Degradation of Antibiotics in Wastewater Using Tetracycline-Resistant Microbes Recovered from an Invitro Hydroponic Phytoremediation System

Aparupa Sengupta, Jenny Isaacson, Zachary Anthony, Rupali Datta & Susan T. Bagley. Michigan Technological University, Houghton, MI 49931

Antibiotic contamination of various environmental resources has been a subject of concern worldwide.  Wastewater treatment facilities are usually not designed to remove antibiotics and hence release these compounds into the environment.  Constant exposure to antibiotics in the environment has been shown to promote the development of antibiotic resistant bacteria which is a potential threat to human health.  In our lab, a “hydroponic system” has been developed using Vetiver grass (Chrysopogon zizanoides L. Nash) to remediate tetracycline from wastewater.  The objective of this project was to assess the role of tetracycline-resistant bacteria in association with the plant in the degradation of tetracycline. Tetracycline-resistant bacteria were isolated from Vetiver roots and contaminated water during the course of remediation (a 30-day period). Tetracycline-resistant bacteria were selected from media with various tetracycline concentrations and transferred to a minimal medium (MSM) with tetracycline as the sole carbon/energy (CE) source.  Colonies observed on the minimal medium were selected for further screening.  Growth and enrichment studies and 96-well plate screening were conducted using MSM medium + tetracycline as a sole CE source to verify that these recovered bacteria can subsist on tetracycline.  Results were analyzed using a spectrophotometer, plate reader and HPLC, respectively. The results indicate that some of these resistant bacteria can subsist solely on tetracycline; work is in progress to confirm the tetracycline degradation and types of metabolites.  These results may be useful in the future in defining specific role of plants and/or microbes in mitigating antibiotic contamination problem from waste water systems.

Note: Presented at Center for Water & Society, at Michigan Tech, Mar 2012.

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Interaction effects of organic acids on the production of ethanol by Scheffersomyces (Pichia) stipitis CBS 6054

Alex Roll, Stephanie Groves, Susan Bagley, Michigan Technological University, Houghton, MI

In addition to liberating the sugars present in lignocellulosic material, dilute acid pretreatment produces compounds that can be inhibitory to yeast metabolism, including ethanol production. Organic acids represent one group of potentially inhibitory compounds that are released during pretreatment. The individual and interaction effects of acetic, formic, and oxalic acid on the production of ethanol by Scheffersomyces (Pichia) stipitis CBS 6054 from xylose was studied using a 23-full factorial design and response surface methodology (RSM). The cultures were grown on liquid media containing xylose supplemented with acetic (0-8 g/L), formic (0-4 g/L), and/or oxalic (0-6 g/L) acid. These acid concentration ranges were representative of those detected in a dilute acid pretreated hemicellulose hydrolysate. Ethanol, xylose, and acid(s) concentrations were monitored every 12 hours during a 72 hour shake-flask fermentation using HPLC equipped with an RID. Acetic acid was observed to have the greatest effect of ethanol production (58%).  Ethanol yields decreased as the concentration of acetic acid increased in all treatments regardless of the presence of the other acids.  Oxalic acid was responsible for 18% of the overall decrease in ethanol production. The presence of formic acid had little effect on ethanol yields (<1%). A synergistic effect between treatments with combinations of the acids was also observed, e.g.,  treatments that combined acetic and oxalic acid at any concentration had a negative impact on ethanol yields. Understanding the individual and interaction effects of these inhibitors may allow for establishing guidelines for optimizing and modeling pretreatments of lignocellulose for ethanol production.

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Preservation of Microorganisms and Organic Compounds within Fluid Inclusions of Halite Crystals from Lake Magic, Western Australia

Amber J. Conner and Kathy C. Benison, Central Michigan University, Mount Pleasant, MI 48859.

