Physiological and Molecular Characterization of Thermophilic Carboxydotrophs

Cisley Cardwell and Sonia M. Tiquia, The University of Michigan-Dearborn, 4901 Evergreen Road, Dearborn, Michigan 48128

Carboxydotrophs are known for their ability to convert CO to butanol through fermentation.  Such a platform offers a promising alternative approach to biofuel production. The main goals of this study were to isolate novel thermophilic microorganisms from composts and characterize the isolates using a set of physiological and molecular assays.  Direct plating and liquid enrichment were employed to isolate thermophilic carboxytrophs from 5 different composts.  The enrichment was carried out in serum bottles containing phosphate-buffered medium gassed with CO, and incubated at 55C.  Aliquots of the enrichment were spread directly on the surface of thioglycollate plates and incubated in an aerobic chamber for 1 week.  Colonies arising on the plates were selected for isolation based on gross morphological features. All in all, 249 thermophilic isolates were recovered from five compost samples.  Gram-staining characteristics, cell morphologies, motility and physiological characterization of these isolates were determined using standard methods.  Overall, 53% of the isolates were gram positive and 47% were gram negative.  Sixty percent of the isolates were facultative anaerobes, the rest are strict anaerobes (38%) and microaerophilic (2%).  Majority of the isolates were motile rods and a few were non-motile cocci. Many of the isolates were capable of fermenting carbohydrates, and many were able to produce 2,3-butanediol through fermentation.  Molecular characterization based on 16S rDNA sequencing will also be carried out to identify these isolates and reveal phylogenetic relatedness to each other, and to known bacterial species from the current database.  This study revealed some phenotypic attributes of thermophilic carboxytrophs from composts.  However, more research work is needed to discover direct biological route for butanol production from CO to strengthen the potential for application of CO-utilizing anaerobic bacteria to synthesis gas bioprocessing.

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Fermentation Capabilities of Mesophilic Anaerobes Isolated from Composts

Shereen Mukhashen , Summar Saad and Sonia M. Tiquia, Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48128

Due to their versatile capabilities, microorganisms have been exploited for their unique biochemical and physiological properties from the earliest times for uses in food preservation, brewing, developing antibiotics, and synthesis of biofuels. Those products are a result of some microorganisms’ ability to complete a process known as fermentation, in which organic substances are broken down into useful products.  In this study, we explored the use of a variety of composts to screen novel microorganisms, which exhibit superb properties in converting organic compounds to useful fermentation products.  The main objectives of this study were to employ enrichment and culturing techniques to isolate novel anaerobic mesophillic microorganisms from various composts; and to examine their fermentation capabilities.  This task was completed through a variety of biochemical and physiological tests with 249 isolates extracted from five different compost samples: (1) dairy manure +straw + woodchips, (2) dairy manure +compost, (3) mature dairy manure, (4) mature leaf compost, and (5) dairy manure+ saw dust. For each compost sample, 49-50 isolates were randomly selected. Of the 249 isolates, 58% were Gram-positive, while 40% were Gram-negative.  A majority of the isolates were facultative anaerobes (67%), while 32% were strict anaerobes and the remaining (1%) were microaerophiles. Only the facultative anaerobes displayed catalase activity. Fermentation assays indicated that 55% of the isolates were capable of fermenting lactose, glucose, and sucrose.  Some (37%) fermented glucose and sucrose only, while others (6%) fermented sucrose and lactose.  Of the isolates that fermented glucose, 86% produced gas and 93% produced acid. The values were similar to the isolates that fermented sucrose.  Of the isolates that fermented lactose, a majority produced acids (62%), while 26% produced gas.  Overall, very little alcohol production was observed from the isolates: only 3% in the glucose fermenters, 1% in the lactose fermenters and none for the sucrose fermenters. The methyl red test illustrated that 22% of the isolates were mixed acid fermenters, and completing the Vogues-Proskaeur test illustrated that only 35% of the isolates successfully produced acetoin.  Based on the data collected, it is significant that a majority of the isolates were capable of fermenting all three types of sugars. The fermentation capabilities displayed by the microorganisms suggest their potential use in the production of natural products. The biochemical and physiological tests conducted helped characterize key properties of the 249 isolates, which are important if these isolates were to be used in the development of various products in the future. As the fermentation capabilities of microbes are fully exploited, we will become closer to producing more economically and naturally efficient by-products to benefit society.

