2021 Montana Biofilm Meeting Virtual Open House

Ask me about: how to deal with zeros, in vitro and clinical trials, 2D and 3D image analysis, quality control of industrial processes, multivariate analysis of ecological communities

Al Parker <parker@math.montana.edu>

I am a professor in MSU's Department of Microbiology & Cell Biology and I also serve as the director of the CBE. My lab uses molecular ecology and physiology to study microbial communities associated with different environments. Laboratory work includes physiology, ecology, and genomics of different organisms relevant to bioremediation, material interactions, groundwater, and biofuels. Ultimately, my driving question is to understand the relationships between structure and function at different scales of biology and the associated ecological and physiological responses. An improved understanding of structure/function relationships will allow predictive modeling and design for a variety of natural and engineered systems. My research projects are currently funded through the Department of Energy, National Science Foundation, the Department of Defense, and private industry.

Matthew Fields <cbedirector@montana.edu>

As the CBE's industrial coordinator, I manage a robust industrial partnership program wherein over 25 companies support research and technology transfer efforts. I work closely with these member companies to assist adoption of biofilm-related technologies. My research focus is on investigations of biofilms in industrial water systems, environmental biofilms, and the development of standardized methods for biofilm analysis.

Paul Sturman <paul_s@montana.edu>

As a senior expert in biofilm science and technology, I have led the Biofilm Control lab at CBE since 1992. My research addresses fundamental mechanisms that protect microbes in biofilms from disinfectants, antibiotics, and the innate immune system as well as practical strategies for managing detrimental biofilms in a wide variety of systems.

Phil Stewart <phil_s@montana.edu>

Stephan Warnat, Assistant Professor of Mechanical Engineering, Montana State University. Stephan Warnat is an early Career Researcher and an expert in system integration of miniaturized sensors and actuators for academic and industrial applications. Warnat worked at the internationally noted Fraunhofer Institute for Silicon Technology, Germany, and developed new sensor integration concepts for innovative systems in collaborations with academic and industrial partners. His interdisciplinary education in electrical engineering, materials science, and mechanical engineering fosters a mission driven development, testing, and integration of new sensing mechanisms. Current research questions address how microfabricated sensors can measure reliably biofilm growth and water quality parameter through an in-situ sensor integration in harsh environmental and industrial applications. Current available systems lack the capability in autonomously measuring changes of bio-geochemical processes that can be utilized to study dynamic processes for extended times. The current projects are related to application such as biofilm growth under microgravity conditions and bio-geochemical parameter detection in soil and ice.

Stephan Warnat <stephan.warnat@montana.edu>

Sequencing technologies can be used to examine microbial community structure and function through analyses of DNA and RNA. Genes, transcripts, single genomes, and community genomes (metagenomes) are rich with information on potential microbial processes, their evolutionary history, and their impact on diverse environments. At the CBE, we investigate genomic and transcriptomic sequences from a wide variety of medical, industrial, and environmental biofilms. Luke specializes in sequence analysis with projects ranging from sulfur cycling at hydrothermal vents to natural gas formation in subsurface coal seams.

Luke McKay <luke.mckay@montana.edu>

Dr. Lauchnor is an associate professor in the Department of Civil Engineering at MSU. Her research group investigates microbially driven processes that contribute to water quality improvement within treatment processes and remediation of contaminated environments. Specifically, her research investigates microbial processes that drive removal of metal contaminants in the environment. Her laboratory and collaborators also investigate microbial contributions to carbon and nitrogen dynamics in engineered wetlands for wastewater treatment.

Ellen Lauchnor <ellen.lauchnor@montana.edu>

When the CBE was originally established, the majority of the focus was on biofilms in industrial processes, like the oil and gas industry. Over time, the field expanded to include health-related and medical biofilms, the built-environment, along with other environmental settings. An opportunity exists for the CBE to further expand biofilm research in other arenas like the food industry, as it stands to reason that where there is moisture and nutrients (i.e., food!), biofilms are likely to be found.

• What are areas in the food industry where there is potential for biofilm growth?

• What technologies exist and what is trending for the future?

We are seeking your input! Share your insights about biofilms in the food industry and ask me how we can help!