Lake Magic in Western Australia is an extreme acid saline lake.  It has pH levels as low as 1.7, salinity levels as high as 32 % total dissolved solids (TDS), and dynamic environmental changes such as desiccation, evaporation, and flooding (Bowen and Benison, 2009). This environment exhibits parallel characteristics with Mars environments, including similarities in minerals, geochemistry, sedimentary textures, sedimentary structures, and diagenetic features. Molecular studies of associated lakes in Western Australia documented novel acidophilic, halophilic microorganisms (Mormile et al., 2009). The purpose of this study is to document microorganisms preserved within fluid inclusions in Lake Magic halite.
        Optical and UV-vis petrography are used to distinguish minerals, prokaryotes, eukaryotes, and organic compounds within fluid inclusions in Lake Magic halite. Suspect microorganisms are 1-3 micron in diameter, with rod and cocci shapes and 4-6 micron in diameter, suggestive of prokaryotic and eukaryotic microorganisms. Exposure to UV visible light produces positive blue-green fluorescence of suspect prokaryotes, eukaryotes, and orange fluorescence of some solids.  Laser Raman spectroscopy indicates the orange-fluorescing solids are beta-carotene.
        Plane light microscopy suggests eukaryotic and prokaryotic features are observed based upon size, morphology, and optical properties. UV-vis petrography exhibits blue-green fluorescence, further suggesting microorganisms within fluid inclusions. Laser Raman analysis identified both beta-carotene and disordered graphite. Our study has shown microorganisms and organic compounds in extreme acid brines are trapped within fluid inclusions in halite and can be observed both optically and chemically.
       
REFERENCES
Bowen, B.B. and Benison, K.C., 2009, Geochemical characteristics of naturally acid and alkaline saline lakes in southern Western Australia: Applied Geochemistry, v. 24, p. 268-284.
Mormile, M.R., Hong, B.-y., and Benison, K.C., 2009, Molecular analysis of the microbial communities of Mars analog lakes in Western Australia: Astrobiology, v. 9, p. 919-930.

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Evaluation of a Wet Weather Treatment System in Removal of Adenoviruses and Enteroviruses during Storm Events Using Cell Culture

Kim Yiseul, Aw Tiong Gim,  Joan B. Rose, Michigan State University, East Lansing, MI 48824

Waterborne pathogens are known to increase during storm events and can be found in high concentrations in combined sewer and storm water flows. In order to reduce waterborne pathogens and protect public health, new treatment processes are needed to handle wet weather-sewage flows. This study is designed to evaluate the effectiveness of a High Rate Clarification (HRC) ballasted flocculation wet weather treatment system in regard to the pathogenic microorganisms. Enteric viruses can cause a wide spectrum of diseases in humans. Cultivatible adenoviruses and enteroviruses were chosen for this study. Both a HRC and conventional wastewater treatment process trains were sampled and examined for the presence of enteric viruses. Samples were collected under rain conditions from three sites at the HRC and conventional treatment processes (raw influent, post-clarification effluent, and final effluent) using a filter adsorption-elution method. Sample volumes collected varied from 4 to 59.6 L. For viral infectivity assay, concentrated samples were analyzed by cell cultures with A549 (for adenoviruses) and buffalo green monkey kidney cells (BGM, for enteroviruses). The development of cytopathic effects (CPE) that are indicative of a viral infection in the cell cultures was monitored. Presence or absence of CPE was confirmed with the second inoculation of samples in new A549/BGM cells and PCR assays. Toxic compounds in environmental samples on BGM cells were observed despite our efforts to minimize it. Based on detection limits and CPE, levels of adenoviruses (MPN/L) for conventional treatment were shown to be < 8.6, 4.7, and < 5.0 in influent, post-clarification, and final effluent, respectively. For HRC, levels of adenoviruses (MPN/L) were shown to be < 21.6 in influent and 3.6 in post-clarification and final effluent. Larger volume collection and reduction in toxicity will be focused on for the next set of samples.

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The Localization of GFP-tagged CpnB and CpnE in Dictyostelium discoideum

Janet E. Price, Jordan D. Dix and Cynthia K. Damer, Central Michigan University, Mount Pleasant, MI 48859

Little is known about the calcium-dependent membrane-binding family of proteins known as copines.  Copines are found in many diverse eukaryotic organisms, including humans.  To study copine function, we are using the model organism Dictyostelium discoideum.  Of the six copines in Dictyostelium, copine A (CpnA) is this only one that has been studied.  A green fluorescent protein (GFP) tagged version of CpnA was previously shown to be cytosolic; however, GFP-CpnA was observed to associate transiently with the plasma membrane, endosomes, lysosomes, phagosomes, and contractile vacuoles.  Our project is to determine the subcellular localization of the other copines within Dictyostelium by also tagging the proteins with green fluorescent protein (GFP). DNA constructs were made to express GFP fused to the N and to the C-terminus of copine B (CpnB) and copine E (CpnE).  First, the cDNAs of cpnB and cpnE genes were amplified using PCR with primers that added restriction sites to the ends of the fragments.  The cDNAs were then cloned into a TOPO cloning vector.  Next, the cDNAs were subcloned from the TOPO cloning vectors and inserted into the pTX-GFP vector upstream and downstream of the GFP gene. These newly made plasmids were then transformed into wild-type Dictyostelium cells.  We are currently using fluorescence microscopy to determine the localization of CpnB and CpnE in Dictyostelium cells.  Our preliminary results suggest that both CpnB and CpnE are found in the cytoplasm and the nucleus.  Nuclear localization suggests a new function for copines in Dictyostelium.