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The Isolation of Anaerobic Thermophilic Bacteria by Using Carbon Monoxide as the Sole Source of Energy

Fatime Ala, Sarah Mendoza, Sukayna Ismail and Sonia M. Tiquia, The University of Michigan- Dearborn, MI

Microorganisms capable of metabolizing carbon monoxide (CO) as their sole carbon and energy source are potentially useful in production of biofuels from synthesis gas feestocks.  Although one-carbon metabolic pathways necessary for CO utilization have been observed in several species of anaerobic bacteria, relatively few are capable of growth on CO alone, particularly in 100% CO atmosphere.  Thus, this study was conducted to isolate new thermophiles that use CO or syngas as a substrate from composts.  Five different compost samples were tested: (1) dairy manure+straw+ woodchips, (2) dairy manure+compost, (3) mature dairy manure, (4) mature leaf compost, and (5) dairy manure+sawdust.  A total of 260 anaerobic thermophilic isolates displaying unique morphologies were recovered from the 5 compost samples.  The isolates were screened for their ability to utilize CO as carbon source by monitoring growth using a spectrophotometer set at 660 nm.  The cells (initial O.D.=0.03-0.05) were inoculated in 26-ml sealed pressure tubes containing 10 ml of phosphate-buffered medium under a 100% CO gas headspace.  The tubes were incubated at 55C in the dark.  Culture turbidities were recorded every 24 hours for 6 days.  Since Butyribacterium methylotrophicum is known to use CO, it was used as a positive control to confirm that our protocol was efficient.  The majority of the isolates grew on phosphate-buffered medium containing glucose as a carbon source.  So far, out of the 173 isolates tested, 137 isolates grew in CO.  To assess the biochemical basis for their ability to produce biofuel (butanol) from CO, the level of activities of carbon monoxide dehydrogenase, formate dehydrogenase, and hydrogenase enzymes will for these 137 isolates. These results will be reported at the meeting.   This project is part of a larger effort to contribute to understanding the microbiology of synthesis gas fermentation for biofuel production.

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Evaluation of an automated DNA extraction for environmental samples

Sangeetha Srinivasan and Joan B Rose, Michigan State University, East Lansing, MI

DNA extraction plays a crucial role in any molecular investigation. Extraction out of environmental samples requires in-depth evaluation as its efficiency could be affected based on the matrix, kits used and even the type of target organisms. As a new method or technology becomes available, it is ideal to assess and compare it with the existing ones before being applied for regular use. Automated DNA extractors are found to be successful in extracting DNA clinical specimens such as blood, sputum, serum and body fluids due to its efficient recovery, lack of cross contamination and ease of performance. The aim of our study was to evaluate the performance of Roche MagnaPure LC Instrument for extracting DNA from samples such as raw sewage and cow feces. We also compared the concentrations of E.coli and enterococci by qPCR from DNA extracted by commercial kits that are routinely used for these types of samples as well as by automated extractor. It was found that the machine failed in extracting DNA directly from such concentrated samples. Therefore, an external lysis method was performed and DNA extraction was carried out from the lysate. The results showed that the autoextractor performed as efficiently or even better as compared to the manual kits. DNA from the auto extractor showed slightly higher qPCR equivalent cell concentrations of E.coli and enterococci, except for cow fecal DNA where qPCR equivalent cell concentrations of enterococci were found to be 0.44 log higher in the manually extracted cow fecal DNA than auto extracted DNA.There was a log10 difference of 0.306 and 0.335 between the machine and manual DNA extractions for concentrations of E.coli from raw sewage and cow feces respectively. For enterococci, there was a log10 difference of 0.26 from raw sewage but the mean log concentration of enterococci from cow feces by manual extraction was 0.44 log10 higher than by the autoextraction. There was a good correlation between machine and the manual extractions for E.coli concentrations from cow feces and enterococci from raw sewage (r2= 0.96 and 0.88 respectively) but no correlation was found between both the extraction methods for E.coli concentrations from raw sewage and enterococci from cow feces (r2= 0.31 and 0.09 respectively). Our results indicate the need to evaluate DNA extraction procedures for different environmental samples.  It was also observed in our study that the external lysis step plays a crucial role in DNA recovery from any samples especially if the target has a tough cell wall such as that for Gram positive bacteria.

