Fundamental Research, Education, or Technology Advancement Barriers

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1 Biofilm Enabled Permeability Reduction in Sands ERC Team Members CBBG Faculty Graduate Students Rebecca Parales, UC Davis Jordan Greer, UC Davis Douglas Nelson, UC Davis Charles Graddy, UC Davis Jason DeJong, UC Davis Collaborators Mary Roth, Faculty, Lafayette College Laurie Caslake, Faculty, Lafayette College Ziqi Chen, Undergraduate Student, Lafayette College Vivian Chen, Undergraduate Student, Lafayette College Rebecca Kandel, Undergraduate Student, UC Davis Ruixing Wang, Visiting Professor, Southeast University (China) Project Goals The Year 2 project goals were to improve bio-film formation uniformity and demonstrate treatment potential in a bench scale 2-D model. Also, to learn the fundamentals behind biofilm formation and how it can be a reliable and repeatable form of seepage control. The Project s Role in Support of the Strategic Plan The project is involved in infrastructure construction with use cases as temporary low permeability barriers for reduced groundwater pumping in dewatering efforts during short term excavation work and localized seepage reduction of high flow zones through dams/levees/walls through localized upstream treatment. It also pertains to Environmental Remediation and Protection as temporary low permeability barriers for mediation of flow regimes during environmental remediation. Biofilm formation is a natural biologically-mediated process that is sometimes considered problematic. Here we are using it in a positive way to decrease the permeability of soils. This project utilizes bio-stimulation, to stimulate the growth of bacteria already present in the soil, although bio-augmentation will also be considered. This technology has the potential to contribute to resilience and sustainability as it would replace the use of carbon footprint intensive materials. In addition, this technology would allow the subsurface environment to return to its natural condition instead of being permanently altered. Fundamental Research, Education, or Technology Advancement Barriers The primary fundamental research barrier is in understanding, and then controlling, the spatial distribution of biofilm formation as well as the repeatability and resilience of biofilm formation. The spatial distribution of bacteria, oxygen, and nutrients during bacterial stimulation and during biofilm formation must be better understood for upscaling of the technology to be possible. These studies will provide a fundamental base of knowledge against which the potential use and effectiveness of quorum sensing inhibitors can be evaluated. Collectively, this will result in the

2 knowledge and ability to control the first order factors that dictate the spatial distribution of biofilm formation, and hence permeability reduction. Many other factors have been identified, explained in the section Achievements in the past year. Any research aspect that involves foreign collaborations, especially indicating the length of time US faculty or students spent abroad conducting their work, and vice versa, and the value added of that work to the student s/faculty work. Dr. Ruixing Wang, a professor in Materials Science and Engineering at Southeast University in Nanjing, China visited UC Davis from September 1 st 2016 to September 1 st During his stay, he participated in lab work and meetings for the biofilm project. He offered an interesting perspective during the planning of the experiment as he drew connections from the research he performs at Southeast University. Dr. Wang works on a project that involves recycling steel slag into bricks, giving him a unique view on sustainability efforts in both the short term and long term stages of the biofilm project. Achievements in previous years The current biofilm project started in year 2 of the ERC (official start date of January 1, 2017). Previous research was performed at UC Davis and Lafayette College. Exploratory bench-scale work was performed and biofilm growth was shown to reduce the permeability of sand by 100- fold based on studies carried out by former graduate student Clayton Proto at UCD. Also, biostimulation was shown to work as well as bio-augmentation. However, the previous research left unanswered many questions concerning the spatial variability and reproducibility of biofilm formation. Achievements in past year Figure 1: UC Davis graduate student Jordan Greer (left) and visiting professor Ruixing Wang at UC Davis Round Table Poster Presentation. In the past year there have been many discoveries made through the three sets of column tests performed at UC Davis. There have also been developments in the analysis of biofilm formation and the role of quorum sensing inhibitors. More details are included below. Column Test 1 & 2: Capability Development In the first two column tests, the goals were to replicate the results of prior biofilm work. An experimental set-up was created in the lab that was automated and programmable to allow the maximum number of test treatments (Figure 2). The columns were bottom fed and the graduated cylinders were set-up to be used as falling head permeameters. A concentrated stock of nutrients was connected to the system, and diluted with water before being pumped into the columns.