Diane Walker <dianew@montana.edu>

Diverse fungi have demonstrated the ability to colonize surfaces and develop biofilms, either as single species or in association with other fungi or other microbes including algae and bacteria. Fungi have been detected within biofilms present in different industrial, agricultural, medical and natural environments. My research focuses on the study of fungal systems and their potential use in different engineering applications. Some of the current projects that I am involved with include the application of fungal-based systems for: (i) water treatment, (ii) biomanufacturing of engineered mycelium composite construction materials, and (iii) bioconversion of lignocellulose waste materials into added-value products. Further, my research also addresses the challenge of developing the next generation of fungal-based systems for wastewater treatment, which are envisioned to exploit the natural association and synergistic interactions that exist between fungi and other microbes in natural environments

Erika Espinosa-Ortiz <erika.espinosaortiz@montana.edu>

Robin will be available to provide an overview of the activities in the Gerlach Research Group in the Bio- and Biofilm-Technology Laboratory, which ranges from Biofilms, Algae and their Microbiomes in Engineered Biomineralization, Urinary Tract Stone Formation, Methane Cycling and Biofuel Production to various smaller projects. The overview is accompanied with a brief list of the most commonly and most recently used experimental and analytical approaches.

Robin Gerlach <robin_g@montana.edu>

The Medical Biofilms Laboratory, led by Dr. Garth James, does research in support of NIH and DOD funded grants in areas such as chronic wounds and tolerance of biofilms to antimicrobial agents as well collaborating with small business on SBIR and STTR funded grants. In addition, the MBL performs custom testing projects for CBE industrial associates and other companies. These projects have included in-vitro evaluation of microbial attachment and biofilm formation on a wide variety of medical devices, including venous access catheters (PIV, PICC, CVC) and needle-free connectors, urinary catheters, and various implantable medical devices (pacemakers, neurostimulators, cochlear implants, surgical mesh, orthopedic implants and spinal fixation devices, dental implants). The MBL has also evaluated antimicrobial lock solutions for both venous access and urinary catheters as well as surgical and wound lavages, antimicrobial wound dressings, toothpastes and mouth washes, and endodontic irrigants. In addition to in-vitro work, the MBL performs ex-vivo analysis of tissues and explanted devices in support of preclinical animal studies and human clinical trials. MBL personnel have expertise in general and molecular microbiology, cell biology, immunology, and virology techniques, as well as scanning confocal laser microscopy, scanning electron microscopy, tissue cryoembedding and sectioning, and fluorescent in-situ hybridization. MBL personnel have extensive experience in designing and adapting in-vitro model systems to perform microbial attachment and biofilm growth experiments under conditions to mimic in-vivo conditions.

Garth James <gjames@montana.edu>

If you are one of the folks who enjoys regulatory science, are curious about what method would be best for your particular biofilm challenge, or would just like to chat about how the CBE has rocked the last 30 years as the first, oldest, and best biofilm center, I am here to discuss!

I've been the PI for the CBE's Standardized Biofilm Methods Lab since 2000; and during that time I have facilitated the development and approval of five standard test methods with #6 in the works. I've researched biofilms in a range of environments including recreational water, beer draught lines, oil fields, and just about every hard surface in the built environment and industry where biofilm is cause for concern. My lab is dedicated to the creation, establishment, and transfer of quantitative biofilm methods for the benefit of academia, government, and industry. Our goal is design, build, and test laboratory reactors that incorporate the relevant engineering specifications of field systems to recreate a growth environment for predicting the efficacy of biofilm control strategies. In 2020, the CBE capitalized on our lab's methods development expertise and expanded our role to include regulatory science.

Darla Goeres <darla_g@montana.edu>

Welcome! I'm a professor in MSU's Department of Chemical and Biological Engineering and have affiliations with the CBE, Thermal Biology Institute, and the Department of Microbiology and Cell Biology. My research group studies biofilms relevant to medical, environmental, and bioprocess fields. Our focus is on the systems biology of chronic wound consortia, microalgal growth and nutrient cycling with bacterial partners, and synthetic consortia engineering for enhanced biocatalytic platforms. One of our most exciting projects right now is looking at fungal biomat production for NASA food applications for potential ISS, lunar, and Martian missions (in collaborations with Nature’s Fynd).