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Cyanobacterial Populations from a Michigan Grassland Alvar

N.A. Pudlo, K.A. Tipton, A.J. Stockero and G.M. Colores, Central Michigan University, Mount Pleasant, MI 48859

The Great Lakes Region harbors nearly all of the North American alvars. Alvars are communities named after similar areas in the Baltic regions of Sweden, Estonia and to a limited extent, western Russia. Alvars are open areas of calcareous bedrock, typically limestone or dolomite, with little to no soil cover or organic matter. This exposed landscape is characterized by extreme seasonal temperature differences and low moisture retention. Although this habitat is inhospitable for most woody growth, undisturbed alvars with thin soils are able to sustain plant communities. The Maxton Plains on Drummond Island, MI was chosen as a study site because it is globally represented as one of the largest, undisturbed alvars. Although there has been research performed on the flora and fauna of North American alvars, little has been done to investigate the microorganisms populating these rare habitats. Since soil and organic matter conditions are poor on alvars, we chose to examine cyanobacterial presence based on their abilities to fix carbon and in some species, Nitrogen. Various soil-substrate samples were obtained, stored and also enriched in BG-11/BG-11o for each sample. Additionally, a microscopic examination was performed on enrichment cultures. DNA was extracted from soil and enrichment samples and PCR amplified using cyanobacteria-specific primers. Mixed PCR products were separated via Denaturing Gradient Gel Electrophoresis (DGGE). Single unique bands from DGGE were sequenced and subjected to a BLASTn search to determine the closest relatives. BLASTn searches accompanied by microscopy revealed the presence of several cyanobacteria including Calothrix sp., Chroococcidiopsis sp., Leptolyngbya sp., Microcoleus sp., Nostoc sp., Petalonema sp., Phormidium sp., Scytonema sp. and uncultured cyanobacteria. Based on our results, the harsh conditions of the Maxton Plains Alvar appear to select for a diverse range of cyanobacteria.

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Inositol Compounds as Nutritional Mediators in the Sinorhizobium meliloti - Alfalfa Nitrogen Fixing Symbiosis

Petra R. A. Kohler*, Elke Schoffers$, and Silvia Rossbach*,*Department of Biological Sciences, $Chemistry Department, Western Michigan University, Kalamazoo, MI 49008

The sugar alcohol inositol has nine possible stereoisomeres. The myo-, scyllo-, D- and L-chiro isomeric forms are naturally occurring inositols, of which the myo-form is the most abundant. The ability to use myo-inositol has been closely associated with the competition process for nodule colonization in the nitrogen fixing symbiosis between several members the bacterial family Rhizobiaceae and legume plants. The rhizobial inositol pathway has been linked to plant-bacteria interactions based on its role in the catabolism of a group of nutritional mediators known as rhizopines. The rhizopines produced by several symbiotic S. meliloti and R. leguminosarum bv. viciae strains are inositol derivatives, scyllo-inosamine and L-3-O-methyl-scyllo-inosamine. We found through mutant analysis that the inositol genes (iol genes) of Sinorhizobium meliloti are required for the growth with myo-, D-chiro and scyllo-inositol as sole carbon source. The regulation and possible catabolite repression of the inositol pathway succinate and glucose, was investigated with a NAD(H) dependent dehydrogenase and a ß-glucuronidase assay. We found the inositol pathway not to be subjected to catabolite repression on the genetic level, but the expression of the iol genes requires induction through a pathway intermediate. An in vitro plant assay revealed that inositol catabolism and its regulation are essential for successfully competing during nodule occupancy. Our findings support the notion that the inositol catabolic pathway of S. meliloti is involved in the establishing and maintaining of the nitrogen fixing symbiosis, and that the pathway itself serves nutritional or signaling purposes.

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Transport of E. coli in Soil Aggregates with Different Agricultural Management Practices