3 The results of the first two column tests are shown in Figures 3 and 4. Figure 3 shows the reduction in permeability of four columns compared to a control column that was fed only water. The columns were treated four times a day with a solution containing 0.1 g/l yeast extract, 0.05 g/l casein peptone and 0.5 g/l glucose as opposed to twelve times with one-fifth the amount of glucose (in Proto s work) and the reduction in permeability took about three times longer to occur. The permeability decreased to approximately the same value as in the study by Proto, but the initial permeability was lower, primarily due to differences in the porosity of the sand used. In test 1, tap water was used in the system, which was found to contain chlorine that could have been toxic to the bacteria. In the second test do-ionized water was used instead of tap water and the permeability decreased as seen in Figure 4. Figure 3: Biofilm Column Test Set-up with automated feeding Figure 2: Column Test 1 Results An issue that arose was the apparent clogging in the permeameter system. After treatment began, bacteria started forming biofilm in the tubing that delivers the nutrients to the columns. This was problematic as it was unknown how much biofilm formation occurred in the tubing versus the columns and whether movement of preformed biofilm from the tubing could have affected the permeability results. When dissecting the columns, a bacterial seal at the airwater interface at the top of the column was found that had burst through; this could have contributed to the sudden jump in permeability in test 1 at Day 16 (Figure 3). In the second column test, the tubing was changed on Day 14, and as seen in Figure 4, this resulted in a Figure 4: Column Test 2 Results small increase in permeability. However, the permeability remained lower than at the start of the study, leading to the conclusion that there indeed was clogging occurring in the soil. The overarching question of the spatial variability of the biofilm formation throughout the columns was highlighted by the observation of a large buildup of biofilm on the inlet porous stone at the inlet after dissecting the columns. Column Test 3: Quorum Sensing Iteration

4 In certain bacteria, biofilm formation is under quorum sensing control. Quorum sensing is a process by which bacteria communicate with each other and work as a group, typically by coordinating gene expression. The process involves production and accumulation of small signaling chemicals called autoinducers. When these acylhomoserine lactone (AHSL) signals build up to a critical concentration, large sets of genes, including genes for biofilm formation, are turned on. It was hypothesized that by initially adding quorum sensing inhibitor to the columns, the bacteria will be unable to communicate with each other and will grow more evenly throughout the column. Once the quorum sensing inhibitor feeding is stopped, the bacteria will form a spatially uniform biofilm throughout the column. Column test three was therefore set up to examine permeability reduction when quorum-sensing inhibitors were added. The quorum sensing inhibitor that was chosen was Furanone 56. This chemical is known to target certain bacteria, including Pseudomonas aeruginosa; an additional type of quorum sensing inhibitor was investigated by the microbiology lab (see more details below). Figure 5 shows the results of the third column test, which consisted of 8 columns total, with two replicates of each treatment. The sand used for column test 3 was collected more recently than that used in tests 1 & 2. In addition, in an attempt to shorten the lag time before permeability reduction began, the sand was seeded with soil from column 1 (5% of total mass) in the second column test. The legend to Figure 5 describes the Day 10: Furanone ends Day 11: DMSO starts treatments for each column. In contrast to column tests 1 and 2, in Figure 5: Column Test 3 Results which columns were completely filled with sand and fed from the bottom, these columns were fed from the top and the porous stone was replaced with a layer of gravel and liquid at the top. This was done in an effort to prevent clogging at the inlet. Surprisingly, the columns that received the quorum sensing inhibitor showed decreased permeability almost immediately, in contrast to the other columns. In addition, when the furanone was stopped on day 10, the columns returned to their initial permeability values. The furanone was dissolved in DMSO, so to eliminate the possibility that DMSO was causing the permeability reduction, possibly by acting as an alternative electron acceptor for anaerobic respiration, we began adding an equivalent amount of DMSO to columns C4 and C7 on Day 11. The results of this test are still being analyzed, but it appears that DMSO did not have any effect on permeability reduction. The columns are currently continuing to run and we plan to carry out additional controls (ie., re-feed furanone-56) to try to understand these results.