Ross Carlson <rossc@montana.edu>

Wastewater treatment is in the midst of a biotechnological revolution in many parts of the developed world, even as approximately half of the world’s wastewater continues to be discharged untreated into the environment. Aerobic granular sludge (AGS) is one of the new competing biofilm-based technologies with potential to transform and expand wastewater treatment around the world. AGS consists of spherical biofilm aggregates of diverse microbial communities several millimeters in diameter. The size and structure of the granules promote metabolic diversity and simultaneous biochemical conversions of soluble wastewater constituents like carbon, nitrogen and phosporus due to oxygen and substrate gradients within the biofilm. The mass of each granule facilitates rapid settling and biomass retention. These innovations reduce the footprint of the treatment plant as well as operations and maintenance costs compared to conventional activated sludge systems. My research group is exploring how the complex matrix of extracellular polymeric substances (EPS) making up the granule structure interacts with recalcitrant organics found in wastewater like pharmaceuticals and per-polyfluoroalkyl substances (PFAS), and how the presence of such compounds influences bioconversion of conventional wastewater constituents. High performance liquid chromatography (HPLC) coupled with quantitative time-of-flight mass spectrometry (QTOF-MS) allows us to assess and quantify the presence of the target compounds in the aqueous phase and adsorbed to the granular biomass. Fluorescence confocal scanning laser microscopy (CLSM) and nuclear magnetic resonance (NMR) imaging is used to compare the morphology and structure of granules exposed to the target compounds versus control granules. Soon, we will include targeted high throughput sequencing (metabarcoding) of the 16S ribosomal RNA genes and transcripts to identify and monitor active members of the microbial community in the AGS over the duration of the study. We aim to identify the primary mechanisms by which the biofilm matrix of the AGS contributes to removal of recalcitrant organics in municipal wastewater.

Catherine Kirkland <catherine.kirkland@montana.edu>

Aerobic granular sludge is a novel wastewater treatment biotechnology in which numerous bacterial species coexist in a spherical biofilm. Oxygen and nutrient gradients throughout each granule allow complete wastewater treatment in a single reactor, and extracellular polymeric substances (EPS) in granules provide a diffusive barrier that protects bacteria from toxic shocks and improves granule settleability. However, many knowledge gaps remain regarding biofilm structure within granules; in particular, structural differences between lab-grown and environmentally-grown granules are largely unexplored. To that end, selective EPS staining, as well as fluorescent in situ hybridization (FISH), were performed on three different granule samples: those grown in lab-scale sequencing batch reactors and those from full-scale treatment facilities in Utrecht, NL and Rockford, IL. Methods developed during this research will be used to evaluate the impacts of common pharmaceutical compounds on granular sludge structures, as granular sludge shows great promise as a pharmaceutical treatment biotechnology.

Kylie Bodle <kyliebodle@montana.edu>

Algae have been used for the production of biofuels for decades. A strain of alga, Chlorella sorokiniana str. SLA-04, was recently isolated from Soap Lake, WA that can grow over a range of pH 7-11. This strain is very efficient at using the dissolved inorganic carbon that is more bioavailable in the lake due to the alkaline conditions. The alkali-tolerance of the strain can reduce the costs of biofuel production by reducing the need for CO2 sparging and the need for an algal pond to be in close proximity of a CO2 source (ex, coal plants). This study uses a combination of in silico predictions and experiments to analyze the changes in biomass composition in response to light availability. Low light predictions suggest that the majority of carbon will be stored in the form of starch, while high light favors the production of lipids. These predictions have been used to design ongoing experiments to confirm the behavior of SLA-04 under light stress conditions. It is additionally hypothesized that the extra carbon available to the cultures under alkaliphilic conditions will increase SLA-04’s tolerance to high light stress, a potential benefit for industrial production of biofuels.

Charles Holcomb <CharlesHolcomb@montana.edu>

Methanotrophs are organisms that can use methane as their sole carbon and energy source. They play a crucial role in carbon cycling in the environment and are also of economic interest, as they can consume methane, a cheap carbon source that is often flared from industrial sites. In this study, metabolic modeling was used to investigate the production of value-added byproducts from methane by a type II methanotroph. These byproducts range from chemical feedstocks like methanol and formaldehyde to reduced carbon compounds like acetate and ethanol. Even the biomass itself can be utilized as a protein source. In our predictions, we manipulate cultivation conditions like nitrogen source and O2 availability to increase production of these byproducts. We also examine the tradeoffs between consumption of methane and production of nitrous oxide, a potent greenhouse gas that methanotrophs are known to release under some cultivation conditions. These in silico predictions provide information that can be used to for industrial design and to understand methanotroph interactions in the environment.