Mustafa A. Mazher, Chung, Hyen C., Smucker, Alvin J.M., Kravechenko, Sasha and Rose, Joan B., Michigan State University, East Lansing, MI, 48823

abstract: In Michigan, agriculture plays a significant role in the economy. With the predicted expansion of land use due to increased interest in biofuels, non-point pollution is becoming of public health  concern. Universally, E. coli is used as an indicator for microbial water quality and public health risk. However, the transport of E. coli after application of manure to agricultural lands is not well understood. The objective of this study is to examine the effect of soil porosity on the retention and spatial arrangement of native bacteria and spiked E. coli both statically and in flow columns. Dried aggregates used for the study came from soils that had 1) conventional tillage corn-soybean-wheat rotation with conventional chemical inputs, 2) no-till corn-soybean-wheat rotations with conventional chemical inputs and 3) native successional plot abandoned from agricultural use after spring 1989. To extract bacteria, aggregates were vortexed, suspended in phosphate buffer water and membrane filtered onto mENDO and TSA media for examination of CFU/g of aggregate. No growth was observed on the mENDO plates indicating no naturally occurring E. coli in dried aggregates, however, treatments 1, 2 and 3 contained an average of 3.02 to 3.76 x10^5 CFU/ g of heterotrophic bacteria cultured on TSA with no difference between the soil treatments. Recovery of spiked E. coli from soil aggregates was good even with low concentrations applied to the aggregate. Spiked concentrations of 3.73x10^3, 5.70 x10^2 , 5.70 x10^1, 9.25 x10^1, 9.25, 4.40 CFU/ aggregate yielded 121.70%, 84.80%, 99.42%, 61.26%, 162.16%, 75.76 averaging 100.85% recovery rate. No E. coli found when less than 1 CFU/ soil aggregate (4.40x10^-1) was applied. Flow columns results indicated apparent retention differences between treatment 1 and treatment 3. After flushing the flow column with 6ml of CaCl2 at vacuum pressure, treatment 1 soil retained 63.02% of the seeded E. coli. On the other hand, treatment 3 retained only 9.45% of seeded E. coli after being flushed with the same volume. Our experiments indicate that although we can recover approximately 100% of the seeded E. coli directly from the whole soil aggregate, flow column experiments show variable retention depending on the agricultural management practice. The latter experiments maybe a more accurate reflection of real life scenario where soils receive a constant flux of water through rainfall events, and under different agricultural treatments, the porosities may affect how much of a pathogen is filtered through the soil and how much leaches through into the groundwater. Future experiments will investigate E. coli spatial arrangement and retention at varying temperatures and after conducting flow column experiments.

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Detection and Enumeration of rRNA group I Pseudomonas in Metalworking Fluids and Biofilm formation using Fluorescent in situ Hybridization (FISH)

Ratul Saha and Susan Bagley, Michigan Technological University, 1400 Townsend Drive, Houghton, MI-49931

Metalworking fluids (MWFs), used in different machining operations, are highly prone to microbial contamination. Species of rRNA group I Pseudomonas often dominate in contaminated MWFs, but their actual distribution is difficult to study using standard culturing techniques as most fail to grow on the commonly used Pseudomonas Isolation Agar, thus underestimating their abundance. To overcome this, fluorescent in situ hybridization (FISH was used to study the distribution and diversity of Pseudomonas rRNA group I species along with biofilm formation by P. fluorescens MWF-1 and the newly described P. oleovorans subsp. lubricantis. From analyzing the available 16S rRNA sequences, a fluorescent molecular probe was designed targeting a conserved signature sequence common to all rRNA group I Pseudomonas using the ARB software package. The designed molecular probe (Pseudo120) was fluorescently labeled with Cy3; the universal bacterial probe EUB338 was labeled with FAM. The CELLC software was used for image analysis. The probe’s specificity was evaluated using hybridization experiments with whole cells of six Pseudomonas rRNA group 1 species using a stringency of 40% formamide at 46°C. The stringency of the hybridization reaction was 30% formamide for the study of biofilm development. Biofilm formation was also investigated by using methylene blue. The probe’s sensitivity was determined to be 103 CFU/ml. It successfully detected and enumerated the abundance and distribution of Pseudomonas indicating levels between 3.2 (±1.1) x 106 and 5.0 (± 2.3) x 106 CFU/ml in different industrial MWF samples (using stringency of 30% formamide at 46 °C). Biofilm formation was found under stagnant and dynamic conditions for both P. fluorescens MWF-1 and P. oleovorans subsp. lubricantis stained with methylene blue and Pseudo120.  On the basis of these observations, the molecular probe can be successfully used for rapid detection, enumeration and study of spatial distribution of rRNA group I Pseudomonas in contaminated MWFs along with biofilm formation by these bacteria.

