Scholarly Work - Center for Biofilm Engineering
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/9335
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Item Experimental Designs to Study the Aggregation and Colonization of Biofilms by Video Microscopy With Statistical Confidenc(Frontiers Media SA, 2022-01) Pettygrove, Brian A.; Smith, Heidi J.; Pallister, Kyler B.; Voyich, Jovanka M.; Stewart, Philip S.; Parker, Albert E.The goal of this study was to quantify the variability of confocal laser scanning microscopy (CLSM) time-lapse images of early colonizing biofilms to aid in the design of future imaging experiments. To accomplish this a large imaging dataset consisting of 16 independent CLSM microscopy experiments was leveraged. These experiments were designed to study interactions between human neutrophils and single cells or aggregates of Staphylococcus aureus (S. aureus) during the initial stages of biofilm formation. Results suggest that in untreated control experiments, variability differed substantially between growth phases (i.e., lag or exponential). When studying the effect of an antimicrobial treatment (in this case, neutrophil challenge), regardless of the inoculation level or of growth phase, variability changed as a frown-shaped function of treatment efficacy (i.e., the reduction in biofilm surface coverage). These findings were used to predict the best experimental designs for future imaging studies of early biofilms by considering differing (i) numbers of independent experiments; (ii) numbers of fields of view (FOV) per experiment; and (iii) frame capture rates per hour. A spreadsheet capable of assessing any user-specified design is included that requires the expected mean log reduction and variance components from user-generated experimental results. The methodology outlined in this study can assist researchers in designing their CLSM studies of antimicrobial treatments with a high level of statistical confidence.Item Systematic Statistical Analysis of Microbial Data from Dilution Series(Springer Science and Business Media LLC, 2020-05) Christen, J. Andrés; Parker, Albert E.In microbial studies, samples are often treated under different experimental conditions and then tested for microbial survival. A technique, dating back to the 1880's, consists of diluting the samples several times and incubating each dilution to verify the existence of microbial Colony Forming Units or CFU's, seen by the naked eye. The main problem in the dilution series data analysis is the uncertainty quantification of the simple point estimate of the original number of CFU's in the sample (i.e., at dilution zero). Common approaches such as log-normal or Poisson models do not seem to handle well extreme cases with low or high counts, among other issues. We build a novel binomial model, based on the actual design of the experimental procedure including the dilution series. For repetitions we construct a hierarchical model for experimental results from a single lab and in turn a higher hierarchy for inter-lab analyses. Results seem promising, with a systematic treatment of all data cases, including zeros, censored data, repetitions, intra and inter-laboratory studies. Using a Bayesian approach, a robust and efficient MCMC method is used to analyze several real data sets.Item Effect of temperature, nitrate concentration, pH and bicarbonate addition on biomass and lipid accumulation in the sporulating green alga PW95(Elsevier BV, 2020-12) Corredor, L.; Barnhart, E.P.; Parker, A.E.; Gerlach, Robin; Fields, Matthew W.The mixed effects of temperature (20 °C, 25 °C and 30 °C), nitrate concentration (0.5 mM and 2.0 mM), pH buffer, and bicarbonate addition (trigger) on biomass growth and lipid accumulation were investigated in the environmental alga PW95 during batch experiments in standardized growth medium. PW95 was isolated from coal-bed methane production water and classified as a Chlamydomonas-like species by morphological characterization and phylogenetic analysis (18S, ITS, rbcL). A factorial experimental design tested the mixed effects on PW95 before and after nitrate depletion to determine a low cost, high efficiency combination of treatments for biomass growth and lipid accumulation. Results showed buffer addition affected growth for most of the treatments and bicarbonate trigger had no statistically significant effect on growth and lipid accumulation. PW95 displayed the highest growth rate and chlorophyll content at 30 °C and 2.0 mM nitrate and there was an inverse relation between biomass accumulation and lipid accumulation at the extremes of nitrate concentration and temperature. The combination of higher temperature (30 °C) and lower nitrate level (0.5 mM) without the use of a buffer or bicarbonate addition resulted in maximal daily biomass accumulation (5.30 × 106 cells/mL), high biofuel potential before and after nitrate depletion (27% and 20%), higher biofuel productivity (16 and 15 mg/L/d, respectively), and desirable fatty acid profiles (saturated and unsaturated C16 and C18 chains). Our results indicate an important interaction between low nitrate levels, temperature, and elevated pH for trade-offs between biomass and lipid production in PW95. This work serves as a model to approach and advance the study of physiological responses of novel microalgae to diverse culture conditions that mimic environmental changes for outdoor biofuel production. The most promising conditions for growth and biofuel production were identified for PW95 and this approach can be implemented for other microalgal production systems.Item Evaluation of the Antimicrobial Efficacy of N-Acetyl-l-Cysteine, Rhamnolipids, and Usnic Acid—Novel Approaches to Fight Food-Borne Pathogens(MDPI, 2021) Chlumsky, Ondrej; Smith, Heidi J.; Parker, Albert E.; Brileya, Kristen; Wilking, James N.; Purkrtova, Sabina; Michova, Hana; Ulbrich, Pavel; Viktorova, Jitka; Demnerova, KaterinaIn