CBE 2021 Regulatory Biofilm Meeting

Of the life support considerations used in manned space missions, the management of water is a primary concern. On the International Space Station (ISS), potable water is recovered from wastewater in the water processor assembly (WPA). While the water processor assembly (WPA) successfully recovers water for potable water, intermittent fouling events present challenges to the water recovery on station and often require equipment component replacements to return to normal function. As space travel and missions increase in both distance and duration, equipment replacement may not be feasible. Improved control of biofilm formation in the WPA and future water processing systems is of interest to NASA. Of interest for this project is biofouling control in the wastewater tank of the WPA where past fouling has occurred. Here we have developed an approach to study nutrient limitation and biocide treatment to improve biofilm control in current and future water processing systems in space.

Liz Sandvik

Center for Biofilm Engineering, Montana State University, Bozeman, MT

Alginate is a biocompatible polysaccharide that is abundantly derived from seaweed. Interestingly, it can also be produced by bacteria and incorporated into their biofilms. The dual compatibility of alginate with bacteria as well, as in the human digestive tract makes it an excellent candidate to deliver probiotics. In this study, we encapsulated a model bacterium, Escherichia coli, into an alginate-based nanofiber system ideal for the delivery of active ingredients into the gut. To create our nanofibers, we systemically studied the solution properties of our precursor solution comprised of three components: the biopolymer sodium alginate (SA), a carrier polymer polyethylene oxide (PEO), and the FDA-approved surfactant polysorbate 80 (PS80). This enabled us to understand the relevance of each property on the formation of smooth nanofibers formed via electrospinning. Next, we added bacteria to the solution. As nanofibers are drawn from the solution during electrospinning, bacteria will be pulled along the fiber and encapsulated within them. However, the addition of bacteria changed the solution properties causing a shift toward beaded fibers, which tend to indicate a less stable system. Finally, we were able to adjust our precursor solution recipe to account for changes in solution properties and again form smooth fibers. Electrospun mats manufactured using our optimized 2.5/1.5/3 wt% SA/PEO/PS80 solution containing 10^8 CFU/mL of E. coli produced smooth alginate-based nanofibers loaded with 2.74 x 10^5 CFU/g of viable bacteria. Ongoing studies will focus on optimizing the crosslinking of the fibers to ensure a controlled release of the bacteria in the gut.

Emily Diep

University of Massachusetts, Department of Chemical Engineering

Microtiter plate methods are commonly used for biofilm assessment. However, their data have often been difficult to reproduce. A ring trial was performed in 5 different laboratories to evaluate the reproducibility and responsiveness of three biofilm quantification methods in 96-well microtiter plates: crystal violet, resazurin, and CFU counts. Experiments were divided into two main groups: control and treatment. An inter-lab protocol was developed for the study. This was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for CFU counts. In the treatment experiments, CFU counts had the best reproducibility with respect to responsiveness, making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor which exhibits good repeatability and reproducibility for different types of assessment methods.

Jontana Allkja

LEPABE, Faculty of Engineering University of Porto, Portugal

Methanotrophs are organisms that use methane as their sole carbon and energy source. These organisms are found in diverse habitats and play an important role in global carbon cycling. Methanotrophs are also of industrial interest, as they are able to oxidize methane to key chemical feedstocks like methanol and formaldehyde. In this study, core metabolic models of a type I and a type II methanotroph were constructed and analyzed via elementary flux mode analysis and flux balance analysis. Type I methanotrophs assimilate methane via the ribulose monophosphate pathway, while type II methanotrophs use the serine pathway. Metabolic modeling facilitates comparison of core carbon and energy metabolism between the methanotroph types. Byproduct excretion in particular was analyzed, as metabolic byproducts determine the industrial potential of methanotrophs and create the possibility of syntrophic community interactions in the environment between methanotrophs and other organisms.

Adrienne Arnold

Center for Biofilm Engineering, Montana State University

Bacteria and fungi readily grow together in the natural environment (e.g. soil), where they perform critical work to maintain the functioning of the ecosystem, such as breaking down organic matter for nutrient recycling. By engineering systems where pollutant-degrading fungi and bacteria are integrated, as they commonly are in nature, we can potentially create enhanced systems for the degradation of pollutants. Most research reports the use of single cultures or communities of either fungi or bacteria for the remediation of soil and water. However, there is a lack of information on the use of multi-domain cultures, particularly fungal-bacterial biofilms. Mixed cultures are more robust than monocultures and, due to their varying metabolic functions, have a division of labor, which could in turn make them more efficient at removing pollutants from soil or water. Furthermore, there is some evidence that fungal-bacterial systems can have improved removal efficiencies compared to their monocultures. This project aims to establish fungal-bacterial biofilms with pollutant-degrading microorganisms and to better understand the associations and interactions between their microbial partners. This presentation will summarize our efforts to co-cultivate environmentally relevant microorganisms including the bacterium Pseudomonas species (P. putida or P. stutzeri) and the fungus Phanerochaete chrysosporium in a continuous drip flow reactor to form fungal-bacterial biofilms. Future experiments to explore the effect of culture conditions (e.g. order of inoculation, temperature, and pH) on fungal-bacterial biofilm development will also be discussed. The obtained fungal-bacterial biofilms in this study will be used for the bioremediation of selenium in acid mine drainage in future projects.

