Release from Streambed to Water Column during Baseflow Periods: A Modeling Study.

Streambed sediments can harbor large populations that are released into the water column during high-flow events. Few studies have been conducted on the rates of transfer from streambed sediment to water column in low-flow conditions in natural streams. The aim of this work was to apply the watershed-scale model SWAT (Soil and Water Assessment Tool) to a natural stream to evaluate the need to account for the release from streambed sediments during baseflow periods and to compare the results of simulating such a release by assuming predominantly passive transport, driven by groundwater influx, against simulations assuming predominantly active transport of random or chemotaxis-driven bacteria movement. concentrations in water during baseflow periods were substantially underestimated when release from the streambed was attributed only to streambed sediment resuspension. When considered in addition to the release due to sediment resuspension at high flows, the active and passive release assumptions provided 42 and 4% improvement, respectively, in the RMSE of logarithms of concentrations. Estimated fluxes to water column during the baseflow periods from June to November ranged from 3.3 × 10 colony-forming units (CFU) m d in the game land area to 1.4 × 10 CFU m d in the mixed pasture and cropland. Results demonstrate that release of from streambed sediments during baseflow periods is substantial and that water column concentrations are dependent on not only land management practices but also on in-stream processes.

Modeling the interannual variability of microbial quality metrics of irrigation water in a Pennsylvania stream.

Knowledge of the microbial quality of irrigation waters is extremely limited. For this reason, the US FDA has promulgated the Produce Rule, mandating the testing of irrigation water sources for many farms. The rule requires the collection and analysis of at least 20 water samples over two to four years to adequately evaluate the quality of water intended for produce irrigation. The objective of this work was to evaluate the effect of interannual weather variability on surface water microbial quality. We used the Soil and Water Assessment Tool model to simulate E. coli concentrations in the Little Cove Creek; this is a perennial creek located in an agricultural watershed in south-eastern Pennsylvania. The model performance was evaluated using the US FDA regulatory microbial water quality metrics of geometric mean (GM) and the statistical threshold value (STV). Using the 90-year time series of weather observations, we simulated and randomly sampled the time series of E. coli concentrations. We found that weather conditions of a specific year may strongly affect the evaluation of microbial quality and that the long-term assessment of microbial water quality may be quite different from the evaluation based on short-term observations. The variations in microbial concentrations and water quality metrics were affected by location, wetness of the hydrological years, and seasonality, with 15.7-70.1% of samples exceeding the regulatory threshold. The results of this work demonstrate the value of using modeling to design and evaluate monitoring protocols to assess the microbial quality of water used for produce irrigation.

Enrichment of stream water with fecal indicator organisms during baseflow periods.

Fecal indicator organisms (FIOs) are generally believed to be present in surface waters due solely to direct deposition of feces or through transport in runoff. However, emerging evidence points toward hyporheic exchange between sediment pore water and the overlying water column during baseflow periods as a source of FIOs is surface waters. The objective of this work was to (a) propose a mass balance-based technique for estimating changes of FIO concentrations in the same volume of water (or "slug") from the inlet to outlet of stream reaches in baseflow conditions and (b) to use such enumeration to estimate rate of the FIO release to stream water column. Concentrations of Escherichia coli (E. coli) and enterococci were measured in the slug while simultaneously monitoring the movement of a conservative tracer, Br that labeled the slug. Concentrations of E. coli in the slug were significantly larger (P = 0.035, P = 0.001, and P = 0.001, respectively) at the outlet reach in all three replications, while enterococci concentrations were significantly larger in two of three replications (P = 0.001, P < 0.001, and P = 0.602). When estimated without accounting for die-off in water column, FIO net release rates across replications ranged from 36 to 57 cells m-2 s-1 and 43 to 87 cells m-2 s-1 for E. coli and enterococci, respectively. These release rates were 5 to 20% higher when the die-off in water column was taken into account. No diurnal trends were observed in indicator concentrations. No FIO sources other than bottom sediment have been observed during the baseflow period. FIOs are released into stream water column through hyporheic exchange during baseflow periods.

Can E. coli or thermotolerant coliform concentrations predict pathogen presence or prevalence in irrigation waters?