the food industry, the increasing antimicrobial resistance of food-borne pathogens to conventional sanitizers poses the risk of food contamination and a decrease in product quality and safety. Therefore, we explored alternative antimicrobials N-Acetyl-L-cysteine (NAC), rhamnolipids (RLs), and usnic acid (UA) as a novel approach to prevent biofilm formation and reduce existing biofilms formed by important food-borne pathogens (three strains of Salmonella enterica and two strains of Escherichia coli, Listeria monocytogenes, Staphylococcus aureus). Their effectiveness was evaluated by determining minimum inhibitory concentrations needed for inhibition of bacterial growth, biofilm formation, metabolic activity, and biofilm reduction. Transmission electron microscopy and confocal scanning laser microscopy followed by image analysis were used to visualize and quantify the impact of tested substances on both planktonic and biofilm-associated cells. The in vitro cytotoxicity of the substances was determined as a half-maximal inhibitory concentration in five different cell lines. The results indicate relatively low cytotoxic effects of NAC in comparison to RLs and UA. In addition, NAC inhibited bacterial growth for all strains, while RLs showed overall lower inhibition and UA inhibited only the growth of Gram-positive bacteria. Even though tested substances did not remove the biofilms, NAC represents a promising tool in biofilm prevention.Item Drip flow reactor method exhibits excellent reproducibility based on a 10-laboratory collaborative study(Elsevier BV, 2020) Goeres, Darla M.; Parker, Albert E.; Walker, Diane K.; Meier, Kelsey; Lorenz, Lindsey A.; Buckingham-Meyer, KelliA standard method for growing Pseudomonas aeruginosa biofilm in the Drip Flow Biofilm Reactor was assessed in a 10-laboratory study. The mean log density was 9.29 Log10(CFU/cm2). The repeatability and reproducibility SDs were equal to 0.22 and 0.24, respectively, providing statistical confidence in data generated by the method.Item Multiscale Flux-Based Modeling of Biofilm Communities(Society for Industrial & Applied Mathematics, 2020-01) Zhang, T.; Parker, A.; Carlson, R.P.; Stewart, Philip S.; Klapper, I.Models of microbial community dynamics generally rely on a subscale description for microbial metabolisms. In systems such as distributed multispecies communities like biofilms, where it may not be reasonable to simplify to a small number of limiting substrates, tracking the large number of active metabolites likely requires measurement or estimation of large numbers of kinetic and regulatory parameters. Alternatively, a largely kinetics-free framework is proposed combining cellular level constrained, steady state flux analysis of metabolism with macroscale microbial community models. This multiscale setup naturally allows coupling of macroscale information, including measurement data, with cell scale metabolism. Further, flexibility in methodology is stressed: choices at the microscale (e.g., flux balance analysis or elementary flux modes) and at the macroscale (e.g., physical-chemical influences relevant to biofilm or planktonic environments) are available to the user. Illustrative computations in the context of a biofilm, including comparisons of systemic and Nash equilibration as well as an example of coupling experimental data into predictions, are provided.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 Bayesian estimation and uncertainty quantification in models of urea hydrolysis by E. coli biofilms(Informa UK Limited, 2021-02) Jackson, Benjamin D.; Connolly, James M.; Gerlach, Robin; Klapper, Issac; Parker, Albert E.Urea-hydrolysing biofilms are crucial to applications in medicine, engineering, and science. Quantitative information about ureolysis rates in biofilms is required to model these applications. We formulate a novel model of urea consumption in a biofilm that allows different kinetics, for example either first order or Michaelis-Menten. The model is fit it to synthetic data to validate and compare two approaches: Bayesian and nonlinear least squares (NLS), commonly used by biofilm practitioners. The shortcomings of NLS motivate the Bayesian approach where a simple Markov Chain Monte Carlo (MCMC) sampler is applied. The model is then fit to real data of influent and effluent urea concentrations from experiments on biofilms of Escherichia coli. Results from synthetic data aid in interpreting results from real data, where first order and Michaelis-Menten kinetic models are compared. The method shows potential for general applications requiring biofilm kinetic information.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 Design and fabrication of biofilm reactors(2020) Goeres, Darla M.; Pedersen, Stephen; Warwood, B. K.; Walker, Diane K.; Parker, Albert E.; Mettler, Madelyn; Sturman, Paul J.Laboratory biofilm reactors are tools that researchers use to grow biofilms that exhibit characteristics sufficiently similar to the environment of interest. Numerous biofilm reactors that model various fluid dynamics are described in scientific literature, each with its associated list of advantages and limitations. This chapter focuses on the process used to design and fabricate biofilm reactors with the stated goal of generating a commercial product. The process begins with identifying the environment of interest and key attributes the reactor should include or model. A prototype is then designed, built, and tested in the laboratory. Modifications are made based upon laboratory performance until a design is achieved that is affordable, practical, operationally simple, and relevant and that provides repeatable, convincing results. This process was used to design the industrial surfaces biofilm reactor, developed to model cooling tower biofilms but suitable to study biofilms grown under low shear, high gas transfer, and intermittently wet conditions.