Gretchen Gutenberger

Montana State University, Center for Biofilm Engineering

The recent outbreak of the COVID-19 pandemic, caused by the SARS-CoV-2 virus, underscores the need for testing capabilities relevant to novel virus threats. Although the major transmission route for SARS-CoV-2 is believed to be person to person contact via respiratory droplets, contact transmission via surfaces may still play a role. In general, determining the survival time of specific viruses on surfaces and most effective disinfectants are important for helping control the spread of viruses. Ideally, testing should be performed on the actual virus of concern, however, in the case of SARS-CoV-2 this requires BSL-3 facilities. The use of surrogate viruses enables work in BSL-2 facilities to reduce costs and timelines for technology development. The method developed by the CBE is based on the accepted EPA test method for high-level anti-viral disinfectants for nonporous surfaces ASTM E2197–1, the “quantitative carrier test” (QCT) using the canine coronavirus (ATCC VR-2068). This project creates an opportunity for the CBE to enter new testing and research areas relevant to SARS-CoV-2 and other human viruses.

Kelly Kirker

Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA

Multiphase flow in porous media occurs naturally in many industrial and environmental systems. Understanding the fundamental flow physics in such systems is essential for many real-life applications. Among others, capillary pressure is an important parameter for multiphase flow in porous media, which was traditionally modeled only as a function of saturation, and this relationship in turn was found to be hysteretic. Extensive research has been going on for decades to investigate and mitigate the hysteresis in capillary pressure-saturation curves. Recently it has been theoretically shown that a unique relation is possible with the inclusion of a few additional variables such as interfacial area, interfacial curvature, and Euler characteristic in the functional form. It is also suggested that such a functional form would work for both equilibrium and non-equilibrium conditions. However, systematic and quantitative experimental investigations and validations of such a functional form are still lacking. To this end, capillary pressure along with saturation and other geometric variables are experimentally quantified for a multiphase flow in 2D micromodels.

Fabricated 2D micromodel is a powerful tool to perform the aforementioned studies as it offers excellent control over porous structures, great repeatability, and excellent optical access. Employing fluorescence microscopy coupled with a high-speed camera, flow configurations, as well as its dynamics, are captured, which are then analyzed using advanced image processing algorithms. In this poster, techniques for 2D micromodel fabrication and simultaneous measurements of capillary pressure, saturation, interfacial area, and Euler Characteristic have been delineated, thus providing a general method for 2D micromodel validation of novel theories related to capillary pressure hysteresis. The results will provide new insight into the hysteretic behavior of capillary pressure as well as validations of new functional forms.

Razin Molla,

Mechanical & Industrial Engineering, Montana State University, Bozeman, MT, USA.

Capillary pressure and capillarity are central to the description of multiphase flow in porous media. For decades, practical and theoretical descriptions of multiphase flow in porous media have been inevitably relying on empirical relations between capillary pressure and phase saturation, which have long been recognized to be hysteretic. Extensive studies have been devoted to understanding and mitigating such hysteresis in hope of achieving a unique description of the state of the porous medium flow system. Although, a direct in-situ measurement of pore-scale capillary pressure would be extremely valuable, on-chip measurement of pore-scale capillary pressure is still lacking due to a number of experimental challenges. Only very few proposed designs are suitable for measurement in multiphase flow in porous media. To that end, we aim to design and fabricate an on-chip sensor that enables direct capillary pressure quantification within individual pores in 2D porous micromodels. The micromodel used in the current study is fabricated in polydimethylsiloxane (PDMS) using soft lithography with a thin membrane incorporated which deflects subject to pressure variations in the fluid flow. With this technique, a 2D pressure field can be inferred by means of a pre-calibrated correlation between the membrane deflection in the z-direction and pressure change. A microscope coupled with a high-speed camera is employed to provide optical readout, allowing for possible simultaneous quantification of other flow characteristics, such as velocity fields, phase distribution and interfacial area. By this experiment, we hope to provide a novel method for direct quantification of capillary pressure at the pore scale and this study will lead to a renewed understanding of pore-scale physics of multiphase flow in porous media.