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The Allelopathic Effect of an Invasive Shrub, Autumn Olive, on Soil Function in Microcosms

Elizabeth A Czerwinski, Nicole L Lynn-Bell, and Peter S Kourtev, Central Michigan University, Mt. Pleasant, MI, 48859

Studies in the last decade have shown that invasive plants have a large potential to alter foreign territories to improve conditions for invasive growth.  Here, we have considered the potential of an invasive shrub, Elaeagnus umbellate (autumn olive), to influence microbial communities in the soil.  Microcosms were established in the laboratory, containing soils from three autumn olive invaded and non-invaded areas in a forest edge site and forested wetland site.  Each soil received either an amendment of water or extract prepared from autumn olive leaves.  To assess impacts on microbial community function enzymatic assays were performed for acid and alkaline phosphatase, alpha-glucosidase, alpha-N-acetylglucosaminidase, glycine aminopeptidase, and phenol oxidase, and peroxidase activities.  Changes in the enzymatic expression in the microcosms indicate that chemicals in the foliage of autumn olive are impacting the function of soil microbial communities.  Autumn olive extract appeared to stimulate microbial function, more so in invaded soils.  This suggests that invaded soils may be conditioned to respond to the presence of Autumn Olive by an increase in enzymatic activity.

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Summary of Recreational Outbreaks in the United States 1978 - 2008 in Relation to Precipitation Events (Preliminary Results)

Christopher D. Wendt and Joan B. Rose, Michigan State University, East Lansing, MI, 48824

Background: The association between rainfall and outbreaks has been investigated with respect to drinking water outbreaks (Curriero, F. C., et al. 2001).  However, outbreaks occurring as a result of contamination of water bodies used for recreation are common occurrence in the United States as well.  The goal of this analysis is to quantify the impact, if any, thatprecipitation rates and extreme events have on recreational outbreaks in ambient waters. Methods: Records of 185 outbreaks associated with recreational water bodies from 1978 - 2008 were obtained from the Environmental Protection Agency.  Data were first analyzed for attack rates and locations, as well as categorized by water body type.  Outbreak locations were then plotted to their associated watersheds and matched with climate data to determine patterns associated with precipitation events. Results: There were 157 outbreaks associated with lakes, 12 associated with rivers, 6 with  springs, 6 with oceans, and 6 with other categories (some associated with multiple sources).  Attack rates for all outbreaks show a mean of 42% and a mode of 100%.  The mean number of cases in the outbreaks was 49.13.  Mean case fatality rate was 1%.  The majority of outbreaks had Shigella as an etiological agent (42 outbreaks), followed by Norovirus (22), E. coli O157:H7 and Schistosoma (18), and Cryptosporidium (14).  Additional results related to precipitation analysis and challenges will be presented.

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Possible Link Between Copine A and the Actin Cytoskeleton

Mingxi Han and Cynthia K. Damer, Central Michigan University, Mount Pleasant, MI 48859

Copines make up a multigene family of calcium-dependent, phospholipid-binding proteins. Copine proteins consists of two C2 domains at the N terminus followed by an “A domain” similar to the von Willebrand A domain found in integrins. The C2 domain is a calcium-dependent membrane-binding motif, while the A domain is thought to be a protein-binding domain. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA- cpnF.  Previous research on one of the copine proteins in Dictyostelium, CpnA, suggests that CpnA interacts with actin.  However, it is not clear, whether CpnA can directly bind to actin. The goal of this project is to determine if CpnA is able to bind to actin monomers and/or filaments and if CpnA is able to regulate actin polymerization in vitro.  First, we created a DNA construct to express a GST-tagged CpnA in Dictyostelium and then transformed the newly made plasmid into wild-type Dictyostelium cells. We then used glutathione agarose chromatography to purify GST-CpnA from Dictyostelium cells.  Next, we cleaved GST from CpnA using thrombin and we are currently working on to purify the CpnA from the GST and thrombin.  Once we have purified a sufficient amount of CpnA, we will test whether CpnA is able to bind directly to actin and able to regulate actin polymerization in vitro.  In addition, we can use GST-pull down assays to explore whether other candidate binding proteins are able to bind directly to CpnA in vitro.

