Center for Biofilm Engineering (CBE)
Permanent URI for this communityhttps://scholarworks.montana.edu/handle/1/9334
At the Center for Biofilm Engineering (CBE), multidisciplinary research teams develop beneficial uses for microbial biofilms and find solutions to industrially relevant biofilm problems. The CBE was established at Montana State University, Bozeman, in 1990 as a National Science Foundation Engineering Research Center. As part of the MSU College of Engineering, the CBE gives students a chance to get a head start on their careers by working on research teams led by world-recognized leaders in the biofilm field.
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Item Screening of Additive Formulations Enables Off-Chip Drop Reverse Transcription Quantitative Polymerase Chain Reaction of Single Influenza A Virus Genomes(American Chemical Society, 2021-03) Loveday, Emma Kate; Zath, Geoffrey K.; Bikos, Dimitri A.; Jay, Zackary J.; Chang, Connie B.The miniaturization of polymerase chain reaction (PCR) using drop-based microfluidics allows for amplification of single nucleic acids in aqueous picoliter-sized drops. Accurate data collection during PCR requires that drops remain stable to coalescence during thermocycling and drop contents are retained. Following systematic testing of known PCR additives, we identified an optimized formulation of 1% w/v Tween-20, 0.8 μg/μL bovine serum albumin, 1 M betaine in the aqueous phase, and 3 wt % (w/w) of the polyethylene glycol-perfluoropolyether2 surfactant in the oil phase of 50 μm diameter drops that maintains drop stability and prevents dye transport. This formulation enables a method we call off-chip drop reverse transcription quantitative PCR (OCD RT-qPCR) in which drops are thermocycled in a qPCR machine and sampled at various cycle numbers “off-chip”, or outside of a microfluidic chip. qPCR amplification curves constructed from hundreds of individual drops using OCD RT-qPCR and imaged using epifluorescence microscopy correlate with amplification curves of ≈300,000 drops thermocycled using a qPCR machine. To demonstrate the utility of OCD RT-qPCR, influenza A virus (IAV) RNA was detected down to a single viral genome copy per drop, or 0.320 cpd. This work was extended to perform multiplexed detection of IAV M gene RNA and cellular β-actin DNA in drops, and direct amplification of IAV genomes from infected cells without a separate RNA extraction step. The optimized additive formulation and the OCD-qPCR method allow for drop-based RT-qPCR without complex devices and demonstrate the ability to quantify individual or rare nucleic acid species within drops with minimal processing.Item Next-generation physiology approaches to study microbiome function at single cell level(Springer Science and Business Media LLC, 2020-02) Hatzenpichler, Roland; Krukenberg, Viola; Lange Spietz, Rachel K.; Jay, Zackary J.The function of cells in their native habitat often cannot be reliably predicted from genomic data or from physiology studies of isolates. Traditional experimental approaches to study the function of taxonomically and metabolically diverse microbiomes are limited by their destructive nature, low spatial resolution or low throughput. Recently developed technologies can offer new insights into cellular function in natural and human-made systems and how microorganisms interact with and shape the environments that they inhabit. In this Review, we provide an overview of these next-generation physiology approaches and discuss how the non-destructive analysis of cellular phenotypes, in combination with the separation of the target cells for downstream analyses, provide powerful new, complementary ways to study microbiome function. We anticipate that the widespread application of next-generation physiology approaches will transform the field of microbial ecology and dramatically improve our understanding of how microorganisms function in their native environment.Item High Potential for Biomass-Degrading Enzymes Revealed by Hot Spring Metagenomics(Frontiers Media SA, 2021-04) Reichart, Nicholas J.; Bowers, Robert M.; Woyke, Tanja; Hatzenpichler, RolandEnzyme stability and activity at elevated temperatures are important aspects in biotechnological industries, such as the conversion of plant biomass into biofuels. In order to reduce the costs and increase the efficiency of biomass conversion, better enzymatic processing must be developed. Hot springs represent a treasure trove of underexplored microbiological and protein chemistry diversity. Herein, we conduct an exploratory study into the diversity of hot spring biomass-degrading potential. We describe the taxonomic diversity and carbohydrate active enzyme (CAZyme) coding potential in 71 publicly available metagenomic datasets from 58 globally distributed terrestrial geothermal features. Through taxonomic profiling, we detected a wide diversity of microbes unique to varying temperature and pH ranges. Biomass-degrading enzyme potential included all five classes of CAZymes and we described the presence or absence of genes encoding 19 glycosyl hydrolases hypothesized to