An increase in food-borne illnesses in the United States has been associated with fresh produce consumption. Irrigation water presents recognized risks for microbial contamination of produce. Water quality criteria rely on indicator bacteria. The objective of this review was to collate and summarize experimental data on the relationships between pathogens and thermotolerant coliform (THT) and/or generic E. coli, specifically focusing on surface fresh waters used in or potentially suitable for irrigation agriculture. We analyzed peer-reviewed publications in which concentrations of E. coli or THT coliforms in surface fresh waters were measured along with concentrations of one or more of waterborne and food-borne pathogenic organisms. The proposed relationships were significant in 35% of all instances and not significant in 65% of instances. Coliform indicators alone cannot provide conclusive, non-site-specific and non-pathogen-specific information about the presence and/or concentrations of most important pathogens in surface waters suitable for irrigation. Standards of microbial water quality for irrigation can rely not only on concentrations of indicators and/or pathogens, but must include references to crop management. Critical information on microbial composition of actual irrigation waters to support criteria of microbiological quality of irrigation waters appears to be lacking and needs to be collected.

Whole genome sequencing of diverse Shiga toxin-producing and non-producing Escherichia coli strains reveals a variety of virulence and novel antibiotic resistance plasmids.

The genomes of a diverse set of Escherichia coli, including many Shiga toxin-producing strains of various serotypes were determined. A total of 39 plasmids were identified among these strains, and many carried virulence or putative virulence genes of Shiga toxin-producing E. coli strains, virulence genes for other pathogenic E. coli groups, and some had combinations of these genes. Among the novel plasmids identified were eight that carried resistance genes to aminoglycosides, carbapenems, penicillins, cephalosporins, chloramphenicol, dihydrofolate reductase inhibitors, sulfonamides, tetracyclines and resistance to heavy metals. Two of the plasmids carried six of these resistance genes and two novel IncHI2 plasmids were also identified. The results of this study showed that plasmids carrying diverse resistance and virulence genes of various pathogenic E. coli groups can be found in E. coli strains and serotypes regardless of the isolate's source and therefore, is consistent with the premise that these mobile elements carrying these traits may be broadly disseminated among E. coli.

Survival of Manure-borne and Fecal Coliforms in Soil: Temperature Dependence as Affected by Site-Specific Factors.

Understanding pathogenic and indicator bacteria survival in soils is essential for assessing the potential of microbial contamination of water and produce. The objective of this work was to evaluate the effects of soil properties, animal source, experimental conditions, and the application method on temperature dependencies of manure-borne generic , O157:H7, and fecal coliforms survival in soils. A literature search yielded 151 survival datasets from 70 publications. Either one-stage or two-stage kinetics was observed in the survival datasets. We used duration and rate of the logarithm of concentration change as parameters of the first stage in the two-stage kinetics data. The second stage of the two-stage kinetics and the one-stage kinetics were simulated with the model to find the dependence of the inactivation rate on temperature. Classification and regression trees and linear regressions were applied to parameterize the kinetics. Presence or absence of two-stage kinetics was controlled by temperature, soil texture, soil water content, and for fine-textured soils by setting experiments in the field or in the laboratory. The duration of the first stage was predominantly affected by soil water content and temperature. In the model dependencies of inactivation rates on temperature, parameter estimates were significantly affected by the laboratory versus field conditions and by the application method, whereas inactivation rates at 20°C were significantly affected by all survival and management factors. Results of this work can provide estimates of coliform survival parameters for models of microbial water quality.

Irrigation waters and pipe-based biofilms as sources for antibiotic-resistant bacteria.

The presence of antibiotic-resistant bacteria in environmental surface waters has gained recent attention. Wastewater and drinking water distribution systems are known to disseminate antibiotic-resistant bacteria, with the biofilms that form on the inner-surfaces of the pipeline as a hot spot for proliferation and gene exchange. Pipe-based irrigation systems that utilize surface waters may contribute to the dissemination of antibiotic-resistant bacteria in a similar manner. We conducted irrigation events at a perennial stream on a weekly basis for 1 month, and the concentrations of total heterotrophic bacteria, total coliforms, and fecal coliforms, as well as the concentrations of these bacterial groups that were resistant to ampicillin and tetracycline, were monitored at the intake water. Prior to each of the latter three events, residual pipe water was sampled and 6-in. sections of pipeline (coupons) were detached from the system, and biofilm from the inner-wall was removed and analyzed for total protein content and the above bacteria. Isolates of biofilm-associated bacteria were screened for resistance to a panel of seven antibiotics, representing five antibiotic classes. All of the monitored bacteria grew substantially in the residual water between irrigation events, and the biomass of the biofilm steadily increased from week to week. The percentages of biofilm-associated isolates that were resistant to antibiotics on the panel sometimes increased between events. Multiple-drug resistance was observed for all bacterial groups, most often for fecal coliforms, and the distributions of the numbers of antibiotics that the total coliforms and fecal coliforms were resistant to were subject to change from week to week. Results from this study highlight irrigation waters as a potential source for antibiotic-resistant bacteria, which can subsequently become incorporated into and proliferate within irrigation pipe-based biofilms.

