Scholarly Work - Center for Biofilm Engineering
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Item Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta(2010-12) Gardner, Robert D.; Peters, P.; Peyton, Brent M.; Cooksey, Keith E.Algal-derived biodiesel is of particular interest because of several factors including: the potential for a near-carbon-neutral life cycle, the prospective ability for algae to capture carbon dioxide generated from coal, and algae’s high per acre yield potential. Our group and others have shown that in nitrogen limitation, and for a single species of Chlorella, a rise in culture medium pH yields triacylglycerol (TAG) accumulation. To solidify and expand on these triggers, the influence and interaction of pH and nitrogen concentration on lipid production was further investigated on Chlorophyceae Scenedesmus sp. and Coelastrella sp. Growth was monitored optically and TAG accumulation was monitored by Nile red fluorescence and confirmed by gas chromatography. Both organisms grew in all treatments and TAG accumulation was observed by two distinct conditions: high pH and nitrogen limitation. The Scenedesmus sp. was shown to grow and produce lipids to a larger degree in alkaliphilic conditions (pH >9) and was used to further investigate the interplay between TAG accumulation from high pH and/or nitrate depletion. Results given here indicate that TAG accumulation per cell, monitored by Nile red fluorescence, correlates with pH at the time of nitrate depletion.Item Microbial and algal alginate gelation characterized by magnetic resonance(2012-10) Fabich, H. T.; Vogt, Sarah J.; Sherick, Matthew L.; Seymour, Joseph D.; Brown, Jennifer R.; Franklin, Michael J.; Codd, Sarah L.Advanced magnetic resonance (MR) relaxation and diffusion correlation measurements and imaging provide a means to non-invasively monitor gelation for biotechnology applications. In this study, MR is used to characterize physical gelation of three alginates with distinct chemical structures; an algal alginate, which is not O-acetylated but contains poly guluronate (G) blocks, bacterial alginate from Pseudomonas aeruginosa, which does not have poly-G blocks, but is O-acetylated at the C2 and/or C3 of the mannuronate residues, and alginate from a P. aeruginosa mutant that lacks O-acetyl groups. The MR data indicate that diffusion-reaction front gelation with Ca2+ ions generates gels of different bulk homogeneities dependent on the alginate structure. Shorter spin–spin T2 magnetic relaxation times in the alginate gels that lack O-acetyl groups indicate stronger molecular interaction between the water and biopolymer. The data characterize gel differences over a hierarchy of scales from molecular to system size.Item Surprises and insights from long-term aquatic datasets and experiments(2012-08) Dodds, W. K.; Robinson, C. T.; Gaiser, E. E.; Hansen, G. J. A.; Powell, H.; Smith, J. M.; Morse, N. B.; Gregory, S. V.; Bell, Tisza A. S.; Kratz, T. K.; McDowell, W. H.Long-term research on freshwater ecosystems provides insights that can be difficult to obtain from other approaches. Widespread monitoring of ecologically relevant water-quality parameters spanning decades can facilitate important tests of ecological principles. Unique long-term data sets and analytical tools are increasingly available, allowing for powerful and synthetic analyses across sites. Long-term measurements or experiments in aquatic systems can catch rare events, changes in highly variable systems, time-lagged responses, cumulative effects of stressors, and biotic responses that encompass multiple generations. Data are available from formal networks, local to international agencies, private organizations, various institutions, and paleontological and historic records; brief literature surveys suggest much existing data are not synthesized. Ecological sciences will benefit from careful maintenance and anlayses of existing long-term programs, and subsequent insights can aid in the design of effective future long-term experimental and observational efforts. Long-term research on freshwaters is particularly important because of their value to humanity.Item Abandoned well CO2 leakage mitigation using biologically induced mineralization: Current progress and future directions(2013-02) Cunningham, Alfred B.Methods of mitigating leakage or re-plugging abandoned wells before exposure to CO2are of high potential interest to prevent leakage of CO2 injected for geologic carbon sequestration in depleted oil and gas reservoirs where large numbers of abandoned wells are often present. While CO2resistant cements and ultrafine cements are being developed, technologies that can be delivered via low viscosity fluids could have significant advantages including the ability to plug small aperture leaks such as fractures or delamination interfaces. Additionally there is the potential to plug rock formation pore space around the