be involved with cellulose, hemicellulose, and oligosaccharide degradation. Our results highlight hot springs as a promising system for the further discovery and development of thermostable biomass-degrading enzymes that can be applied toward generation of renewable biofuels. This study lays a foundation for future research to further investigate the functional diversity of hot spring biomass-degrading enzymes and their potential utility in biotechnological processing.Item Low-Temperature Biosurfactants from Polar Microbes(MDPI AG, 2020-08) Trudgeon, Benjamin; Dieser, Markus; Balasubramanian, Narayanaganesh; Messmer, Mitch; Foreman, Christine M.Surfactants, both synthetic and natural, are used in a wide range of industrial applications, including the degradation of petroleum hydrocarbons. Organisms from extreme environments are well-adapted to the harsh conditions and represent an exciting avenue of discovery of naturally occurring biosurfactants, yet microorganisms fromcold environments have been largely overlooked for their biotechnological potential as biosurfactant producers. In this study, four cold-adapted bacterial isolates from Antarctica are investigated for their ability to produce biosurfactants. Here we report on the physical properties and chemical structure of biosurfactants from the genera Janthinobacterium, Psychrobacter, and Serratia. These organisms were able to grow on diesel, motor oil, and crude oil at 4 C. Putative identification showed the presence of sophorolipids and rhamnolipids. Emulsion index test (E24) activity ranged from 36.4–66.7%. Oil displacement tests were comparable to 0.1–1.0% sodium dodecyl sulfate (SDS) solutions. Data presented herein are the first report of organisms of the genus Janthinobacterium to produce biosurfactants and their metabolic capabilities to degrade diverse petroleum hydrocarbons. The organisms’ ability to produce biosurfactants and grow on different hydrocarbons as their sole carbon and energy source at low temperatures (4 C) makes them suitable candidates for the exploration of hydrocarbon bioremediation in low-temperature environments.Item Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis(Frontiers Media SA, 2020-02) Steward, Katherine F.; Eilers, Brian; Tripet, Brian; Fuchs, Amanda; Dorle, Michael; Rawle, Rachel; Soriano, Berliza; Balasubramanian, Narayanaganesh; Copie, Valerie; Bothner, Brian; Hatzenpichler, RolandBioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.Item Activity-based cell sorting reveals responses of uncultured archaea and bacteria to substrate amendment(Springer Science and Business Media LLC, 2020) Reichart, Nicholas J.; Jay, Zachary J.; Krukenberg, Viola; Parker, Albert E.; Lange Spietz, Rachel K.; Hatzenpichler, RolandMetagenomic studies have revolutionized our understanding of the metabolic potential of uncultured microorganisms in various ecosystems. However, many of these genomic predictions have yet to be experimentally tested, and the functional expression of genomic potential often remains unaddressed. In order to obtain a more thorough understanding of cell physiology, novel techniques capable of testing microbial metabolism under close to in situ conditions must be developed. Here, we provide a benchmark study to demonstrate that bioorthogonal non-canonical amino acid tagging (BONCAT) in combination with fluorescence-activated cell sorting (FACS) and 16S rRNA gene sequencing can be used to identify anabolically active members of a microbial community incubated in the presence of various growth substrates or under changing physicochemical conditions. We applied this approach to a hot spring sediment microbiome from Yellowstone National Park (Wyoming, USA) and identified several microbes that changed their activity levels in response to substrate addition, including uncultured members of the phyla Thaumarchaeota, Acidobacteria, and Fervidibacteria. Because shifts in activity in response to substrate amendment or headspace changes are indicative of microbial preferences for particular growth conditions, results from this and future BONCAT-FACS studies could inform the development of cultivation media to specifically enrich uncultured microbes. Most importantly, BONCAT-FACS is capable of providing information on the physiology of uncultured organisms at as close to in situ conditions as experimentally possible.Item Physical and chemical mechanisms that influence the electrical conductivity of lignin-derived biochar(2021-10) Kane, Seth; Ulrich, Rachel; Harrington, Abigail; Stadie, Nicholas P.; Ryan, Cecily A.Lignin-derived biochar is a promising, sustainable alternative to petroleum-based carbon powders (e.g., carbon black) for polymer composite and energy storage applications. Prior studies of these biochars demonstrate that high electrical conductivity and good capacitive behavior are achievable. However, these studies also show high variability in electrical conductivity between biochars (– S/cm). The underlying mechanisms that lead to desirable electrical properties in these lignin-derived biochars are poorly understood. In this work, we examine the