The Possible Role of Bacteriophage in Inflammatory Bowel Diseases

Rebecca Allen and John Wertz, Calvin College, Grand Rapids, MI

Inflammatory Bowel Diseases (IBD), such as Crohns Disease (CD) affects many people around the world, and an estimated 1.4 million people in the US (CDC). It is believed these diseases are caused by dysbiosis; the irregular balance of “helpful” and “harmful” bacteria in the human gut. (Tamboli et al., 2004) We believe this dysbiosis could be due to bacteriophage, a virus that only infects bacteria. Recent evidence from patients with Ulcerative Colitis demonstrated a 100 fold increase in free bacteriophage versus healthy individuals. (Lepage et al. 2008)

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The effect organic acids produced from the dilute acid pretreatment of lignocellulose on biomass and ethanol production in Pichia stipitis CBS 6054

Alexander Roll, Stephanie Groves, and Susan Bagley, Michigan Technological University, Houghton, MI 49931

The pretreatment of lignocellulose waste streams releases fermentable sugars but can also produce potentially inhibitory compounds. As detected by HPLC analysis, known inhibitory compounds included a mixture of organic acids and aldehydes. Furfural and 5-HMF were present at concentrations well below the inhibitory levels while acetic, formic, and oxalic acid were present at known inhibitory levels. The goal of this research was to understand the effect of these acids, individually and in a mixture, on biomass and ethanol production by Pichia stipitis CBS 6054. A synthetic dilute acid hydrolysate (DAH) medium with an organic acid mixture was used to assess the impact of the organic acids. A known range of concentrations of each acid was also used with the synthetic DAH. To test the synergistic effects, acetic, formic, and oxalic acids, at levels found in the DAH, were added to a synthetic hydrolysate medium. In both studies, fermentations were carried out over a period of 72 hours under microaerophilic conditions. The individual and synergistic inhibition study results were compared to those obtained from fermentations of a synthetic dilute acid hydrolysate and DAH. Acetic and formic acids were inhibitory to both biomass and ethanol production at concentrations above 2 g/L. Oxalic acid had no inhibitory effects on biomass and ethanol production below 6 g/L. A synergistic effect was observed in the treatments that contained the acid mixture compared to the control. The treatments with DAH, however, had more of an inhibitory effect than the acid mixture for both biomass production and ethanol yield. Although, organic acids do play important role in the inhibitory nature of the hydrolysate, this study shows that they are not the only compounds present in the DAH that effect biomass and ethanol production. Understanding how these inhibitory compounds change the biomass and ethanol production could help increase the efficiency and total ethanol recovery in various bioprocess systems. This data will be used to develop a model for assessing the toxicity of a DAH hydrolysate.

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Adaptation of Escherichia coli KO11 from Dilute Acid Pretreated Industrial Effluent

Elizabeth Hubsky, Stephanie Groves and Susan Bagley, Michigan Technological University, Houghton, MI 49931

Escherichia coli KO11, a previously constructed genetically modified ethanologenic E. coli strain, was used to convert dilute acid pretreated industrial effluent into ethanol. E. coli KO11 is able to convert both pentose and hexose sugars to ethanol. It contains the genes encoding for pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis. In initial fermentation studies with E. coli KO11, low product yields were achieved as a result of the inhibitory compounds present after the dilute acid pretreatment of industrial waste effluent. Adaptation is one method to address this challenge. The feasibility of improving ethanol yields from E. coli KO11 on dilute acid pretreated industrial effluent using repeated cell subculture was investigated. Unadapted E. coli KO11 was placed in 100% dilute acid hydrolysate (neutralized to a pH of 6.0) and was unable to produce ethanol, though it was able to produce biomass. It was then grown on increasing concentrations of dilute acid pretreated hydrolysate supplemented with Luria-Bertani medium (20mg/L), Xylose (20g/L) and chloramphenicol (100 mg/L). By selecting for improved strains that are able to grow and produce ethanol at high concentrations of hydrolysate, the product yield can be enhanced. Biomass production, substrate utilization, and ethanol yield were used to measure the efficiency of E. coli KO11 during this process. Through adaptation, E. coli KO11 will increase ethanol yield on dilute acid pretreated industrial effluent.