5 Two of the columns in test 3, C3 and C8, are shorter columns with piezometers built in to measure head loss throughout the column (Figure 6). Knowing the head loss in certain areas throughout the column gives insight to the spatial variability of the biofilm formation. The initial piezometer Figure 6: Sketch of and data collected from a column fitted with piezometers readings showed that (P1, P2) for monitoring head loss throughout the column in column test 3. most of the head loss occurred in the bottom section of the columns rather than evenly throughout the columns, even in the first feedings. Figure 6 shows a graph of the different permeabilities occurring in C3, computed from head loss, throughout the column. It is hypothesized that the fines migrating in the column are effecting the head loss distribution. This will be further studied, as it is currently unknown how fines effect where biofilm formation occurs. Analysis of biofilm formation and the role of quorum sensing inhibitors In order to understand the function of biofilm inhibitors in our column studies, we wanted to be able to quantify biofilm formation in the presence and absence of different quorum sensing inhibitors. We chose furanone 56 (CAS-Number: ; C5H3BrO2 from Adipogen), which is known to inhibit biofilm formation by Pseudomonas aeruginosa PAO1, and was proposed to be used by the Lafayette group in column studies. Furanones are thought to target the LuxR receptor proteins, most likely by preventing binding of the autoinducer molecules (acylhomoserine lactones; AHSLs) that build up in high-density bacterial cultures. We also obtained from Amy Schaefer at the University of Washington an E. coli expression clone that can be used to purify the B. thuriengensis AHSL lactonase enzyme AiiA, which cleaves lactone rings of a wide variety of AHSLs and thus is capable of inhibiting quorum sensing and biofilm formation by a different mechanism. Lafayette undergraduate Vivian Chen and UCD graduate student Charles Graddy grew up several liters of E. coli carrying the cloned lactonse gene and purified three batches of lactonase. After testing activity of the lactonase, it was determined that the amount of purified protein needed to run bench-scale column studies was prohibitive in the short amount of time available to the summer students, so the lactonase was used in small scale microtiter plate tests. A microtiter plate test was developed to monitor biofilm formation and examine the ability of furanone 56 and AiiA to inhibit biofilm formation by the model organism Pseudomonas aeruginosa PAO1 using the method of O'Toole (2011). Under the conditions used, with the same medium that was being used to feed the columns, a clear reduction in PAO1 biofilm formation was observed with furanone-56, but not with lactonase for reasons that are not

6 yet understood. Ultimately, we plan to use this assay to monitor the presence of biofilm forming organisms in the columns by taking samples over time from different locations in the columns to monitor the distribution of biofilm forming cells and determine whether early treatment of the columns with quorum sensing inhibitor results in more uniform biofilm distribution. Summary of other relevant work being conducted within and outside of the ERC and how this project is different Inside the ERC there is formal linkage but no overlap with the following projects: - Electrokinetic Sub-surface Transport for Soil Remediation and Mineral Precipitation Torres/ASU - Microbially Enhanced Iron-modified Zeolite Permeable Reactive Barrier Papelis/NMSU - Microbial Ecology of Stimulated Ureolytic Biocementation Nelson/UCD Outside of the ERC we have been actively collaborating with researchers Mary Roth (Professor of Civil and Environmental Engineering), and Laurie Caslake (Professor of Biology) at Lafayette College, an Undergraduate Research College. Professors Roth and Caslake have been awarded a NSF grant ( RUI: Reducing Permeability in Sands Using Biofilm-Forming Bacteria and Quorum Sensing Inhibitors to Create Uniform Growth ) to engage undergraduate students in geotechnical research. Our groups have participated in joint monthly webinars since January 2017 and have exchanged many s back and forth. The experiments at both colleges are complementary, and the discoveries made at each site have benefitted Figure 7: Lafayette REU student Ziqi Chen (left) and UC Davis graduate student Jordan Greer (right) in UC Davis lab both groups. In Summer 2017 two undergraduates from Lafayette college spent 7 weeks carrying out research at UC Davis. Ziqi Chen (pictured in Figure 8) is a rising senior in Civil Engineering who worked with Jordan Greer on the design, performance and data analysis of bench-scale column experiments. Vivian Chen is a rising senior studying microbiology, who has been working with Professor Caslake since January This summer she worked with graduate student Charles Graddy in the microbiology laboratory. Both undergraduate students are currently in the process of applying to graduate school. Plans for the next year There have been many unanticipated challenges in creating an effective biofilm to influence permeability reduction due to the amount of pore volume that requires the amount of EPS produced to be substantial. The plans for next year are based on discoveries at UC Davis and Lafayette in the past year. Using information from the first three column tests performed at Davis, the team is considering the many variables that influence biofilm formation for