causes of the variation in conductivity of lignin-derived biochar to optimize the electrical conductivity of lignin-derived biochars. To this end, we produced biochar from three different lignins, a whole biomass source (wheat stem), and cellulose at two pyrolysis temperatures (900 °C, 1100 °C). These biochars have a similar range of conductivities (0.002 to 18.51 S/cm) to what has been reported in the literature. Results from examining the relationship between chemical and physical biochar properties and electrical conductivity indicate that decreases in oxygen content and changes in particle size are associated with increases in electrical conductivity. Importantly, high variation in electrical conductivity is seen between biochars produced from lignins isolated with similar processes, demonstrating the importance of the lignin’s properties on biochar electrical conductivity. These findings indicate how lignin composition and processing may be further selected and optimized to target specific applications of lignin-derived biochars.Item Antimicrobial activity of naturally occurring phenols and derivatives against biofilm and planktonic bacteria(2019-10) Walsh, Danica J.; Livinghouse, Tom; Goeres, Darla M.; Mettler, Madelyn; Stewart, Philip S.Biofilm-forming bacteria present formidable challenges across diverse settings, and there is a need for new antimicrobial agents that are both environmentally acceptable and relatively potent against microorganisms in the biofilm state. The antimicrobial activity of three naturally occurring, low molecular weight, phenols, and their derivatives were evaluated against planktonic and biofilm Staphylococcus epidermidis and Pseudomonas aeruginosa. The structure activity relationships of eugenol, thymol, carvacrol, and their corresponding 2- and 4-allyl, 2-methallyl, and 2- and 4-n-propyl derivatives were evaluated. Allyl derivatives showed a consistent increased potency with both killing and inhibiting planktonic cells but they exhibited a decrease in potency against biofilms. This result underscores the importance of using biofilm assays to develop structure-activity relationships when the end target is biofilm.Item Dynamic processing of DOM: Insight from exometabolomics, fluorescence spectroscopy, and mass spectrometry(2018-06) Smith, Heidi J.; Tigges, Michelle M.; D'Andrilli, Juliana; Parker, Albert E.; Bothner, Brian; Foreman, Christine M.Dissolved organic matter (DOM) in freshwater environments is an important source of organic carbon, supporting bacterial respiration. Frozen environments cover vast expanses of our planet, with glaciers and ice-sheets storing upwards of 6 petagrams of organic carbon. It is generally believed that DOM liberated from ice stimulates downstream environments. If true, glacial DOM is an important component of global carbon cycling. However, coupling the release of DOM to microbial activity is challenging due to the molecular complexity of DOM and the metabolic connectivity within microbial communities. Using a single environmentally relevant organism, we demonstrate that processing of compositionally diverse DOM occurs, but, even though glacially derived DOM is chemically labile, it is unable to support sustained respiration. In view of projected changes in glacier DOM export, these findings imply that biogeochemical impacts on downstream environments will depend on the reactivity and heterogeneity of liberated DOM, as well as the timescale.Item Light-Based 3D Printing of Hydrogels with High-Resolution Channels(2019-01) Benjamin, Aaron D.; Abbasi, Reha; Owens, Madison; Olsen, Robert J.; Walsh, Danica J.; LeFevre, Thomas B.; Wilking, James N.Hydrogels are soft, water-based gels with widespread applications in personal care products, medicine and biomedical engineering. Many applications require structuring the hydrogel into complex three-dimensional (3D) shapes. For these applications, light-based 3D printing methods offer exquisite control over material structure. However, the use of these methods for structuring hydrogels is underdeveloped. In particular, the ability to print hydrogel objects containing internal voids and channels is limited by the lack of well-characterized formulations that strongly attenuate light and the lack of a theoretical framework for predicting and mitigating channel occlusion. Here we present a combined experimental and theoretical approach for creating well-defined channels with any orientation in hydrogels using light-based 3D printing. This is achieved by the incorporation of photoblocker and the optimization of print conditions to ensure layer-layer adhesion while minimizing channel occlusion. To demonstrate the value of this approach we print hydrogels containing individual spiral channels with centimeter-scale length and submillimeter-scale cross-section. While the channels presented here are relatively simple, this same approach could be used to achieve more complex channel designs mimicking, for example, the complex vasculature of living organisms. The low cytotoxicity of the gel makes the formulation a promising candidate for biological applications.