wellbore in particularly problematic situations. We are carrying out research on the use of microbial biofilms capable of inducing the precipitation of crystalline calcium carbonate using the process of ureolysis. This method has the potential to reduce well bore permeability, coat cement to reduce CO2–related corrosion, and lower the risk of unwanted upward CO2 migration. In this spotlight, we highlight research currently underway at the Center for Biofilm Engineering (CBE) at Montana State University (MSU) in the area of ureolytic biomineralization sealing for reducing CO2 leakage risk. This research program combines two novel core testing systems and a 3-dimensional simulation model to investigate biomineralization under both radial and axial flow conditions and at temperatures and pressures which permit CO2 to exist in the supercritical state.This combination of modeling and experimentation is ultimately aimed at developing and verifying biomineralization sealing technologies and strategies which can successfully be applied at the field scale for carbon capture and geological storage (CCGS) projects.Item Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum(2013-08) Valenzuela, Jacob J.; Carlson, Ross P.; Gerlach, Robin; Cooksey, Keith E.; Peyton, Brent M.; Bothner, Brian; Fields, Matthew W.Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.Item Investigation of coal-associated bacterial and archaeal populations from a diffusive microbial sampler (DMS)(2013-08) Barnhart, Elliott P.; Bowen De León, Kara; Ramsay, Bradley D.; Cunningham, Alfred B.; Fields, Matthew W.The Powder River Basin (PRB) in southeastern Montana and northeastern Wyoming contains massive coal deposits with biologically generated coal bed methane (CBM). The microbial ecology of an area within a coal bed influenced by recent groundwater recharge was sampled with a diffusive microbial sampler (DMS). The DMS contained native coal material and was incubated in situ (57 m depth) to allow colonization of the coal particles. Pyrotag sequence analyses of SSU rRNA gene sequences from the coal contained within the post-incubation DMS detected methylotrophic and hydrogenotrophic methanogenic archaea along with diverse bacterial communities. Microbial enrichments (coal or acetate/H2) were established from the DMS, and the enriched bacterial and archaeal communities were characterized via clone library analysis. The in situ bacterial communities were more diverse than the archaeal communities, and the archaeal populations differed between coal incubated in situ and in laboratory enrichments. In addition, bacterial diversity was higher for laboratory enrichments with coal compared to enrichments without coal. The elucidation of relationships between microorganisms involved in coal degradation and metabolite (acetate, H2) utilization within coal-dependent microbial communities is crucial to understanding and improving in situ coal bed methane production.Item Pathways of 2,4,6-trinitrotoluene transformation by aerobic yeasts(2013-08) Ziganshin, Ayrat M.; Gerlach, RobinThe production and use of various highly persistent synthetic compounds lead to environmental pollution. Among such compounds, 2,4,6-trinitrotoluene (TNT) is the one which is commonly used as an explosive. Synthesis and wide use of TNT in ammunition have resulted in the contamination of soil, air, surface water, and groundwater. TNT and its nitro group reduction products are highly toxic, potentially mutagenic and persistent contaminants which can persist in the environment for a long time (Spain et al. 2000; Stenuit et al. 2005; Smets et al. 2007; Singh et al. 2012). The U.S. Environmental Protection Agency has classified TNT as one of the most dangerous pollutants in the biosphere. Hence, remediation of TNT-contaminated sites is urgently warranted at places of its production and use (Keith and Telliard 1979; Fiorella and Spain 1997).Human exposure to TNT or its nitro group reduction metabolites can lead to the development of diseases, such as aplastic anemia, cataracts, impaired liver function and the formation of tumors in the urinary tract (Hathaway 1985; Yinon 1990; Leung et al. 1995). Hence, it is inevitable to work out strategies targeting the degradation of TNT.Decontamination of sites contaminated with explosives, especially with TNT, is possible with application of various physical, chemical, and biological methods. The main advantages of bioremediation are environmental friendliness and involvement of low cost (Rodgers and Bunce 2001).Item Cyanidiales diversity in Yellowstone National Park(2013-08) Skorupa, Dana J.; Reeb, V.; Castenholz, R. W.; Bhattacharya, D.; McDermott, Timothy R.The Cyanidiales are unicellular red algae that are unique among phototrophs. They thrive in acidic, moderately high-temperature habitats typically associated with geothermally active regions, although much remains to be learned about their distribution