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Characterization of Microorganisms Recovered from Lignocellulosic Industrial Waste Stream Effluent

Zhicong Liu, Aparupa Sengupta, Stephanie Groves, and Susan T. Bagley, Department of Biological Sciences, Michigan Technological University, Houghton, MI

The goal of this study was to isolate and recover microorganisms able to ferment the sugars remaining in a lignocellulosic industrial waste stream; as such isolates may be better adapted to the waste stream than laboratory cultures and able to ferment over a wider temperature range. Enrichment studies were used to examine for C6 (e.g., glucose) and. C5 (e.g., xylose) sugar use. Enrichment samples were incubated from 4°C- 50°C using three media: YPG (with glucose), YPX (with xylose), and R2A liquid media (with glucose). When growth was apparent in the flasks, the contents were transferred to YPG, YPX and R2A agar plates. A total of 34 pure cultures were obtained (16 bacteria and 18 yeast) based on differences in colony morphotypes. These strains were characterized using standard microbiological techniques; it was verified that they could grow with glucose and/or xylose as the sole carbon and energy sources. Four yeast and two bacterial isolates able to grow with both sugars and also able to grow at the higher temperature range (35°C - 45°C) were subjected to evaluation for sugar utilization, biomass production, and ethanol production (analyzed using HPLC). The cultures that produce ethanol yields close to theoretical yields will be subjected to further analysis and adaptation and could be the model organisms for biofuel production in the future.

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The Use of Quantitative Microbial Risk Assessment and Transport Models to Determine Beach Closures

Brian M. Panzl, Mark H. Weir, and Joan B. Rose, Michigan State University

Natural water recreation locations are a well known source of exposure for gastrointestinal infection outbreaks. Therefore a means of predicting not only potential microbial impacts to recreational beaches, but risk of gastrointestinal infection associated with these impacts is a useful tool. This work is underway in an effort to develop a tool for beach managers to quickly identify the risk level associated with recreational exposure to waterborne pathogens in the environment, such as Salmonella, E. coli O157:H7 or Shigella. The tool allows for a predictive estimate of risk using transport models for two Great Lakes beaches, based on meteorological and surface water quality data. Transport models were selected from the open literature for two test beaches; Silver Beach in Saint Joseph, MI and Washington Park Beach in Michigan City, IN. Excel was chosen as the initial platform for the tool, based on the large experience base within the potential user group. Visual Basic macros were also implemented in order to provide the functionality of executing the transport models. A built in aspect of this tool is the capability to auto update from the internet, which greatly reduces the demand on the user to generate the values independently. The immediate next step in the evolution of this tool is to bring the functionality of the Excel spreadsheet to a program that can handle a more demanding computational system, which would then provide a more descriptive risk assessment. A more distant objective is to transport this tool onto a smart phone. This would create a dynamic aspect that would provide any potential user with the ability to use it on location without the need of a computer interface. This device is currently in the early stages of its development, but the ambition of this project is to provide beach managers with a portable tool that can quickly give effective analysis of any danger posed by a given outbreak and thus improve the safety of recreational beach users.

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Inositol Catabolism in Sinorhizobium meliloti is negatively regulated by the IolR transcriptional regulator

Petra R. A. Kohler, Ee-Leng Choong, Silvia Rossbach, Department of Biological Sciences, Western Michigan University, Kalamazoo, Michigan 49008-5410

We recently showed that Sinorhizobium meliloti, the nitrogen fixing symbiont of alfalfa, catabolizes myo-, scyllo- and D-chiro-inositol and that plant derived inositols serve as nutritional mediators in symbiotic nitrogen fixation. S. meliloti requires the functional inositol catabolic pathway and its regulation for successfully competing during host plant nodulation. Thus, the function of inositol extends beyond being a carbon source and seems to be involved in the signal exchange between micro- and macrosymbiont. In this study we elucidates the transcriptional organization and regulation of the inositol catabolic pathway in S. meliloti using reverse transcriptase PCR (RT-PCR), electrophoretic shift assays (EMSA), as well as reporter gene and dehydrogenase assays. The S. meliloti inositol catabolism (iol) genes are organized three transcriptional units: the on pSymB located idhA gene, the chromosomal iolYRCDEB operon and the monocistronically iolA gene. The iol genes were weakly constitutively expressed, and expression was strongly induced by inositol. The EMSA demonstrated that IolR recognizes a conserved palindromic sequence (5’-GGAA5-11TTCC-3’) in its own promoter as well as in the promoters of idhA, iolY and iolC. The idhA gene is monocistronically transcribed. The iolYRCDEB operon is transcribed from the PiolY and contains two internal operators upstream of iolR and iolC. The iolA promoter does not contain the IolR-binding motif and was independently regulated of IolR. In addition, iolA was constitutively expressed, but expression increase in the presence of inositol. This indicates that the iolA gene product is not part of the core inositol catabolic pathway, but seems to play a more central role in the general metabolism of S. meliloti. As a matter of fact, a growth study in minimal medium revealed that a functional iolA gene is also required for valine metabolism.