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A Novel Tauopathy Model Utilizing Dictyostelium discoideum

Kristi E. Miller, A.L. Erwin, S.L. Hall, M.L. Steinhilb, and C.K. Damer, Central Michigan University, Mount Pleasant, MI 48858, USA

Alzheimer’s disease is a chronic, progressive brain disorder, affecting approximately 35 million people worldwide. Pathologically, Alzheimer’s disease is characterized by the accumulation of two types of brain lesions: senile plaques and neurofibrillary tangles. Neurofibrillary tangles are found within neurons and are formed by the aggregation of paired helical filaments, in which the main component is tau, a microtubule associated protein. The mechanisms by which tau aggregates into filaments remains uncertain but studies have shown that phosphorylation and calpain proteolysis are major contributors to tau mediated toxicity. In this study, we will utilize Dictyostelium discoideum as a model organism to monitor tau toxicity and its effect on the Dictyostelium life cycle. Dictyostelium cells will be transformed with wild-type (tauWT) and mutant forms of human tau (phosphorylation-incompetent, tauAP; calpain-resistant, tauCR; tau 17kD fragment, tau17kD). Preliminary results indicate the successful expression of tauAP in Dictyostelium wild-type cells. However, the constitutive expression of tauWT, tauCR, and tau17 was sufficiently toxic to induce cell death. Therefore, further research will utilize an inducible vector system, pDM310, in which transcription of the tau gene can be reversibly turned on with the addition of doxycycline. If successful, we will then characterize the effect of tau and mutant tau expression on Dictyostelium survivability, cell division and development. Confocal microscopy will be used to observe the mechanisms of tau toxicity and lambda phosphatase treatment will be performed to determine the importance of phosphorylation on serine and threonine sites for proteolysis by calpain.

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Identification and Analysis of Breaks in Human Common Fragile Site FRA3B Using Saccharomyces cerevisiae

William J Fitzsimmons and Anne Casper, Eastern Michigan University, Ypsilanti, MI 48197

It is important to understand the genetic changes causing uncontrolled cell proliferation that leads to tumor formation.   It has been shown that common fragile sites, areas of high instability in the genome, can break under stressful conditions resulting in inactivation of tumor suppressor genes.  One such fragile site is FRA3B, which is located within the fragile histidine triad protein (FHIT) tumor suppressor gene.  We have a yeast artificial chromosome (YAC) inserted into Saccharomyces cerevisiae that contains a 500 Kb segment of FHIT, containing FRA3B. We have shown that under conditions of replication stress, the fragile site insert breaks exclusively within small region located within intron 5 of the FHIT gene.  Here we document the narrowing of break positioning within intron 5 using telomere PCR and sequencing.

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Exploring the Role of Origin Paucity in DNA Replication Stress and Fragile Site Breaks

Katherine A. Dziuba and Anne M. Casper, Eastern Michigan University, Ypsilanti, MI

Fragile sites are portions of individual chromosomes that tend to break when replication stress is introduced to the cell. Low levels of folate and thymidylate can cause replication stress by changing nucleotide pools.  Replication stress can also be caused by reducing levels of an enzyme called DNA polymerase alpha that functions to add primers DNA during replication. It has been proposed that a regional lack of origin sites on the chromosome in fragile site regions is responsible for breaks. With fewer origin sites present, it is likely that each DNA replication fork would have to travel a very long distance for DNA replication to be completed due to the large amount of DNA that needs to be replicated. Any delay of DNA synthesis would lead to incomplete DNA replication in the fragile site region before the cell enters mitosis. If the cell does not completely replicate DNA, then the chromosome may break at the fragile site when daughter cells are formed in the process of mitosis. We have identified break locations on a yeast artificial chromosome (YAC), a chromosome that carries an insert of human DNA from fragile site FRA3B. This YAC has only one origin of replication.  The breaks we have mapped are located in the region of the insert farthest from the origin.  We are now in the process of inserting another origin on the YAC.   According to the ‘origin paucity’ hypothesis, we expect that when a second origin site is inserted, the breaks in the YAC will occur closer to the center of the insert due to the fact that the inserted origin will efficiently replicate all the DNA found in the distal end of the insert. There is also a possibility that with the insertion of another origin site DNA replication will occur efficiently with no breaks.