and diversity within such extreme environments. We focused on Yellowstone National Park (YNP), using culture-dependent efforts in combination with a park-wide environmental polymerase chain reaction (PCR) survey to examine Cyanidiales diversity and distribution in aqueous (i.e., submerged), soil and endolithic environments. Phylogenetic reconstruction of Cyanidiales biodiversity demonstrated the presence of Cyanidioschyzon and Galdieria lineages exhibiting distinct habitat preferences. Cyanidioschyzon was the only phylotype detected in aqueous environments, but was also prominent in moist soil and endolithic habitats, environments where this genus was thought to be scarce. Galdieria was found in soil and endolithic samples, but absent in aqueous habitats. Interestingly, Cyanidium could not be found in the surveys, suggesting this genus may be absent or rare in YNP. Direct microscopic counts and viable counts from soil samples collected along a moisture gradient were positively correlated with moisture content, providing the first in situ evidence that gravimetric moisture is an important environmental parameter controlling distribution of these algae.Item Direct measurement and characterization of active photosynthesis zones inside wastewater remediating and biofuel producing microalgal biofilms(2014-03) Bernstein, Hans C.; Kessano, M.; Moll, Karen M.; Smith, Terrence; Gerlach, Robin; Carlson, Ross P.; Miller, Charles D.; Peyton, Brent M.; Cooksey, Keith E.; Gardner, Robert D.; Sims, R. C.Microalgal biofilm based technologies are of keen interest due to their high biomass concentrations and ability to utilize light and CO2. While photoautotrophic biofilms have long been used for wastewater remediation, biofuel production represents a relatively new and under-represented focus area. However, the direct measurement and characterization of fundamental parameters required for industrial control are challenging due to biofilm heterogeneity. This study evaluated oxygenic photosynthesis and respiration on two distinct microalgal biofilms cultured using a novel rotating algal biofilm reactor operated at field- and laboratory-scales. Clear differences in oxygenic photosynthesis and respiration were observed based on different culturing conditions, microalgal composition, light intensity and nitrogen availability. The cultures were also evaluated as potential biofuel synthesis strategies. Nitrogen depletion was not found to have the same effect on lipid accumulation compared to traditional planktonic microalgal studies. Physiological characterizations of these microalgal biofilms identify fundamental parameters needed to understand and control process optimization.Item Enzymatic cleaning of biofouled thin-film composite reverse osmosis (RO) membrane operated in a biofilm membrane reactor(2014-06) Khan, Mohiuddin M. T.; Danielsen, S.; Johansen, K.; Nelson, Sara E.; Camper, Anne K.Application of environmentally friendly enzymes to remove thin-film composite (TFC) reverse osmosis (RO) membrane biofoulants without changing the physico-chemical properties of the RO surface is a challenging and new concept. Eight enzymes from Novozyme A/S were tested using a commercially available biofouling-resistant TFC polyamide RO membrane (BW30, FilmTech Corporation, Dow Chemical Co.) without filtration in a rotating disk reactor system operated for 58 days. At the end of the operation, the accumulated biofoulants on the TFC RO surfaces were treated with the three best enzymes, Subtilisin protease and lipase; dextranase; and polygalacturonase (PG) based enzymes, at neutral pH (~7) and doses of 50, 100, and 150 ppm. Contact times were 18 and 36 h. Live/dead staining, epifluorescence microscopy measurements, and 5 µm thick cryo-sections of enzyme and physically treated biofouled membranes revealed that Subtilisin protease- and lipase-based enzymes at 100 ppm and 18 h contact time were optimal for removing most of the cells and proteins from the RO surface. Culturable cells inside the biofilm declined by more than five logs even at the lower dose (50 ppm) and shorter incubation period (18 h). Subtilisin protease- and lipase-based enzyme cleaning at 100 ppm and for 18 h contact time restored the hydrophobicity of the TFC RO surface to its virgin condition while physical cleaning alone resulted in a 50° increase in hydrophobicity. Moreover, at this optimum working condition, the Subtilisin protease- and lipase-based enzyme treatment of biofouled RO surface also restored the surface roughness measured with atomic force microscopy and the mass percentage of the chemical compositions on the TFC surface estimated with X-ray photoelectron spectroscopy to its virgin condition. This novel study will encourage the further development and application of enzymes to remove biofoulants on the RO surface without changing its surface properties.
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