Adrienne Arnold <adriennearnold@montana.edu>

Primary motivations for studying the subsurface are to expand the knowledge of Earth’s microbial diversity and the subsurface microorganisms under low nutrient conditions that significantly impact C, S, N, P and mineral cycles. One such biogeochemical cycle of importance to groundwater systems is microbial denitrification, the reduction of nitrate (NO3-) from organic and inorganic sources back to atmospheric nitrogen (N2). However, little is known about the extent of microbially-mediated denitrification in groundwater environments. The key to harnessing microbial potential is to find the optimal set of parameters that promotes enhanced rates of denitrification. In anaerobic environments, oxygen is not readily available for respiration, therefore microbes must use alternative electron acceptors such as NO3- to respire, reducing NO3- to N2. To investigate the environmental parameters that influence denitrification this work uses a co-culture of Rhodanobacter sp. R12 and Acidovorax sp. 3H11 that when grown together, can complete full biotic denitrification. Batch experiments mimicking field conditions were run using the Rhodanobacter sp. R12 and Acidovorax sp. 3H11 co-culture under varying pH values , dissolved oxygen concentrations, carbon sources, and amino acids. Samples were analyzed for growth performance, nitrate reduction, and single cell analysis including the integration of stable isotope probing with Raman Microspectroscopy and the identification of individual microbial cells and fluorescent in-situ hybridization (FISH). This will quantitatively track the abundance of individual organisms across treatments. Higher rates of denitrification are expected to occur when the organisms are grown together and in anaerobic conditions at a pH of 7.

Uve Strautmanis <2000.uve@gmail.com>

Treatment Wetlands (TW) require little mechanical and electrical input yet still treat water as effectively as conventional systems, but TW implementation has been limited by the lack of knowledge of their treatment capacity and design standards and recommendations. This project aims to increase further TW installation by understanding the complex and interrelated biogeochemical processes that drive water treatment in TW. The basic mechanisms of TW are similar to conventional systems because they both rely on microbes to perform processes such as nitrification, denitrification and decomposition of organic matter. These processes can be tracked through Chemical Oxygen Demand (COD) and nitrate levels.

To begin with, samples of the treated water were taken one hour, three days, seven days and fourteen days after feeding but data consistently showed that essentially all of the COD and nitrate treatment was happening in the first three days. With this information, a more detailed time analysis was performed on the COD treatment group. Samples were collected at hours zero, one, five, twenty-four, forty-eight and seventy-two. Within the first hour of feeding, 54%-71% of COD was removed in every plant. This is likely an indication of sorption rather than chemical degradation. Treatment does not vary much between plants, providing evidence for the consistency and reliability of TW. This research is ongoing and the current focus is on pushing the limits of sorption so that we can calculate rates of treatment.

Nina Denny <nina.joy.denny@gmail.com>

Poly-perfluoroalkyl substances (PFAS) are a class of man-made chemicals used as surfactants, fire retardants, and coating materials that include perfluorooctanoic acid (PFOA), perfluoro octane sulfonate acid (PFOS) among other chemicals. PFAS compounds are very persistent in the environment and can lead to adverse health outcomes in humans. PFAS can migrate from consumer products and enter the influent of wastewater treatment facilities (WWTF) where they are poorly removed by conventional wastewater treatment methods making effluent from WWTF a major source of PFAS in the environment. Aerobic Granular Sludge (AGS) is a novel microbial community that can be used for the treatment of wastewater. The extracellular polymeric substance (EPS) structure in AGS may facilitate the removal of PFOA/PFOS via sorption. AGS will be used to remove PFAS since AGS is both cost-effective and energy-efficient compared to conventional activated sludge. It also has excellent settleability, high biomass retention, and tolerance to toxicity. Removal mechanisms such as sorption and biodegradation may contribute to PFAS removal efficiency. Liquid chromatography with mass spectrometry (LC-MS/MS) will be used to assess the extent to which PFOA and PFOS partition to the sludge phase using a mass balance approach. Microscopy will be used to monitor morphological and structural changes. Other nutrients in wastewater such as carbon, nitrogen, phosphorus will be measured to see how PFAS influence the conventional treatments of wastewater. It is expected that EPS in granules will improve the sorption of PFAS relative to activated sludge and reduce the PFAS load from wastewater.