7 permeability reduction. One important aspect we are going to look into is how the type of soil influences biofilm formation. The type of soil affects biofilm formation due to the size and gradation of particles (which collectively affect the pore space structure) as well as existing bacteria present. Additional column tests will be carried out to establish the effects of bottom vs. top feeding, as well as different tubing configurations to reduce unwanted clogging. Small scale growth tests (both aerobic and anaerobic) will be carried out to optimize the nutrient solution. One hypothesis is that with the current nutrient mixture, phosphate may be limiting in the columns, and addition of phosphate will stimulate growth and biofilm formation. We assume that aerobic respiration is occurring near the inlet of the columns and fermentation deeper into the column where oxygen is depleted. We plan to examine the inclusion of alternative electron acceptors, such as nitrate, in the nutrient solution, as anaerobic respiration is much more efficient than fermentation and should result in faster growth of bacteria. The use of quorum sensing inhibitors will be further investigated to determine their effectiveness in developing uniform biofilm throughout the column. There will also be continued collaboration with Lafayette, including biweekly meetings and continuous exchange. Two undergraduate students from Lafayette will again spend the summer carrying out research at UC Davis. Expected milestones and deliverables for the project - Advancements in biofilm quantification and small scale assays in biology lab through use of quorum sensing inhibitors and fluorescence microscopy. - Develop a protocol for biofilm quantification, including sampling and analysis process. - Develop a protocol for measuring the uniformity of biofilm formation using piezometers and sampling techniques - Demonstrate effectiveness of permeability reduction using biofilms across a range of soil types - Expand research to include physical seepage model (e.g. sheet pile model) to explore additional complexities in treatment method and effectiveness in 2-D space - Identify and bring in industry partner to review and comment as technology moves towards 2-D model testing Member company benefits The biofilm project currently has no official industry partners as it is in the first year and working primarily in the fundamental technology plane. However, in the coming year it will be beneficial to bring in industry partners as we try to begin upscaling of the technology. For industry member companies participation would provide an opportunity to become engaged in a new, sustainable technology for groundwater flow reduction and/or modification that could be useful for dewatering, seepage control, and groundwater remediation technologies. If relevant, commercialization impacts or course implementation information

8 Biofilm enabled permeability reduction has the potential to be a more cost-effective, sustainable, and possibly reversible technology compared to the chemical or cement based technologies currently in use today. References Proto, C. (2013). Bio-mediated permeability reduction of saturated sands (Master s Thesis). University of California, Davis, California. O'Toole, G. A. (2011). Microtiter dish biofilm formation assay. Journal of Visualized Experiments: JoVE, (47). Shrout, J. D., Chopp, D. L., Just, C. L., Hentzer, M., Givskov, M., & Parsek, M. R. (2006). The impact of quorum sensing and swarming motility on Pseudomonas aeruginosa biofilm formation is nutritionally conditional. Molecular Microbiology, 62: