particularly important for maintaining functional connectivity, or in contrast, at risk of becoming isolated. Functional connectivity is an important component when assessing population vulnerability because it describes the ability of genetic material to flow through the landscape. A loss of gene flow can lead to isolation of population segments, reduction of effective population size, loss of genetic diversity and subsequent erosion of evolutionary potential, and ultimately metapopulation collapse. However, other factors including habitat area and quality, affect effective population size and population stability. Genetic diversity is the material upon which evolution acts. In the face of rapid environmental change, species must either shift their range in order to track their ecological niche, or adapt in situ to the new environment. The former strategy requires sufficient available habitat as well as dispersal ability, which is unlikely for American pikas. Genetic diversity, therefore, is an important indicator of long-term population viability. In Chapter 3, I quantified genetic diversity within thirteen study sites and investigated the relationship between genetic diversity and environmental variables related to climate as well as habitat configuration and quality. As expected, habitat area was important at both the local and regional scales. Temperature was also a significant predictor of genetic diversity, with hotter sites having lower genetic diversity. However, and somewhat surprisingly, precipitation was a better predictor of genetic diversity than temperature, with sites receiving moderate levels of precipitation having higher genetic diversity. I also compared population differentiation among study sites and identified sites that are particularly distinct. In the process of the above analyses, I also identified a previously undescribed contact zone between the northern and southern Rocky Mountain genetic lineages, recognized as separate subspecies, within Rocky Mountain National Park. Climate change is predicted to affect habitat quality and landscape permeability, both of which have detectable genetic consequences. In Chapter 4, I quantified genetic diversity and structure within two study sites, Yosemite and Lassen Volcanic National Parks, from two sampling periods separated by approximately a century in time. I extracted DNA from historic study skins housed in the University of California Berkeley Museum of Vertebrate Zoology (MVZ) and compared multilocus microsatellite genotypes to those generated from modern fecal and tissue samples. Additionally, the historic survey data, including specimens, field journals, and other artifacts, were used in a series of systematic resurveys and subsequent species distribution modelling by other researchers. The nature of national parks as protected areas affords the opportunity to isolate the effects of climate change to a greater degree than other areas that have also experienced significant land use changes. I found no evidence for a change in genetic diversity within these two sites, consistent with observations from other studies that occupancy has remained relatively stable. I did find some evidence suggesting increasing population differentiation, potentially as a result of eroding landscape permeability. These results provide an important baseline for comparison with other sites, as well as reference points for future genetic monitoring of these populations. In Chapter 5, I provide general synthesis and conclusions, as well as considerations for future research.
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1798. [Article] Effect of selenium source and supplementation rate in ewes on selenium status, passive immunity and growth performance of their lambs
Selenium (Se) is an essential micronutrient of sheep. Supplementation is especially important in young lambs in order to prevent Se-deficiency signs. In the United States, the FDA regulates Se supplementation ...Citation Citation
- Title:
- Effect of selenium source and supplementation rate in ewes on selenium status, passive immunity and growth performance of their lambs
- Author:
- Stewart, Whitney C.
Selenium (Se) is an essential micronutrient of sheep. Supplementation is especially important in young lambs in order to prevent Se-deficiency signs. In the United States, the FDA regulates Se supplementation to ruminant diets at a level of 0.3 mg/kg Se (as fed basis). Questions still exist regarding what chemical form of Se is the most bioavailable supplement and what are the best supplementation rates for optimal productivity. Three interrelated experiments from the same study were conducted 1) to evaluate the effect of Se source and supplementation rate in ewes on Se status of their lambs at birth, 2) to examine the effect of Se source and supplementation rate on IgG concentrations in ewe colostrum and lamb serum, and 3) to evaluate the effects of Se source and supplementation rate on ewe reproductive performance and subsequent vitality and growth performance of their lambs. For these experiments, 240 ewes were divided into 8 treatment groups and drenched weekly (at an amount equal to their summed daily intake) for one year, including during gestation and early lactation, with no Se (deficient); at recommended levels (0.3 mg/kg) with inorganic sodium selenite, sodium selenate, or organic Se-yeast (Se-Y); or at supranutritional levels (0.9 and 1.5 mg/kg) with sodium selenite or Se yeast. In the third experiment, 88 ewes continued the study into year two for an additional 28 weeks. Year two treatments included no Se (deficient) ewes (n = 25); ewes supplemented at recommended levels (0.3 mg/kg) with organic Se-Y (n = 20); ewes supplemented at supranutritional levels (0.9 and 1.5 mg/kg) with organic Se-Y (n = 18 and 27, respectively). In the first experiment, which assessed the effects of Se source and supplementation rate in ewes on Se status of their offspring, we found that Se administered by weekly drenching of ewes during gestation and early lactation was effective at increasing Se concentrations in ewe colostrum and milk at 30 days in milk (DIM), and in improving the Se status of lambs (whole-blood and serum Se at birth, and skeletal-muscle Se at 14 days of age) (all P < 0.001). Selenium concentrations in lacteal secretions and lambs were higher in ewes drenched with Se yeast compared to ewes drenched with inorganic Se sources (P < 0.01). Selenium concentrations in lacteal secretions and in lambs increased linearly with supranutritional concentrations of Se yeast (P < 0.001), whereas Se concentrations did not differ in ewes drenched with 0.9 or 1.5 mg/kg of inorganic sodium selenite (P > 0.05). In summary, weekly oral drenching of ewes with Se yeast during gestation and early lactation was found to be an effective method for improving Se status of their newborn lambs. In the second experiment, which examined the effects of Se source and supplementation rate in ewes on colostral IgG and lamb serum-IgG concentrations, we measured colostral and lamb serum-IgG concentrations at parturition, and lamb serum-IgG concentration again at 48 hours postnatal. Although Se drenching of ewes was effective at increasing whole-blood Se concentrations in ewes and lambs (P < 0.001), and colostral Se concentration (P < 0.001), there was no consistent or significant increase in IgG concentrations in ewe colostrum nor lamb serum at 48 hours of age (P > 0.05) irrespective of Se source, or supplementation rate. Therefore, we conclude that Se supplementation in ewes, and the Se status of newborn lambs, have little effect on IgG concentrations in colostrum and subsequent IgG concentrations in lamb serum at 48 hours postnatal. The purpose of the third experiment was to examine the effect of Se source and supplementation rate on ewe reproductive performance and subsequent vitality and growth performance of lambs, over two consecutive lambing seasons. In year one, lambing percent; number of lambs born per ewe; lamb birth weights; 90- and 120-day BW and ADG; and pounds of lamb weaned per ewe were measured. The purpose of the second year of this study was to continue our observations for the SeY treated ewes, adding additional parameters to monitor ewe reproductive performance (percentage of ewes exhibiting estrus; percentage of ewes exhibiting estrus that lambed; percentage of ewes lambing; and number of lambs born per ewe). Year two lamb performance measures included birth weights; neonatal vigor scores; 10-, 20-, and 60-day BW and ADG; and percentage of lambs surviving to 60 days. In year one, Se supplementation, regardless of supplementation rate, did not affect lambing percent, number of lambs born per ewe, lamb birth weights, 90-day BW and ADG, or pounds of lamb weaned per ewe. However, lambs from ewes in the 1.5 mg/kg SeY group had greater120-day BW (P = 0.10) and ADG (P = 0.06) than lambs from ewes in the 0.3 mg/kg group. This same trend was apparent in a subgroup of lambs from Suffolk ewes receiving Se at 1.5 mg/kg that had greater 120-day BW (P = 0.07) and ADG (P = 0.09) compared to lambs receiving SeY at 0.3 mg/kg. Similarly, a subgroup of lambs reared as twins from ewes receiving Se at 1.5 mg/kg had greater120-day BW (P = 0.07) and ADG (P = 0.08) than lambs reared as twins from ewes in the 0.3 mg/kg group. In year two, there were two ewe reproductive performance measures that were negatively affected by Se supplementation. First, the percentage of ewes exhibiting estrus that lambed in the 0.3 mg/kg SeY group (75%) was lower compared to ewes receiving no Se supplement (100%; P = 0.02). Also, the percentage of ewes lambing was lower (P< 0.04) for ewes receiving 0.3 mg/kg SeY (75%) compared to ewes receiving no Se supplemental (96%). Se supplementation, regardless of source and supplementation rate did not affect percentage of ewes exhibiting estrus, or number of lambs born per ewe. There were three lamb performance measures that were affected by Se supplementation. Lambs from ewes receiving Se at 1.5 mg/kg tended to have greater lamb vigor scores compared to lambs from ewes receiving SeY at 0.3 mg/kg (P = 0.07). Second, the 60-day ADG was greater for lambs from ewes in the 1.5 mg/kg group compared to lambs from ewes in the no Se group (P =0.07). A subgroup of lambs from Suffolk ewes receiving Se at 1.5 mg/kg had greater 60-day BW (P = 0.03) and ADG (P = 0.06) compared to lambs receiving SeY at 0.3 mg/kg. Similarly, a subgroup of lambs reared as twins from ewes receiving Se at 1.5 mg/kg had greater 60-day BW (P = 0.08) and ADG (P = 0.06) than lambs reared as twins from ewes in the 0.3 mg/kg group. Third, there was an increased percentage of lambs surviving to 60 days in lambs from ewes in the 1.5 mg/kg group compared to lambs from ewes in the no Se group (86% vs. 64%, respectively; P =0.04). Se supplementation, regardless of source and supplementation rate, did not affect lamb birth weight nor 10- and 20-day BW and ADG. We conclude that supranutritional Se supplementation of ewes, primarily with SeY, positively affects later lamb performance measures (i.e., 60- and 120-day BW and ADG), and that these effects may be more pronounced for lambs of heavier breed (i.e., Suffolk) and lambs reared as twins.
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The forest alpine tundra ecotone (FTE, also known as alpine treeline or subalpine parkland), is a conspicuous feature of mountain landscapes throughout the world. Climate change-driven increases in temperature ...
Citation Citation
- Title:
- Patterns of tree establishment and vegetation composition in relation to climate and topography of a subalpine meadow landscape, Jefferson Park, Oregon, USA
- Author:
- Zald, Harold Samuel James
The forest alpine tundra ecotone (FTE, also known as alpine treeline or subalpine parkland), is a conspicuous feature of mountain landscapes throughout the world. Climate change-driven increases in temperature are believed to result in FTE movement and tree invasion of subalpine meadows, which have been documented throughout the Northern Hemisphere across a wide range of geographic locations, climatic regimes, forest types, land use histories, and disturbance regimes. Climate-driven FTE movement may have numerous ecological effects such as: positive temperature feedbacks, increased net primary productivity and carbon storage, and declines of plant populations and species. The magnitude of these ecological effects is highly uncertain, but will be largely determined by the rates and spatial extent of FTE movement and meadow invasion. FTE movement and meadow invasion are often considered at global or regional spatial scales in relation to climate, yet they are fundamentally driven by tree regeneration processes that are influenced by a variety of climatic and biophysical factors at micro site, landscape, and regional scales. Much of the research on the FTE has not taken a landscape approach incorporating multi-scale processes. For example, species distribution models used to project climate change effects on future species distributions and plant biodiversity in mountainous landscapes rely on species distribution data that is often sparse and incomplete across FTE landscapes. This dissertation attempts to overcome many of the limitations in FTE research by taking a landscape approach to develop a greater understanding of past spatiotemporal patterns of tree invasion, current spatial patterns of vegetation composition and structure, and potential future patterns of climate-driven tree invasion in the FTE. The setting for this research is Jefferson Park, a 260 ha subalpine parkland landscape in the Oregon High Cascades, USA. This study uses field plots, remotely sensed imagery, airborne Light Detection and Ranging (LiDAR), and simulation modeling to: 1) predictively map current fine-scale species distributions, vegetation structure, and tree ages; 2) reconstruct patterns of tree invasion over the last fifty years in subalpine meadows in relation to climatic conditions, landforms, microtopography, and seed dispersal limitations; and 3) develop a statistical model that projects future patterns of tree invasion into subalpine meadows under different climate scenarios in Jefferson Park. In chapter two, I generated fine-scale spatially-explicit predictions of current vegetation composition, structure, and tree ages in the Jefferson Park study area. Objectives of this chapter were threefold: 1) to characterize spatial patterns of tree ages, vegetation composition, and vegetation structure in a FTE landscape in the Oregon Cascades using predictive mapping; 2) determine how vegetation composition and structure were associated with gradients of environmental factors derived from multispectral satellite imagery and Light Detection and Ranging (LiDAR) data; and 3) determine if predictive mapping characterizations of tree age, vegetation composition, and vegetation structure were improved by the inclusion of LiDAR data. Predictive mapping of vegetation attributes was accomplished using gradient analysis with nearest neighbor imputation; integrating field plots, multispectral SPOT 5 satellite imagery, and LiDAR data. Vegetation composition was best described by SPOT 5 imagery and LiDAR-derived topography, while vegetation structure was best