Solid Manure As a Source of Fecal Indicator Microorganisms: Release under Simulated Rainfall.

Understanding and quantifying microbial release from manure is a precondition to estimation and management of microbial water quality. The objectives of this work were to determine the effects of rainfall intensity and surface slope on the release of Escherichia coli, enterococci, total coliforms, and dissolved chloride from solid dairy manure and to assess the performance of the one-parametric exponential model and the two-parametric Bradford-Schijven model when simulating the observed release. A controlled-intensity rainfall simulator induced 1 h of release in runoff/leachate partitioning boxes at three rainfall intensities (30, 60, and 90 mm h(-1)) and two surface slopes (5% and 20%). Bacterial concentrations in initial release were more than 1 order of magnitude lower than their starting concentrations in manure. As bacteria were released, they were partitioned into runoff and leachate at similar concentrations, but in different volumes, depending on slope. Bacterial release occurred in two stages that corresponded to mechanisms associated with release of manure liquid- and solid-phases. Parameters of the two models fitted to the bacterial release dependencies on rainfall depth were not significantly affected by rainfall intensity or slope. Based on model performance tests, the Bradford-Schijven model is recommended for simulating bacterial release from solid manure.

Release and Removal of Microorganisms from Land-Deposited Animal Waste and Animal Manures: A Review of Data and Models.

Microbial pathogens present a leading cause of impairment to rivers, bays, and estuaries in the United States, and agriculture is often viewed as the major contributor to such contamination. Microbial indicators and pathogens are released from land-applied animal manure during precipitation and irrigation events and are carried in overland and subsurface flow that can reach and contaminate surface waters and ground water used for human recreation and food production. Simulating the release and removal of manure-borne pathogens and indicator microorganisms is an essential component of microbial fate and transport modeling regarding food safety and water quality. Although microbial release controls the quantities of available pathogens and indicators that move toward human exposure, a literature review on this topic is lacking. This critical review on microbial release and subsequent removal from manure and animal waste application areas includes sections on microbial release processes and release-affecting factors, such as differences in the release of microbial species or groups; bacterial attachment in turbid suspensions; animal source; animal waste composition; waste aging; manure application method; manure treatment effect; rainfall intensity, duration, and energy; rainfall recurrence; dissolved salts and temperature; vegetation and soil; and spatial and temporal scale. Differences in microbial release from liquid and solid manures are illustrated, and the influential processes are discussed. Models used for simulating release and removal and current knowledge gaps are presented, and avenues for future research are suggested.

Differences in biofilm formation of produce and poultry Salmonella enterica isolates and their persistence on spinach plants.

Spinach plants were irrigated biweekly with water containing 2.1 log CFU Salmonella/100 ml water (the maximum Escherichia coli MPN recommended by the Leafy Greens Marketing Agreement; LGMA), or 4.1 CFU Salmonella/100 ml water to determine Salmonella persistence on spinach leaves. Green Fluorescent protein expressing Salmonella were undetectable by most-probable number (MPN) at 24 h and 7 days following each irrigation event. This study indicates that Salmonella are unlikely to persist on spinach leaves when irrigation water is contaminated at a level below the LGMA standards. In a parallel study, persistence of Salmonella isolated from poultry or produce was compared following biweekly irrigation of spinach plants with water containing 6 log CFU Salmonella/100 ml. Produce Salmonella isolates formed greater biofilms on polystyrene, polycarbonate and stainless steel surfaces and persisted at significantly higher numbers on spinach leaves than those Salmonella from poultry origin during 35 days study. Poultry Salmonella isolates were undetectable (<1 log CFU/g) on spinach plants 7 days following each irrigation event when assayed by direct plating. This study indicates that Salmonella persistence on spinach leaves is affected by the source of contamination and the biofilm forming ability of the strain.

Effectiveness of vegetated filter strips in retention of Escherichia coli and Salmonella from swine manure slurry.