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Adaptation of Pichia stipitis CBS 6054 to Dilute Acid Pretreated Aspen Hydrolysate for Improved Ethanol Production

Stephanie L. Groves, Jill I. Jensen, Juan Morinelly, and Susan T. Bagley, Michigan Technological University, Houghton, MI 49931

Pichia stipitis CBS 6054 is a natural xylose-fermenting yeast that can be used for the conversion of lignocellulosic sugars to ethanol. Adaptation was used to modify P. stipitis CBS 6054 for increased ethanol yields when grown on high percentages of dilute acid pretreated aspen hydrolysate. The culture was grown in increasing concentrations of hydrolysate until the hydrolysate comprised 80% of the total media volume. Biomass production, substrate utilization, and ethanol production were evaluated. The adapted strain’s fermentation capabilities were considerably improved over the unadapted parent strain. On media containing 80% hydrolysate, the adapted strain produced a maximum of 4.5±0.04 g ethanol/L within 24 hours, using 91% of the available sugars. This resulted in an ethanol yield of 0.45±0.06 g/g. In contrast, the unadapted wild-type strain utilized less then 1% of the available sugars and produced no ethanol over a 72 hour period. The adapted strain was able to produce more biomass compared to the unadapted strain.  The 80% adapted strain was also transferred to media containing lower percentages of hydrolysate (60 – 70%) and showed increased ethanol yields (+0.05-0.1 g/g) compared to strains that were only adapted to these levels.  The increased productivity has not been lost with culture storage.  Although more studies need to be conducted, these data support that adaption rather than acclimation has occurred. This strain performed more efficiently than previously reported P. stipitis adapted CBS 6054 strains.  Adaptation is a useful and inexpensive means of optimizing organisms for the production of ethanol from lignocellulosic sugar mixtures.

This poster was presented at the 2009 Annual Meeting of the Society of Industrial Microbiology in Toronto.

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Structure/Function analysis of the Actin Crosslinking Domain of MARTX from Vibrio cholerae using the model organism Saccharomyces cerevisiae

Samantha Johnston, Mann, Jason, Celmer, A., and Juris, Stephen J.
Central Michigan University, Dow Science 246, Mount Pleasant, MI  48859

MARTX from Vibrio cholerae is a 4545 amino acid member of the multifunctional autoprocessing Repeats-in-Toxin (RTX) family that contains effector domains that inhibit normal cell function, including an actin crosslinking domain (ACD) that covalently crosslinks actin within target cells.  The ACD crosslinking activity is poorly understood: the crosslinking domain does not resemble other proteins with known function and only a few cofactors, including Mg2+ and ATP, are known to be required for crosslinking activity.  We hypothesize that the ACD enzymatically catalyzes the crosslinking of actin via a novel mechanism.  In order to characterize the mechanism of the ACD, we employed a structure/function analysis in the yeast model system Saccharomyces cerevisiae to identify critical amino acids in the ACD required for actin crosslinking.  Much like expression of the ACD is cytotoxic to epithelial cells, we demonstrate that inducible expression of the ACD is cytotoxic to S. cerevisiae.  This cytotoxicity is likely due to disruption of the actin cytoskeletal network in yeast, as rhodamine phalloidin staining in yeast expressing wild-type ACD shows a lack of actin cables.  We have employed random mutagenesis of wild-type ACD located on a 2-micron yeast expression plasmid and transformed this mutant library into yeast containing a centromeric plasmid with the wild-type ACD.  This strategy will allow us to focus efforts on mutant ACD proteins that are able to fold and function properly and outcompete for wild-type ACD substrate within our yeast model system, thus promoting yeast survival.  We are currently screening mutants that we have isolated by this strategy to determine the nature of the mutation and how that mutation has disrupted ACD activity.  These experiments will provide a fundamental knowledge of critical amino acids involved in the actin crosslinking activity of the ACD from V. cholerae and will provide an understanding of the pathogenesis of organisms producing ACD-containing toxins.