described by LiDAR-derived vegetation heights. Predictions of species occurrence were most accurate for tree species, moderate for shrub species and vegetation groups, and highly variable for graminoid species. Tree age, which was the most accurately predicted vegetation structure variable, indicates the study area was largely un-forested in 1600, gradually invaded by trees from 1600 to the 1920's, and rapidly invaded from the 1920's to 1980. Predictive mapping of vegetation structure variables such as basal area and stand density were subject to large amounts of error, possibly resulting from scale incompatibilities between vegetation patterns and plot size, and/or heterogeneous FTE landscapes where forest structure does not develop along consistent trajectories with stand age. This study suggests integrating multispectral satellite imagery, LiDAR data, and field plots can accurately predict fine-scale spatial characterizations of species distributions and tree invasion in the FTE. This study also indicates that sample design can influence spatial patterns of model uncertainty, which needs to be considered if predictive mapping of vegetation and sensitive ecosystems is a component of inventory and monitoring programs. In chapter three, I focused on quantifying spatiotemporal patterns of subalpine parkland tree invasion in Jefferson Park over the past five decades in relation multi-scale climatic and biophysical controls. LiDAR data provided previously unavailable fine-scale spatial characterizations of microtopography and vegetation structure. I utilized LiDAR, georeferenced field plots, and tree establishment reconstructions to quantify spatiotemporal patterns of tree invasion in relation to late season snow persistence, landform types, fine-scale topographic variability, distances from potential seed sources, and climate variation within 130 ha of the subalpine parkland landscape of Jefferson Park. Tree occurrence (i.e. tree presence in 2 m plots and grid cells) occurred in 7.75% of study area meadows in 1950 and increased to 34.7% in 2007. Landform types and finer-scale patterns of topography and vegetation structure influenced summer snow depth, which influenced temporal and spatial patterns of tree establishment. Tree invasion rates were higher on debris flow landforms, which had lower summer snow depth than glacial landforms, suggesting potentially rapid treeline responses to disturbance events. Tree invasion rates were strongly associated with reduced annual snow fall on glacial landforms, but not on debris flows. Tree establishment was spatially constrained to micro sites with high topographic positions and close proximity to overstory canopy, site conditions associated with low summer snow depth. Seed source limitations placed an additional species-specific spatial constraint on where trees invaded meadows. Climate and topography had an interactive effect, with trees establishing on higher topographic positions during both high snow/low temperature and low snow/high temperature periods, but had greater than expected establishment on lower topographic positions during low snow/high temperature periods. Within the context of larger landform types, topography and proximity to overstory trees constrained where trees established in the meadows, even during climate periods with higher temperatures and lower snowfall. Results of this study suggest large scale climate-driven models of vegetation change may overestimate treeline movement and meadow invasion, because they do not account for biophysical controls limiting tree establishment at multiple spatial scales. In chapter four, I used field data and analyses from chapter 3 to parameterize a spatially and temporally explicit statistical model of fine-scale tree invasion within 130 ha of the Jefferson Park study area. The model incorporated both the climatic and biophysical controls found in chapter 3 to influence tree invasion. The model was used in two ways: (1) to spatially project patterns of tree invasion from 1950 to 2007 in response to historical climate; and (2) to project future tree invasion of the study area from 2007 to 2064 under six different annual snowfall scenarios. Modeling addressed the following questions: (1) Can fine-scale (2 m pixel size) patterns of historical tree invasion be accurately predicted? (2) How sensitive is future tree invasion (and therefore meadow persistence) to different future snowfall scenarios? (3) Are non-climatic factors such as landforms and biotic interactions associated with different spatial patterns of tree invasion? From 1950 to 2007, simulated historical meadow area declined from 82% to 65% of the study area. Model outputs of historical area, spatial distributions, and spatial clustering of tree invasion generally agreed with independent validation, and suggest biotic interactions due to young tree establishment facilitation are important on glacial landforms but not debris flows. Simulations of future scenarios indicated meadow declined to 36 to 43% of the study area by 2064. Projected meadow area declined with reduced annual snow fall, but not under prolonged high and low snow fall periods. Meadows persisted under all future scenarios in 2064. This model suggests subalpine meadows may significantly decline under climate warming, but will still persist in 2064. Micro sites and recruitment limitation may be equally or more important factors than climate change in influencing subalpine landscape change, suggesting local high-elevation persistence of subalpine meadows under future climate warming.