Tasnim Ritu <tasnimsultana.ritu@student.montana.edu>

Aerobic granular sludge (AGS) is a novel wastewater treatment technology with several advantages over the conventional activated sludge process, including increased biomass retention, the ability to withstand toxic shock loads, and accelerated treatment rates. Aerobic granules are spherical biofilm aggregates consisting of diverse bacterial populations bound together by extracellular polymeric substances (EPS) in the absence of a carrier material. The EPS matrix is a complex hydrogel structure whose molecular makeup is largely unknown but is the reason behind why biofilms are more resistant and resilient than planktonic cells. Aside from providing structural stability, EPS acts as a buffer between the microbial community and the surrounding bulk fluid, making granules less vulnerable to negative changes in their environment, such as the presence of certain pollutants. Due to their complex EPS structure and microbial diversity, aerobic granules may offer a promising solution to the growing concern over the presence of pharmaceuticals and personal care products (PPCPs) in wastewater. Aerobic granules are expected to better treat for these compounds than in conventional activated sludge systems since bacterial flocs used in conventional systems do not contain EPS and are therefore more susceptible to the negative impacts of PPCPs.

The overarching purpose of this research is to explore the extent to which AGS can remove PPCPs from wastewater and how PPCPs influence the removal of the typical wastewater constituents: carbon, nitrogen, and phosphorus. In this study, enriched planktonic cultures of ammonia- and nitrite-oxidizing bacteria grown from crushed aerobic granules will be exposed to PPCP-laden media. The microbial density and nitrogen removal efficiency of these cultures will be measured in both long- and short-term exposure experiments. Comparisons will later be drawn between these planktonic cultures and aerobic granules cultivated in a laboratory reactor dosed with the same PPCPs. These results will demonstrate the extent to which the EPS matrix aids in the protection from and treatment of PPCPs.

Madeline Pernat <madeline.pernat@student.montana.edu>

Traditionally, the use of single cultures or communities of either fungi and bacteria are used in bioremediation applications, such as remediation of coal mine tailings, chemical waste, and wastewater effluents. However, current findings suggest that mixed-domain systems, e.g. fungal-bacterial cultures, can be more robust compared to their monocultures and can potentially be more efficient at removing pollutants from different environmental matrices. Despite their promising use, there is still a lack of information regarding the potential use of mixed-domain systems for bioremediation. This study aims to establish fungal-bacterial biofilms using relevant pollutant-degrading microbes, including the bacterial species Pseudomonas putida and the fungal species Phanerochaete chrysosporium, both known to be capable to degrade a wide range of pollutants. Single-species and multi-domain biofilms were grown in a Drip Flow Reactor; different cultivation factors (e.g., pH, sequence of inoculation) were tested to assess their effect on the establishment and growth of the biofilms. The obtained biofilms will be used for the bioremediation of selenium in acid mine drainage.

Sandra Kohl <skohl12345@gmail.com>

Aerobic granular sludge (AGS) are compact, spherical biofilm aggregates used in wastewater treatment to simultaneously remove carbon, nitrogen, and phosphate. Previous studies have demonstrated that nuclear magnetic resonance (NMR) imaging can provide important insights into the internal structures of these complex, heterogenous granules. However, these studies have not elucidated how proteins or polysaccharides, the main constituents of the extracellular polymeric substances (EPS), influence the NMR relaxation behavior observed. This study explores AGS structure using alginate beads with added model proteins and polysaccharides to characterize their relaxation behavior, and ultimately uncover details of the composition and structure of the EPS in an actual granule. Preliminary findings have showed that increased protein concentration increased the T2 relaxation times in model AGS infused with model proteins, while increased polysaccharide concentrations had the opposite effect.

Matthew Willett <matthewwillett@montana.edu>