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Characterization of the Recombinant Streptococcal Protein IdeZ and its interaction with Human Protease Inhibitor Cystatin C

Rebecca A. Hyatt and Jennifer L. Hess, Aquinas College, Grand Rapids, MI 49506

The bacterial pathogen Streptococcus zooepidemicus enhances its ability to cause disease and combat the immune system of its host, primarily humans and horses, through the use of the cysteine proteinase, IdeZ (Immunoglobulin G-degrading enzyme of S. zooepidemicus).  This occurs because IdeZ, which has uniquely high substrate specificity for immunoglobulin G (IgG), is capable of evading detection by cleaving the antibody at its hinge region.  In functional studies, IdeZ has yielded similar results to the well characterized homologous enzyme IdeS (Immunoglobulin G-degrading enzyme of Streptococcus).  Previously in this laboratory, site-specific primers were designed to target nucleotide sequences to change the amino acids thought to influence the cleavage of IgG.  These primers were then utilized in the mutation of plasmid DNA containing recombinant IdeZ obtained from E. coli.  In this study, prior research was expanded upon through the creation of additional mutants and experimentation with the protease inhibitor, cystatin C, and the well characterized inhibitor, iodoacetate.  Results from functional assays were consistent with previous studies, and indicated that the purported mutations involving the cysteine and arginine/aspartic acid/glycine residues in the active site of IdeZ prevented cleavage of IgG from occurring.  Additionally, this study verified that, cystatin C enhanced the IgG-cleaving ability of lysates capable of cleaving IgG, including the glutamine mutant (Q125L), the cleaving ability of which appears to have improved.  It is possible that further experimentation with IdeZ will result in a variety of practical applications pertaining to human and veterinary medicine, as well as therapeutic drugs targeting proteolytic enzymes, as has already been demonstrated with homologous enzymes.

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Prokaryotic diversity in the four chambered stomach of white-tailed deer.
Zhu, C., Surowiec, C., Weaver. M.N., Swanson, B.J. and Kourtev, P.S., Biology Department, Central Michigan University, Mt. Pleasant, MI

As a ruminant animal, deer have complex four-chambered stomachs in which they process their food.  Stomach microorganisms are crucial to animals as they decompose the cellulose in their diet, yet they have not been well characterized.  We collected the stomachs of eighteen white tailed deer harvested by hunters during the fall of 2007 in the lower peninsula of Michigan. We analyzed the bacterial and archaeal community in the four chambers of the deer stomachs (rumen, reticulum, omasum and abomasum) using PCR-DGGE of the 16S rRNA gene and sequencing.  Archaeal communities were dominated by a few types (4-6 per sample) and were highly similar across all of the sampled deer and in the four chambers of the ruminant stomach.  Dominant archaeal bands were identified as uncultured microorganisms most closely related to Methanobrevibacter and Methanosphaera.  In contrast, bacterial communities were relatively more diverse (with 6-13 distinct dominant bands per sample).  Bacterial communities varied between deer, but were highly similar within the first three chambers of individual animals.  Abomasums (the fourth chamber, characterized by a low pH) possessed a reduced bacterial diversity and unique bacterial phylotypes not found in the first three chambers. The majority of dominant bands sequenced from DGGE fingerprints were similar to uncultured rumen bacterial clones related to Streptococcus, Prevotella and Treponema, among others.   Our sample size does not allow us to unequivocally determine what factors might be controlling the diversity of prokaryotes in the deer stomach. However, it is clear that deer gut bacterial communities are more variable and diverse than archaeal  communities. It appears that most of the prokaryotic organisms that dominate deer guts have not been cultured.

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Cryptosporidium and Giardia contamination in urban and rural watersheds in the midwestern USA

Christopher Wendt, Rebecca Ives, Rachel McNinch, Shikha Singh, Joan Rose, Michigan State University, 3 Natural Resources Building, East Lansing, MI, 48824

The pathogens Cryptosporidium and Giardia have a history as etiologic agents in many recreational water outbreaks in the United States but the occurrence and risk associated with natural waters impacted by sewage or non-point sources has not been well explored.  To evaluate the differences of parasite contamination in rural and urban watersheds, analyses were conducted at recreational areas and parks as well as in sources including combined stormwater / sewage discharges, and drains associated with agricultural activities throughout the Great Lakes region.    Average concentrations of Giardia for the rural area represented by River Raisin watershed was 0.02137 per liter and 0.50576 per liter for Cryptosporidium. Infectious oocysts were detected in one sample at levels of 2301 per liter from an agricultural tile drain.   Average concentrations at sites in the more urbanized Grand River watershed were 0.05095 per liter for Giardia and 0.03705 per liter for Cryptosporidium.  Comparisons of parasite contamination evaluated in urban and rural waters using samples obtained from Chicago, Illinois and the River Raisin watershed in Michigan will be presented.  Thus far, we have found that Giardia is of greater significance in urban watersheds influenced by sewage and Cryptosporidium is of greater concern in rural waters influenced by animal wastes.

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