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Molecular Characterization of Stream Biofilm Communities in Response to Light and Phosphorous

Alicia Ketcham and Dan Clemans, Eastern Michigan University, Ypsilanti, MI

Microbial communities cultivated on unglazed ceramic tiles in artificial streams were analyzed to examine microbial diversity and dynamics in response to different levels of light and phosphorous. Results from culture independent molecular techniques indicated that the microbial communities consisted largely of bacteria from the Proteobacteria phylum. Another interesting result was the presence of chloroplast hits, and their higher prevalence in streams containing low light and phosphorous conditions. Further molecular analysis will help identify the specific organisms and how they respond to these nutrient and light conditions.

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Comparing DNA Extraction Methods for Environmental Water Samples: A Malawi Case Study

Yolanda M. Brooks, Aslan, Asli, Joan Rose, Michigan State University, East Lansing, MI 48824

Current standards for fecal contamination in surface waters limit source identification and rapid results from nonpoint, and point sources with human fecal effluent. Microbial source tracking methods have been developed to identify human fecal sources from environmental samples. These methods are highly dependent on the extracted DNA quality and quantity and co-extracted inhibitors which limit downstream applications. In this study, we collected 11 environmental water samples from Malawi, East Africa; 1 tap water sample, 1 waste water sample, 2 lake water samples and 7 river water samples (20 ml – 500 ml). Samples were membrane filtered and stored in the dark until DNA extraction. We compared three extraction protocols on the sample filters; Qiamp Stool DNA Mini Kit® (Qiagen), PowerSoil Kit® (Mo Bio Laboratories) and EPA DNA Crude Extraction Method followed by Qiamp DNA Mini Kit® (Qiagen). The DNA extracts were tested for three markers; Escherichia coli uid gene, Enterococci 23S gene and a human specific marker, Bacteroides thetaiotaomicron alpha mannanese gene, with qPCR.The Enterococci, Bacteriodes and E. coli markers from all samples ranged from 1.7*102 -3.1*104 cells/100ml; 1.5*102 -6.1*104 cells/100ml and 6.8*103 -7*105 cells/100ml, respectively. The kits’ extraction efficacy varied according to the indicator. For all water types, E.coli and Bacteroides markers’ amplification from the three kits was not statistically different (p>0.05). However, the Enterococci marker’s amplification from the EPA Extraction combined with Qiamp was significantly lower than the Qiamp Stool and Mo Bio PowerSoil Kits (p<0/05). For the river samples, the E.coli and Bacteroides markers’ amplifications was significantly higher in Mo Bio PowerWater Kit. The Enterococci marker amplification were significantly higher in the Qiamp Stool Kit (p>0.05). The data indicate that Qiamp Stool kit is better at extracting Enteroocci’s gram + DNA, while Mo Bio PowerSoil is better at extracting Bacteroides. Larger dataset trials will be needed to conclusively decide which protocols work best for various environmental water samples and targets.

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Comparative Analysis of Pichia stipitis CBS 6054 Adapted to a Dilute Acid Pretreated Waste Stream for Improved Ethanol Yield

Stephanie Groves1, Jifei Liu2, Alex Roll3, Jill Jensen2,4, David Shonnard2, and Susan Bagley1, 1 Department of Biological Sciences, 2 Department of Chemical Engineering, 3 Department of Civil and Environmental Engineering, Michigan Technological University, Houghton, MI4 Currently at the Idaho National Laboratory, Idaho Falls, ID

Adaptation of yeast to dilute acid pretreated (DAP) hydrolysate has been shown to be a viable method for overcoming inhibition as well as increasing ethanol yields; however, the effect of initial feedstock composition is are not well characterized. The aim of this work was to compare hydrolysates from two different dilute acid pretreated feedstocks and their effects on the adaptation of Pichia stipitis CBS 6054. The yeast was adapted to a DAP hydrolysate of a lignocellulose-containing waste stream. Adaptation was carried out over a period of three – four weeks using repeated sub-culturing in increasing concentrations of hydrolysate (up to 100%) The fermentation capabilities of both the adapted and the unadapted strains were evaluated in order to verify the adaptation resulted in improved product yields, substrate utilization, and biomass production. The adapted strain produced >70% of the theoretical ethanol yield within 72 hours, using close to 100% of the available sugars. In contrast, the unadapted strain utilized < 1% of the available sugars and produced no ethanol over 72 hours. These results were compared to a previous adaptation of P. stipitis to dilute acid pretreated Aspen hydrolysate. Adaptation to the waste stream hydrolysate resulted in lower ethanol yields, which may be attributed to the larger quantity of inhibitory compounds. The current direction of this work is to perform comparative proteomic analysis both sets of unadapted/adapted P. stipitis strains. Determining the molecular and biochemical mechanisms involved in adaptation will benefit biofuels research by providing target proteins/pathways for bio-prospecting and in the design of novel genetically modified organisms.