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1800. [Article] Anaerobic biodegradation of the cyclic nitramines, RDX and HMX, by ovine ruminal microbes
Cyclic nitramines released into all environmental compartments through anthropogenic activities are toxic and possibly carcinogenic and mutagenic. Soils on military ranges, located throughout the world ...Citation Citation
- Title:
- Anaerobic biodegradation of the cyclic nitramines, RDX and HMX, by ovine ruminal microbes
- Author:
- Eaton, Hillary L.
Cyclic nitramines released into all environmental compartments through anthropogenic activities are toxic and possibly carcinogenic and mutagenic. Soils on military ranges, located throughout the world in various climatic regions and close to human activities, are especially susceptible to cyclic nitramine contamination. The properties of soils on military ranges will directly affect the cyclic nitramine contamination and biodegradation; however, studies in this area are limited. Previous related research was mainly focused on monitoring the temporal and spatial changes in nitrate explosive concentrations and various in situ and ex situ remediation techniques. Anaerobic biodegradation of cyclic nitramines in soils is scarce, but appears to be a more efficient means of bioremediation than aerobic techniques; however, most published research has not been applied in situ as there are numerous hindrances to applications in the field. The present research therefore aims to study the feasibility of ruminal bioremediation as an inexpensive, ecologically conscious, and viable means to remediate soils on military ranges contaminated with cyclic nitramines by (1) determining if RDX degradation in whole ovine rumen fluid occurs and isolating and identifying organisms capable of degradation through enrichments; (2) evaluating the ability of 24 commonly isolated bacteria from the rumen to degrade RDX and determining the metabolite pathway by capable isolates, as well as by consortia in whole rumen fluid; and (3) exploring HMX degradation in whole ovine rumen fluid and identifying the HMX-degradation pathway in whole rumen fluid and by capable isolates tested. Bioremediation is of great interest in the detoxification of soil contaminated with residues from explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX). Although there are numerous forms of in situ and ex situ bioremediation, ruminants would provide the option of an in situ bioreactor that could be transported to the site of contamination. Bovine rumen fluid has been previously shown to transform 2,4,6- trinitrotoluene (TNT), a similar compound, in 4 h. In this study, RDX incubated in whole ovine rumen fluid was nearly eliminated within 4 h. Whole ovine rumen fluid was then inoculated into five different types of media to select for archaeal and bacterial organisms capable of RDX biotransformation. Cultures containing 30 μg mL⁻¹ RDX were transferred each time the RDX concentration decreased to 5 μg mL⁻¹ or less. Time point samples were analyzed for RDX biotransformation by HPLC. The two fastest transforming enrichments were in methanogenic and low nitrogen basal media. After 21 days, DNA was extracted from all enrichments able to partially or completely transform RDX in 7 days or less. To understand microbial diversity, 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) finger- printing was conducted. Cloning and sequencing of partial 16S rRNA fragments were performed on both low nitrogen basal and methanogenic media enrichments. Phylogenetic analysis revealed similar homologies to eight different bacterial and one archaeal genera classified under the phyla Firmicutes, Actinobacteria, and Euryarchaeota. After continuing enrichment for RDX degraders for 1 year, two consortia remained: one that transformed RDX in 4 days and one which had slowed after 2 months of transfers without RDX. DGGE comparison of the slower transforming consortium to the faster one showed identical banding patterns except one band. Homology matches to clones from the two consortia identified the same uncultured Clostridia genus in both; Sporanaerobacter acetigenes was identified only in the consortia able to completely transform RDX. This is the first study to examine the rumen as a potential bioremediation tool for soils contaminated with RDX, as well as to discover S. acetigenes in the rumen and its potential ability to metabolize this energetic compound. The ability of ruminal microbes to utilize the explosive compound RDX, in both ovine whole rumen fluid and 24 individual bacterial isolates from the rumen was examined. Compound degradation was determined by high performance liquid chromatography (HPLC) analysis, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of metabolites. Organisms in whole rumen fluid microcosms were able to degrade 180 μM RDX within 4 h. In whole rumen fluid, the concentrations of all mono-, di-, and tri-nitroso intermediates formed due to reduction of the nitro groups on RDX amounted to approximately 9 uM at 24 hours, which represented one pathway to ring cleavage. The appearance of peak m/z 175, hexahydro-1,3-dinitro-1,3,5-triazine, represented a second pathway that RDX followed after reduction to the first nitroso intermediate, MNX. Ruminal isolates were able to degrade RDX between ranges of 34 to 256 μM in 120 hours to unidentified ring cleavage metabolites. Clostridium polysaccharolyticum and Desulfovibrio desulfuricans subsp. desulfuricans were able to degrade RDX when it was supplemented in addition to nitrogen and carbon. Anaerovibrio lipolytica, Prevotella ruminicola and Streptococcus bovis IFO were able to degrade RDX when supplemented as a sole source of nitrogen. This data indicated that while several rumen bacterial strains may have the individual ability to degrade RDX, bioremediation in whole rumen fluid was more efficient. We proposed a pathway of RDX degradation by ruminal microbes under anaerobic conditions that involved the reduction of RDX to MNX, with trace amounts further reduced to DNX and TNX. The majority of the MNX was further degraded via a second pathway to hexahydro- 1,3-dinitro-1,3,5-triazine (peak m/z 175), which appeared to be an unstable molecule that resulted in rapid ring cleavage and degradation to unidentified metabolites. This was the first study to demonstrate that whole rumen fluid would be a more efficient means of bioremediation than individual ruminal isolates, based on the speed and completion of degradation of RDX; and that several ruminal isolates had the ability to degrade RDX in vitro. The octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) molecule is an eight-membered ring of alternating carbon and nitrogen atoms, with a nitro group attached to each nitrogen atom. The ability of ruminal microbes to degrade HMX as consortia in whole rumen fluid and as 23 common ruminal isolates was examined by LC-MS/MS analysis. The initial concentration of HMX was 27 μM and whole rumen fluid was incubated with HMX for 24 hours under anaerobic conditions in the dark, while the isolates were incubated with 17 μM HMX for 120 hours, along with negative controls. All experiments were repeated in triplicate. Our results demonstrated that HMX was nearly completely degraded in whole rumen fluid in four hours. Peaks at m/z 149 and m/z 193 suggest ring cleavage through the mono-nitroso intermediate, (1-NO-HMX), reduction pathway; and via hydroxylamino-HMX derivatives, as a second pathway. None of the 23 ruminal isolates tested were able to degrade HMX when supplemented in either a low carbon basal medium or low nitrogen basal medium in pure culture in 120 hours under the conditions specified. This is the first study to prove ruminal microbes can degrade HMX and to propose metabolic pathways, which could lead to the application of ruminal bioremediation on contaminated military soils. In summary, the present research revealed that ruminal organisms were not only capable of degrading the cyclic nitramines, RDX and HMX, in less than 24 hours, but the efficiency of degradation in the strongly reduced environment of the rumen surpassed other means of remediating explosive compounds through aerobic or alternate anaerobic techniques.