Vegetated filter strips (VFS) are commonly recommended as a best management practice to prevent manure-borne microorganisms from reaching surface water resources. However, relatively little is known about the efficacy of VFS in mitigating bacterial runoff from land-applied swine manure. A field lysimeter study was designed to evaluate the effect of surface soil hydrologic conditions and vegetation on the retention of swine manure-borne Escherichia coli and Salmonella under simulated rainfall conditions. Experimental plots (6.5 m × 3.9 m) were set on a 5% slope lysimeter with loamy topsoil, clay loam or loam subsoil and a controllable groundwater level. Three small flow-intercepting miniflumes were installed 4.5 m from the plot's top, while all remaining runoff was collected in a gutter at the bottom. Plots were divided into bare soil and grass vegetation and upper surface soil moisture before rainfall events was controlled by the subsurface groundwater level. Swine manure slurry inoculated with E. coli and Salmonella, and with added bromide tracer, was applied on the top of the plots and simultaneously initiated the simulated rainfall. Runoff was collected and analyzed every 5 min. No substantial differences between retention of E. coli and Salmonella were found. In initially wet soil surface conditions, there was limited infiltration both in bare and in vegetated plots; almost all bromide and about 30% of bacteria were recovered in runoff water. In initially dry soil surface conditions, there were substantial discrepancies between bare and vegetated plots. In bare plots, recoveries of runoff water, bromide and bacteria under dry conditions were comparable to wet conditions. However, in dry vegetated plots, from 50% to 75% of water was lost to infiltration, while bromide recoveries ranged from 14 to 36% and bacteria recovery was only 5%. Substantial intraplot heterogeneity was revealed by the data from miniflumes. GIS analysis of the plot microtopography showed that miniflumes located in the zones of flow convergence collected the majority of bacteria. Overall, the efficiency of VFS, with respect to the retention of swine manure bacteria, varied dramatically depending upon the hydrologic soil surface condition. Consequently, VFS recommendations should account for expected amounts of surface soil water saturation as well as the relative soil water storage capacity of the VFS.

Integrating waveguide biosensor.

The Integrating Waveguide Biosensor was developed for rapid and sensitive detection of bacterial cells, spores, and toxins. A sandwich format of immunoassay was employed using Salmonella as model. The analyte was immunocaptured on the inner surface of the waveguide and then detected by the antibody conjugated with fluorescent dye. The waveguide was illuminated by an excitation light at a 90 degrees angle. The emitted light from fluorescent labels on the surface of the waveguide was efficiently collected and channeled to a detector at the end of the waveguide, while minimizing interference from the excitation light. Utilizing fluorescent dye Cy5, a 635-nm diode laser for excitation, and a photomultiplier tube detector, the Integrating Waveguide Sensor System was able to detect approximately ten captured cells of Salmonella.

A multiple protocol to improve diagnosis and isolation of Shiga toxin-producing Escherichia coli from human stool specimens.

Many infections caused by Shiga toxin-producing Escherichia coli (STEC) are undiagnosed, particularly non-O157 STEC. We evaluated the use of a multiple protocol approach to improve diagnosis, isolation, and characterization of STEC strains. Among 18 presumptive STEC-positive stool samples received by the INOVA Fairfax Hospital, Falls Church, VA, in 2006, 16 were Shiga toxin positive. From these 16 stool samples, 8 O157:H7 and 5 non-O157 STEC were isolated by plating onto sorbitol MacConkey (SMAC) agar. The remaining 5 stool samples that did not yield colonies on SMAC agar plates were enriched. All enriched samples were Shiga toxin positive, and 2 O157:H7 and 1 non-O157 STEC were subsequently isolated. The 2 remaining enriched samples did not yield isolates; however, based on polymerase chain reaction (PCR) analysis, both samples contained STEC genes. Based on PCR analysis of non-O157 strains, 3 strain types were identified. Samples from 3 patients, received within 2 days of one another, had a similar gene profile-eae and stx(1) negative and stx(2) positive-suggesting that these patients were likely infected with the same strain. Our results indicate that a multiple protocol approach is necessary to reliably diagnose and isolate STEC strains, and that PCR profiling of strains could allow for more rapid identification of outbreaks.

Development of a rapid and sensitive immunoassay for detection and subsequent recovery of Bacillus anthracis spores in environmental samples.

Bacillus anthracis is considered a major threat as an agent of bioterrorism. B. anthracis spores are readily dispersed as aerosols, are very persistent, and are resistant to normal disinfection treatments. Immunoassays have been developed to rapidly detect B. anthracis spores at high concentrations. However, detection of B. anthracis spores at lower concentrations is problematic due to the fact that closely related Bacillus species (e.g., B. thuringiensis) can cross-react with anti-B. anthracis antibodies, resulting in false positive detections. Subsequent polymerase chain reaction (PCR) analysis is required to differentiate virulent strains. We report here on a protocol for the rapid, sensitive detection of B. anthracis spore using the Integrating Waveguide Biosensor followed by a method for the rapid release and germination of immunocaptured spores. A detection limit of ca. 10(3) spores was achieved by incubating spores simultaneously with capture and detection antibodies ("liquid-phase" assay) prior to capture on capillary tubes/waveguides. Subsequent incubation with BHI broth directly in capillary tubes allowed for rapid germination, outgrowth, and release of spores, resulting in vegetative cells for PCR analysis.

Exploring the feasibility and use of accelerometers before, during, and after a camp-based CIMT program for children with cerebral palsy.

PURPOSE: The aim of this pilot study was to determine the feasibility and use accelerometers before, during, and after a camp-based constraint-induced movement therapy (CIMT) program for children with hemiplegic cerebral palsy. METHODS: A pre-test post-test design was used for 12 children with CP (mean = 4.9 yrs) who completed a 30-hour camp-based CIMT program. The accelerometer data were collected using ActiGraph GT9X Link. Children wore accelerometers on both wrists one day before and after the camp and on the affected limb during each camp day. Three developmental assessments were administered pre-post CIMT program. RESULTS: Accelerometers were successfully worn before, during, and directly after the CIMT program to collect upper limb data. Affected upper limb accelerometer activity significantly increased during the CIMT camp compared to baseline (p< 0.05). Significant improvements were seen in all twelve children on all assessments of affected upper limb function (p< 0.05) measuring capacity and quality of affected upper limb functioning. CONCLUSION: Accelerometers can be worn during high intensity pediatric CIMT programs to collect data about affected upper limb function. Further study is required to determine the relationship between accelerometer data, measure of motor capacity, and real-world performance post-CIMT.

Impact of microbial diversity on rapid detection of enterohemorrhagic Escherichia coli in surface waters.

Enterohemorrhagic Escherichia coli (EHEC) are a physiologically, immunologically and genetically diverse collection of strains that pose a serious water-borne threat to human health. Consequently, immunological and PCR assays have been developed for the rapid, sensitive detection of presumptive EHEC. However, the ability of these assays to consistently detect presumptive EHEC while excluding closely related non-EHEC strains has not been documented. We conducted a 30-month monitoring study of a major metropolitan watershed. Surface water samples were analyzed using an immunological assay for E. coli O157 (the predominant strain worldwide) and a multiplex PCR assay for the virulence genes stx(1), stx(2) and eae. The mean frequency of water samples positive for the presence of E. coli O157, stx(1) or stx(2) genes, or the eae gene was 50%, 26% and 96%, respectively. Quantitative analysis of selected enriched water samples indicated that even in samples positive for E. coli O157 cells, stx(1)/stx(2) genes, and the eae gene, the concentrations were rarely comparable. Seventeen E. coli O157 strains were isolated, however, none were EHEC. These data indicate the presence of multiple strains similar to EHEC but less pathogenic. These findings have important ramifications for the rapid detection of presumptive EHEC; namely, that current immunological or PCR assays cannot reliably identify water-borne EHEC strains.

Aggregative adherence fimbriae I (AAF/I) mediate colonization of fresh produce and abiotic surface by Shiga toxigenic enteroaggregative Escherichia coli O104:H4.

The Shiga toxigenic Escherichia coli O104:H4 isolated during the 2011 European outbreak expresses Shiga toxin 2a and possess virulence genes associated with the enteroaggregative E. coli (EAEC) pathotype. It produces plasmid encoded aggregative adherence fimbriae I (AAF/I) which mediate cell aggregation and biofilm formation in human intestine and promote Shiga-toxin adsorption, but it is not clear whether the AAF/I fimbriae are involved in the colonization and biofilm formation on food and environmental matrices such as the surface of fresh produce. We deleted the gene encoding for the AAF/I fimbriae main subunit (AggA) from an outbreak associated E. coli O104:H4 strain, and evaluated the role of AAF/I fimbriae in the adherence and colonization of E. coli O104:H4 to spinach and abiotic surfaces. The deletion of aggA did not affect the adherence of E. coli O104:H4 to these surfaces. However, it severely diminished the colonization and biofilm formation of E. coli O104:H4 on these surfaces. Strong aggregation and biofilm formation on spinach and abiotic surfaces were observed with the wild type strain but not the isogenic aggA deletion mutant, suggesting that AAF/I fimbriae play a crucial role in persistence of O104:H4 cells outside of the intestines of host species, such as on the surface of fresh produce.

Rainfall intensity effects on removal of fecal indicator bacteria from solid dairy manure applied over grass-covered soil.

The rainfall-induced release of pathogens and microbial indicators from land-applied manure and their subsequent removal with runoff and infiltration precedes the impairment of surface and groundwater resources. It has been assumed that rainfall intensity and changes in intensity during rainfall do not affect microbial removal when expressed as a function of rainfall depth. The objective of this work was to test this assumption by measuring the removal of Escherichia coli, enterococci, total coliforms, and chloride ion from dairy manure applied in soil boxes containing fescue, under 3, 6, and 9cmh(-1) of rainfall. Runoff and leachate were collected at increasing time intervals during rainfall, and post-rainfall soil samples were taken at 0, 2, 5, and 10cm depths. Three kinetic-based models were fitted to the data on manure-constituent removal with runoff. Rainfall intensity appeared to have positive effects on rainwater partitioning to runoff, and removal with this effluent type occurred in two stages. While rainfall intensity generally did not impact the parameters of runoff-removal models, it had significant, inverse effects on the numbers of bacteria remaining in soil after rainfall. As rainfall intensity and soil profile depth increased, the numbers of indicator bacteria tended to decrease. The cumulative removal of E. coli from manure exceeded that of enterococci, especially in the form of removal with infiltration. This work may be used to improve the parameterization of models for bacteria removal with runoff and to advance estimations of depths of bacteria removal with infiltration, both of which are critical to risk assessment of microbial fate and transport in the environment.

Modeling fate and transport of fecally-derived microorganisms at the watershed scale: State of the science and future opportunities.

Natural waters serve as habitat for a wide range of microorganisms, a proportion of which may be derived from fecal material. A number of watershed models have been developed to understand and predict the fate and transport of fecal microorganisms within complex watersheds, as well as to determine whether microbial water quality standards can be satisfied under site-specific meteorological and/or management conditions. The aim of this review is to highlight and critically evaluate developments in the modeling of microbial water quality of surface waters over the last 10 years and to discuss the future of model development and application at the watershed scale, with a particular focus on fecal indicator organisms (FIOs). In doing so, an agenda of research opportunities is identified to help deliver improvements in the modeling of microbial water quality draining through complex landscape systems. This comprehensive review therefore provides a timely steer to help strengthen future modeling capability of FIOs in surface water environments and provides a useful resource to complement the development of risk management strategies to reduce microbial impairment of freshwater sources.

Continuous Gradient Temperature Raman Spectroscopy of Oleic and Linoleic Acids from -100 to 50 °C.

We analyzed the unsaturated fatty acids oleic (OA, 18:1n-9) and linoleic (LA, 18:2n-3), and a 3:1 LA:OA mixture from -100 to 50 °C with continuous gradient temperature Raman spectroscopy (GTRS). The 20 Mb three-dimensional data arrays with 0.2 °C increments and first/second derivatives allowed rapid, complete assignment of solid, liquid, and transition state vibrational modes. For OA, large spectral and line width changes occurred in the solid state γ to α transition near -4 °C, and the melt (13 °C) over a range of only 1 °C. For LA, major intensity reductions from 200 to 1750 cm-1 and some peak shifts marked one solid state phase transition at -50 °C. A second solid state transition (-33 °C) had minor spectral changes. Large spectral and line width changes occurred at the melt transition (-7 °C) over a narrow temperature range. For both molecules, melting initiates at the diene structure, then progresses towards the ends. In the 3:1 LA:OA mixture, some less intense and lower frequencies present in the individual lipids are weaker or absent. For example, modes assignable to C8 rocking, C9H-C10H wagging, C10H-C11H wagging, and CH3 rocking are present in OA but absent in LA:OA. Our data quantify the concept of lipid premelting and identify the flexible structures within OA and LA, which have characteristic vibrational modes beginning at cryogenic temperatures.