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Suspect Microbes and Organic Compounds in Permian Acid Saline Lake Halite Deposits: A Case Study from Western Kansas

James J. Zambito, Kathy C. Benison and Amber Connor, Central Michigan University, Mt. Pleasant, MI 48859

Identifying microbes in ancient deposits presents a number of challenges, including ruling out sample contamination or alteration. However, the presence of primary fluid inclusions in halite crystals provides an ideal window for the preservation of hydrosphere, biosphere and atmosphere.  We present preliminary results of our analysis of materials within fluid inclusions in halite from a core drilled in western Kansas containing Middle and Late Permian (~265 million years ago) acid saline lake deposits.  Analyses used include plane-, reflected, and polarized-light petrography, ultraviolet light microscopy (UV-vis microscopy), and laser Raman microscopy.  A number of suspect microbes with varying morphologies were identified using petrography and UV-vis microscopy, including ~2 nm diameter objects with fine hairs radiating from a central body (somewhat similar to previously described 'hairy blobs'), clustered 1 nm translucent spheres, round opaque objects with varying diameters, and orange-red crusts and crystals.  Laser Raman microscopy indicates that some of these suspects are likely minerals diagnostic of complex water chemistries associated with the depositional environment, while other suspects possess Raman spectra indicative of biomarkers.  A suite of analyses is necessary to adequately identify and characterize suspect microbes in ancient halite.

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Recovery of Tetracycline-Resistant Bacteria from Vetiver (Chrysopogan zizanoides L. Nash) Root and Contaminated Water

Jenny Isaacson, Aparupa Sengupta, Zachary Anthony, Rupali Datta & Susan Bagley, Michigan Technological University, Houghton, MI-49931, USA

Antibiotics are regarded as emerging contaminants worldwide.  These compounds are detected in various aquatic systems and not only cause ecological disturbances but also pose potential threat to human health.  Tetracycline, a broad spectrum antibiotic, is one that is commonly detected in different environmental sources.  This antibiotic is commonly used for both human and veterinary purposes.  In our lab, we have been using a hydroponic phytoremediation system using vetiver grass to remediate tetracycline.  The focus of this experiment was to isolate and recover tetracycline-resistant bacteria from vetiver grass and water samples during the remediation trial (over 30 days). After samples were collected, viable counts using spread plate technique were performed on the collected samples.  The plates consisted of R2A agar and ranges of concentrations of tetracycline (0, 10, 25, 50, 100 ppm).  All plates were incubated for 24-72 hours at 30?C. Shannon-Weaver and other diversity indices were performed using the different colony morphotypes that were recorded.  Work is in progress to assess if these tetracycline-resistant bacteria are able to grow on tetracycline as the single carbon source.  Future work involves deciphering the role of these tetracycline-resistant bacteria (that can grow on tetracycline as sole carbon source) in degradation of tetracycline in aquatic systems.

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Anaerobe Clostridium acetobutylicum and Biodiesel Glycerol Byproduct in Undergraduate Research

James Graves, and Vincent V. Vuljaj, University of Detroit Mercy, Detroit, MI 48221

Acetone-butanol-ethanol (ABE) fermentation by anaerobe Clostridium acetobutylicum is a source of biofuel. Growth of the organism in the presence of biodiesel glycerol byproduct was examined in this investigation. The Oxyrase For Broth (Oxyrase, Inc.) enzyme system was used to assist in creating anaerobic conditions. C. acetobutylicum grew well on Schaedler blood agar Oxyplates, brain heart infusion (BHI) agar with Oxyrase, in fluid thioglycollate medium with or without Oxyrase and in phenol red broth with 0.2% agar and Oxyrase. The organism did not grow well in minimal broth or phenol red broth, with Oxyrase. Tube cultures had closed caps and anaerobic plate cultures were incubated in zip-lock bags. In thioglycollate medium with Oxyrase growth was assessed by measuring culture optical density (OD) by use of a Klett-Summerson colorimeter. In cultures containing added glucose or a mixture of glucose and byproduct growth was increased while that in cultures containing byproduct or pure glycerol was similar to that in the base medium. In phenol red broth that contained 0.2% agar and Oxyrase cultures that contained added glucose or a mixture of glucose and byproduct exhibited a positive fermentation reaction in contrast to those containing byproduct or pure glycerol.  Test paper (Hydrion) showed that the pH of cultures containing glucose dropped from 8.0 to 5.0. A glucose oxidase and peroxidase assay (Precision Labs, Inc.) showed that the concentration of glucose had dropped to an undetectable level. An alcohol oxidase and peroxidase assay (AlcoScreen) indicated the presence of alcohol in all cultures. Experiments with glycerol byproduct made by catalyst column or transesterification produced results that appeared similar. It could be profitable to find a use in industry for the waste glycerol byproduct from biodiesel plants.