Nishagar Raventhiran

Mechanical & industrial Engineering, Montana State University, Bozeman

Interest in biofertilizers as sustainable alternatives to chemical fertilizers has developed in recent years as the negative environmental impacts of chemical fertilization such as groundwater contamination and decreased soil quality are becoming increasingly evident. Nitrogen-fixing cyanobacteria are promising biofertilizers as they provide both nitrogen and carbon to the soil, and if successfully established, may reduce long-term fertilizer inputs. However, the effect of biofertilizers on the soil microbiome is largely unknown and unexplored, despite the vital roles soil microorganisms play in driving major soil nutrient cycles. Therefore, we sought to understand how a nitrogen-fixing cyanobacterial biofertilizer (CBF) affected crop growth, nutrient cycling, and the soil microbiome over a three-year field study (2018-2020) with perennial bioenergy crops switchgrass and tall wheatgrass. The field study is located at the MSU Arthur H. Post Research Farm near Bozeman, MT where each year we conducted soil sampling from April-September on plots either fertilized with a locally isolated Nostoc sp. CBF, urea, or left unfertilized. High-throughput sequencing on the Illumina MiSeq platform of 16S and LSU rRNA gene sequences was used to capture the bacterial/archaeal and fungal soil communities respectively, while soil chemical analysis monitored changes in C, N, and pH.

Of particular interest is the influence of the locally isolated Nostoc sp. biofertilizer on the cyanobacterial community of the soil microbiome as well as the persistence of the biofertilizer following application. The persistence of the biofertilizer may be indicative of biocrust formation which could increase its value as an agricultural application. Formation of cyanobacterial biocrusts has been utilized as a restoration strategy for degraded soils due to increased soil aggregation by cyanobacterial exopolysaccharides as well as increased nutrient and moisture retention. In addition, determining biofertilizer establishment is vital to decreasing long-term N and C inputs. Here we present microbial community data representing the effects of two years of biofertilizer application on the bacterial community of the soil crust at our Post Research Farm field site.

Hannah Goemann

Montana State University, Center for Biofilm Engineering, 366 Barnard Hall, Bozeman, MT 59717, USA

Adhesives are essential to automobile, aerospace, electronics, and wood industries. Most adhesives currently in the market use petroleum products and volatile organic compounds (VOCs) as raw materials. Both can be harmful to human health and the environment, raising concerns for both workers and users. These concerns have created a demand for more sustainable adhesives, leading to a trend towards green adhesives in global sealants and adhesives markets. Most of the safer and more sustainable adhesives are bio-based, natural adhesives. Some of the prominent green adhesives are soy and starch adhesives making up 50% of the green adhesives market, with applications in construction, paper, and packaging. Other organic bio-based adhesives include albumin, casein, beeswax, gum Arabic, soybean proteins, and starch for use in specialized applications. However, the use of bio-based adhesives is limited due to their sensitivity to water, low adhesive strength compared to synthetic adhesives, and limited usability on a number of surfaces (their use is limited to mostly wood and paper). To expand the functionality of bio-based adhesives, improve adhesive strength and decrease water sensitivity, a natural adhesive worth exploring is a biomineral composite. The biomineral composite consists of organic polymers and microbially produced calcium carbonate. Calcium carbonate is produced in place during the curing process using a mechanism referred to as ureolysis-induced calcium carbonate precipitation (UICP). UICP occurs according to the following reaction and can be promoted by bacterial cells (MICP: microbially induced calcium carbonate precipitation) or through the free enzyme (EICP: enzymatically induced calcium carbonate precipitation). H2N-CO-NH2(urea) + 2H2O + Ca2+ (urease enzyme --> ) 2NH4+ + CaCO3(s)

The calcium carbonate formed as a result of this reaction along with microbial cells (or free enzyme), its products, and organic additives is referred to as a biomineral composite. To expand the application range of bio-based adhesives beyond wood and paper in this work, the substrates tested are glass and steel. So far, guar gum and soy protein have been used as additives to make biomineral composites. We show that the adhesive strength of the composite produced with soy protein is greater than guar gum composites, soy protein or guar gum alone. MICP composites with and without additives have been previously studied for a broad array of applications, including soil stabilization, concrete remediation, creating subsurface barriers and remediation of radionuclides. Field applications of this process by our lab group have shown the potential of using MICP with lower cost bulk chemicals. We are building on this knowledge to develop novel composites for adhesive and similar applications.