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Enteric Bacteria and Biodiesel Glycerol Byproduct in Undergraduate Research

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

Escherichia coli and Klebsiella species have become important in biofuel research because strains have been engineered that produce ethanol, butanol or microdiesel.These bacteria which naturally utilize pure glycerol were assessed for potential to utilize biodiesel glycerol byproduct made by transesterification or catalyst column.To determine if byproduct contained inhibitory components E. coli was cultured in nutrient broth to which byproduct had been added. Culture optical density (OD) was measured by use of a Klett – Summerson colorimeter. Growth in culture containing byproduct from transesterification was slightly decreased while that in culture containing byproduct from catalyst column was increased. Bacteria were grown in minimal broth to evaluate byproduct as an individual carbon source. Byproduct made by transesterification was pH adjusted due to high alkalinity. There was not a considerable difference between culture ODs produced from glycerol and byproduct from catalyst column. Byproduct from transesterification did not support growth as well as that from catalyst column. K. pneumoniae appeared to utilize glycerol and byproduct better than E. coli. Both organisms grew significantly better on glucose than glycerol. A rapid solid phase glucose oxidase and peroxidase based assay (Precision Labs, Inc.) was used to detect glucose in cultures. Both E. coli and K. pneumoniae lowered added glucose to an undetectable level. A rapid solid phase alcohol oxidase and peroxidase based assay (AlcoScreen) was used to detect alcohol. E. coli showed a positive test for the presence of alcohol in cultures produced from glucose or glycerol. It was unknown if alcohol was made from byproduct because stock solutions showed a positive test for the presence of alcohol. These observations indicate that enteric bacteria may be able to utilize biodiesel glycerol byproduct as an industrial feedstock.

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Effects of Glyphosate on Wetland Soil Microbial Community Composition

Jennifer K. Kirk, Kristin E. Judd, and Daniel L. Clemans, Eastern Michigan University, Ypsilanti, Michigan 48197

Phragmites australis,known as the common reed, is an invasive plant that has strong negative impacts on wetland ecosystems. These effects include increasing plant primary production and litter mass, decreasing water flow, and reducing bio-diversity. Microorganisms are central to this ecosystem as they facilitate processes essential to wetland function through nutrient cycling, detritus breakdown, and by providing a base for the food web. To control Phragmites, the broad-spectrum herbicide glyphosate is often applied. While its toxicity effect on macro-organisms and specific model microorganisms has been well researched, effects on microbial community structure have not. Because microbes are important regulators of nutrient cycling in wetlands, and changes in their composition can alter this function, the purpose of this study was to determine whether differences in microbial community structure occur after glyphosate treatment. We hypothesized that glyphosate addition would cause an overall shift in the microbial community composition given that some microbial groups would be enriched based on their ability to use glyphosate as a nutrient source. We collected a series of soil samples from a local wetland prior to and following glyphosate application. Using terminal restriction fragment length polymorphism (T-RFLP) analysis, a bacterial community profile was constructed through time. Preliminary analysis indicates that one year following glyphosate treatment, microbial community structure appears distinct while three years later communities closely resemble untreated sites. Given microbes’ role in wetland ecosystems, our results suggest that glyphosate may impact ecosystem function through shifts in microbial community structure.

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Characterizing coaggregation among strains of human gastrointestinal bacteria

Lindsey Kolar and Daniel Clemans, Eastern Michigan University, Ypsilanti, MI

Coaggregation is the process by which genetically distinct bacteria adhere to each other by means of specific adhesin and receptor molecules. This adhesion process is thought to be an integral part of biofilm formation, and in the human gastrointestinal tract coaggregation may be responsible for host benefits such as increased microbial production of the anti-cancer agent butyrate. Visual, chemical, and heat coaggregation assays were performed through pair-wise mixtures of 16 gut microbes. The results of this study reveal 16 coaggregation interactions, with Lactobacillus rhamnosus ATCC 53103, L. acidophilus NCFM, and L. plantarum ATCC 39542 being the most promiscuous coaggregators. An attempt is made to identify specific adhesin and receptor molecules, and a potential model for a human gastrointestinal biofilm is constructed..

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