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The ehxCABD Operon of Locus of Enterocyte Effacement Negative Shiga Toxin-Producing Escherichia coli is Regulated by the Histone-Like Nucleoid Structuring Protein

Miles T. Rogers and M.E. Scott, Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49001

Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens that cause diarrhea and hemorrhagic colitis with the potential to cause life-threatening hemolytic uremic syndrome. Two distinct groups of STEC exist, those that carry the Locus of Enterocyte Effacement (LEE) and STEC that do not have LEE. Most STEC have a large virulence plasmid that contains the ehxCABD operon encoding the enterohemolysin toxin (ehxA). Previous studies suggest that gene-products within the LEE control expression of ehxCABD in LEE-positive STEC. Here we reveal the mechanism behind H-NS repression of ehxCABD carried by LEE-negative STEC isolates. A transposon mutant of STEC O91:H21 was generated causing deletion of the histone-like nucleoid-structuring protein (H-NS). The mutant, 34.7, was defective in adherence to human colonic epithelial cells and motility but was hyperhemolytic. Levels of EhxA increased as determined by SDS-PAGE, Western blot, and RT-PCR analysis of the mutant. DNA upstream of ehxCABD used in electrophoretic mobility shift assay revealed that H-NS bound to this fragment of DNA and transcriptional fusion with promoterless lacZ gene localized the promoter between -100 and -1 bp upstream of the translational start site of ehxC. Thus, the ehxCABD promoter contains a H-NS cis-acting element required for repression of ehxCABD in LEE-negative STEC.

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Effects of Fragile Site FS2 on Mitotic Crossovers in the Yeast Saccharomyces cerevisiae

Danielle Rosen, Ellen Younkin, Shaylynn Miller and Anne Casper, Eastern Michigan University Ypsilanti, MI  48197

Detection of Campylobacter under Modified Conditions

Andrew Bruce, Kaitlyn Delancey, Zach Geurin, Zack Gould and Joan Rose, Michigan State University, 3 Natural Resources, 480 Wilson Rd, East Lansing, MI 48823

This study aimed to develop improved methods for detecting Campylobacter spp.  In waste water. Problems have occurred in the standard operating procedure (SOP) normally used by our laboratory to detect Campylobacter. Following enrichment of the sample culture, PCR analysis usually resulted in higher detected concentrations of Campylobacter than biochemical testing. Three modified methods addressing oxygen exposure, nutrient dilution, and PCR sensitivity were analyzed alongside an unmodified procedure. Limiting oxygen exposure and adding enrichment media both resulted in higher recovery percentages over unmodified protocol. We conclude the diluted nutrients inhibited the culture growth more than the over-exposure to oxygen. No PCR positives were found when using the modified method prior to enrichment of the initial Campylobacter present. The PCR analysis was not sensitive enough to detect Campylobacter without a pre-enrichment. Thus the PCR results after cultivation were indicative of live bacteria. Key to this detection was the enrichment procedure. Further research is recommended to experiment with varying volumes of additional enrichment media, and levels of oxygen.

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Evaluation of vetiver grass (Chrysopogon zizanoides L. Nash) as a novel lignocellulosic feedstock for ethanol production by Scheffersomyces (Pichia) stipitis 

Emily M. Geiger, Stephanie L Groves, Susan T. Bagley, Rupali Datta, Michigan Technological University, Houghton, MI 49931

The limited supply of crude oil has caused a shift towards alternative fuel sources, such as bioethanol produced from microbial sugar fermentation.  Identification of plant species that can be used as novel lignocellulosic feedstocks is imperative. Vetiver grass (Chrysopogon zizanoides L. Nash) is a robust sub-tropical plant candidate for lignocellulosic ethanol due to its high biomass productivity and low lignin content.  In this study, the effects of several factors for the successful pretreatment and of vetiver were examined to produce a highly fermentable hemicellulosic hydrolysate for fermentation by Scheffersomyces (Pichia) stipitis.  Each pretreatment factor was evaluated for its effect on characteristics that maximize ethanol yield from fermentative processes, including high sugar yield and minimal production of inhibitors. Statistical analysis of percent yields concluded the optimal pretreatment conditions were milled post-senescence feedstock pretreated with 1% sulfuric acid (w/v) at 115C for 60 minutes.  The optimized dilute acid pretreatment of vetiver grass leads to the liberation of C5 and C6 monosaccharides, including xylose, arabinose, glucose, galactose and mannose, from the hemicellulosic fraction of the plant.  The utilization of substrates within the hemicellulosic hydrolysate and their conversion to ethanol by the native xylose-fermenting yeast S. stipitis CBS 6054 was examined following dilute acid pretreatment of vetiver.  Monosaccharide and ethanol concentrations were measured using HPLC equipped with a RID over a 60-hour period.  The fermentations were carried out on a gyratory shaker set at 160 rpm and 30oC.  Percent theoretical yields and volumetric productivity were calculated for statistical analysis using R. The dilute acid pretreatment achieved a 73.7% theoretical yield of total sugars hydrolyzed; fermentation of the vetiver hydrolysate achieved a 68.6% theoretical yield and volumetric productivity equal to 0.14g/V-t. This indicates that vetiver has the potential for use as a viable feedstock for lignocellulosic ethanol production.

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Role of RecQ Helicase SGS1 in Repair of Double Strand Breaks at Common Fragile Site FS2

Rachel C Parent, Kayleigh Watson and Anne M Casper, Eastern Michigan University, Ypsilanti, MI 48197

The ability of a cell to repair DNA is crucial for survival. A common form of DNA damage that can lead to mutations is double-strand DNA  (dsDNA) breaks. Many of the translocations, amplifications, and deletions seen in cancer cells result from repeated dsDNA breaks in particular regions of the genome. Common fragile sites are one type of region with frequent dsDNA breaks and alterations in cancer cells.  There are many proteins needed for the repair of dsDNA breaks, one of which is Sgs1p. Sgs1p is a non-replicative helicase that functions in unwinding secondary structure and in 5’-3’ resection of a dsDNA break, leaving a 3’ Overhang to be used for repair.  We have shown that the lack of SGS1 leads to an increase in instability at a fragile site in yeast, FS2, and it also alters the way in which breaks at FS2 are repaired.

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Escherichia coli Association with Filamentous Green Algae in Lakes Huron and Michigan

Christopher T Finch and Elizabeth Alm, Central Michigan University, Mount Pleasant, Michigan 48859

Cladophora, a filamentous green algae, has been indicated as an environmental reservoir for fecal indicator bacteria and several species of pathogens. This study attempted to determine whether a relationship exists between the presence/absence of the fecal indicator, Escherichia coli, within filamentous green algae mats and abiotic water quality measurements at the sample sites.  Algae samples were collected from random locations throughout the Lake Huron and Lake Michigan shorelines during summer 2011. Water parameters measured were pH, water temperature, conductivity, ChlA, turbidity, D.O., redox potential, and depth of collection. Samples were then taken and placed on ice until they are brought back to the laboratory where they were processed within 6 hours of collection using at EPA standards. A Principle Component Analysis (PCA), a Discriminate Function Analysis (DFA) and a Multi-Response Permutation Procedures (MRPP) were run to determine if any relationships existed between abiotic data and the presence or absence of E. coli within the algal mats. The PCA using Principal component1 (PC1) and PC2 were able to account for 64% of the variation in the abiotic data. When the PCA was graphed with the E. coli presence/absence data it appeared to separate the E. coli present/absent sites from each other with minor overlap. The DFA was graphed using LD1 with E. coli presence/absence and there was no overlap between groups on the graph. A MRPP was ran to test the groups, p-value=0.0783 and the A-value=0.0592. The PCA shows the data driving the presence/absent groups apart by productivity. It seems that productive sites are more likely to have E. coli present than non-productive sites. The DFA was able to completely separate the present/absent groups with no overlapping.  The MRPP A-value describes within-group homogeneity compared to random expectation.
Therefore according to preliminary data that the MRPP used for calculations, these groups are not different from one another via the abiotic data and currently the collection of water parameters to predict E. coli is not an alternative to standard E. coli counts for water quality.

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