Sobia Anjum, <sobia.anjum@montana.edu>

Montana State University

We isolated and characterized a green alga, Chlorella sorokiniana SLA-04, capable of growing at high pH (~10.2) and high alkalinity (>50mEq). High pH/high alkalinity algal cultivation has the potential to drastically reduce the cost and increase the range of possible locations for industrial scale algal biomass production for biofuel and high value product generation. The high pH and high alkalinity conditions provide non-limiting concentrations of inorganic carbon for photosynthesis and the effective scavenging of atmospheric CO2, thereby allowing for high productivity algal growth (pilot biomass productivity >16 g/m2/day) in the absence of concentrated CO2 sources. Additionally, alkaliphilic strains thrive under high pH-high alkalinity conditions inhibitory to many competing mesophilic microalgae, bacteria, archaea, viruses and predatory zooplankton. We have begun characterizing the microbial community in these algal cultures using microscopic and phylogenetic approaches, which has allowed us to identify potentially beneficial and detrimental interactions of algae and other microorganisms.

Using state-of-the-art DNA sequencing technologies, we were able to detect bacterial phyla as well as potential grazers (amoeba and ciliates) in high pH-high alkalinity SLA-04 cultures. We are also in the process of characterizing the physiology of strain SLA-04 and its interactions with associated microorganisms including 19 bacterial strains isolated from indoor and outdoor cultures of SLA-04. We recently obtained an axenic culture of SLA-04 and other high pH-adapted algae through repeated antibiotic treatments and are sequencing their genomes. Algal-prokaryotic interactions are being characterized using metagenomic and metatranscriptomic sequencing in combination with activity-based and metabolomic analyses (BONCAT, NanoSIMS, and Raman confocal microspectroscopy). These data are providing the foundation for developing metabolic network models, which will guide both microbiome and algal genome engineering approaches (using e.g. CRISPR-Cas9-based approaches) for overall cultivation improvement. Our goal is to understand and exploit the synergistic effects of algae-microbiome interactions for maximum benefit in high pH/high alkalinity cultivations and provide a framework for controlling inter-organismal interactions in other algal cultures important for biofuel and bioproduct generation.

Huyen Bui

Center for Biofilm Engineering, Montana State University, Bozeman, MT, USA

Electrochemical impedance spectroscopy (EIS) is a powerful technique for characterizing bulk and interfacial properties in aqueous, solid and gas systems. The technique is based on applying an oscillating voltage at a single-frequency to a device under test (DUT) and measuring the complex electrical current. Varying the frequency and calculating the complex resistance/impedance allows modeling of the DUT using electrical equivalent circuits. Changes to the recorded spectra indicate in situ biofilm formation and an increase of microbial concentrations in the media. We have developed microfabricated EIS sensors that are small (~ 9 x 26 mm), low cost and amendable to use in a variety of environments, providing exciting opportunities for spatially resolved, real-time monitoring of biofilm in industrial settings.

This poster presents preliminary results of the use of these sensors in metalworking fluids, where microbial contamination is a significant factor in their degradation, causing biofouling and corrosion of equipment, the imperilment of product quality, and posing occupational safety risks.

Matthew McGlennen

Montana State University

Algal biofilm reactors (ABRs) have some possible advantages for biomass production due to the concentrated nature of the resulting biomass potentially requiring less energy for water removal via settling as compared to suspended culture growth. Current biofilm systems often have many moving belts, disks or scrapers for biomass harvesting with some requiring complex mechanical systems for proper operation. Biomass productivity for Chlorella vulgaris was investigated in a novel ABR that uses reverse-flow aeration as a harvesting mechanism for biofilm detachment. Effect of different medium compositions was also examined. Calcium (Ca2+) is well known to stabilize biofilms by crosslinking extracellular polysaccharide (EPS) cross linkers (Cooksey 1981). The effects of Ca2+ and sodium bicarbonate on biomass and lipid productivity (Gardner, Cooksey et al. 2012) were evaluated in the ABR. Addition of Ca2+ and bicarbonate resulted in the fastest growth of C. vulgaris biofilms. Areal biomass productivities, measured as ash free dry weight (AFDW), ranged from 0.278 ± 0.013 to 0.700 ± 0.159 g m-2 day-1 and were the highest in treatments with additional calcium and bicarbonate. Supplementing the media with only bicarbonate resulted in an AFDW productivity of 0.39 g m-2 day-1. Harvesting with aeration resulted in an average biomass detachment of 69.04 ± 13.9 %. End point lipid content was determined after harvesting the biomass at steady state areal cell density. Lipid production in the biofilm was limited to membrane lipids (7-10 % wt/wt fatty acid methyl esters (FAME)) and bicarbonate had no effect on lipid productivity.

Muneeb Rathore

Montana State University, Center for Biofilm Engineering and Chemical & Biological Engineering