Name: bacterial cellulose spheres that encapsulate solid materials
Uploaded: 2021-02-26T15:00:00
Description: Watch this Scientific Journal Video about Bacterial Cellulose Spheres that Encapsulate Solid Materials at JoVE.com

This work is a continuation of a Montana Tech Research Assistant Mentorship Program project by Adolfo Martinez, Catherine Mulholland, Tyler Somerville, and Laurel Bitterman. Research was sponsored by the National Science Foundation under Grant No. OIA-1757351 and the Combat Capabilities Development Command Army Research Laboratory (Cooperative Agreement Number W911NF-15-2-0020). Any opinions, findings and conclusions, or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation or the Army Research Lab. We would also like to thank Amy Kuenzi, Lee Richards, Katelyn Alley, Chris Gammons, Max Wohlgenant, and Kris Bosch for their contributions.

1. Creation and maintenance of bacterial cellulose starter culture
<ol>
	<li>Obtain a starter culture of bacterial cellulose, approximately 50 g, in the form of a SCOBY. It can be purchased commercially (e.g., from Cultures for Health). Place the SCOBY into a 1 L beaker, covered with a paper towel.</li>
	<li>Boil 700 mL of deionized water, transfer it to a separate vessel from the one containing the SCOBY, and add 85 g of sucrose.</li>
	<li>Once the sucrose has dissolved, add 2 bags of black tea (4.87 g). Steep the tea...

<ol>
	<li>Mohainin Mohammad, S., Abd Rahman, N., Sahaid Khalil, M., Rozaimah Sheikh Abdullah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Overview+of+Biocellulose+Production+Using+Acetobacter+xylinum+Culture.">An Overview of Biocellulose Production Using Acetobacter xylinum Culture.</a> Advances in Biological Research. 8 (6), 307-313 (2014).</li><li>Dufresne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cellulose.">Bacterial cellulose.</a> Nanocellulose. , 125-146 (2017).</li><li>Czaja, W., Romanovicz, D., Brown, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+investigations+of+microbial+cellulose+produced+in+stationary+and+agitated+culture.">Structural investigations of microbial cellulose produced in stationary and agitated culture.</a> Cellulose. 11 (3-4), 403-411 (2004).</li><li>Hu, Y., Catchmark, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+characterization+of+spherelike+bacterial+cellulose+particles+produced+by+acetobacter+xylinum+JCM+9730+strain.">Formation and characterization of spherelike bacterial cellulose particles produced by acetobacter xylinum JCM 9730 strain.</a> Biomacromolecules. 11 (7), 1727-1734 (2010).</li><li>Goh, W. N., Rosma, A., Kaur, B., Fazilah, A., Karim, A. A., Bhat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+and+physical+properties+of+microbial+cellulose+produced+during+fermentation+of+black+tea+broth+(kombucha).">Microstructure and physical properties of microbial cellulose produced during fermentation of black tea broth (kombucha).</a> International Food Research Journal. 19 (1), 153-158 (2012).</li><li>Toyosaki, H., Naritomi, T., Seto, A., Matsuoka, M., Tsuchida, T., Yoshinaga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+of+Bacterial+Cellulose-producing+Acetobacter+Strains+Suitable+for+Agitated+Culture.">Screening of Bacterial Cellulose-producing Acetobacter Strains Suitable for Agitated Culture.</a> Bioscience, Biotechnology, and Biochemistry. 59 (8), 1498-1502 (1995).</li><li>Shi, Z., Zhang, Y., Phillips, G. O., Yang, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utilization+of+bacterial+cellulose+in+food.">Utilization of bacterial cellulose in food.</a> Food Hydrocolloids. 35, 539-545 (2014).</li><li>Holland, M. C., Eggensperger, C. G., Giagnorio, M., Schiffman, J. D., Tiraferri, A., Zodrow, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Postprocessing+Alters+the+Permeability+and+Selectivity+of+Microbial+Cellulose+Ultrafiltration+Membranes.">Facile Postprocessing Alters the Permeability and Selectivity of Microbial Cellulose Ultrafiltration Membranes.</a> Environmental Science and Technology. 54 (20), 13249-13256 (2020).</li><li>Le Hoang, S., Vu, C. M., Pham, L. T., Choi, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+physical+characteristics+of+epoxy+resin/+bacterial+cellulose+biocomposites.">Preparation and physical characteristics of epoxy resin/ bacterial cellulose biocomposites.</a> Polymer Bulletin. 75 (6), 2607-2625 (2018).</li><li>Vu, C. M., Nguyen, D. D., Sinh, L. H., Pham, T. D., Pham, L. T., Choi, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmentally+benign+green+composites+based+on+epoxy+resin/bacterial+cellulose+reinforced+glass+fiber:+Fabrication+and+mechanical+characteristics.">Environmentally benign green composites based on epoxy resin/bacterial cellulose reinforced glass fiber: Fabrication and mechanical characteristics.</a> Polymer Testing. 61, 150-161 (2017).</li><li>Pavaloiu, R. D., Stoica, A., Stroescu, M., Dobre, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+release+of+amoxicillin+from+bacterial+cellulose+membranes.">Controlled release of amoxicillin from bacterial cellulose membranes.</a> Central European Journal of Chemistry. 12 (9), 962-967 (2014).</li><li>Trovatti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biocellulose+membranes+as+supports+for+dermal+release+of+lidocaine.">Biocellulose membranes as supports for dermal release of lidocaine.</a> Biomacromolecules. 12 (11), 4162-4168 (2011).</li><li>Trovatti, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cellulose+membranes+applied+in+topical+and+transdermal+delivery+of+lidocaine+hydrochloride+and+ibuprofen:+In+vitro+diffusion+studies.">Bacterial cellulose membranes applied in topical and transdermal delivery of lidocaine hydrochloride and ibuprofen: In vitro diffusion studies.</a> International Journal of Pharmaceutics. 435 (1), 83-87 (2012).</li><li>Shaviv, A., Mikkelsen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled-release+fertilizers+to+increase+efficiency+of+nutrient+use+and+minimize+environmental+degradation+-+A+review.">Controlled-release fertilizers to increase efficiency of nutrient use and minimize environmental degradation - A review.</a> Fertilizer Research. 35 (1-2), 1-12 (1993).</li><li>Eggensperger, C. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+living+filtration+membranes.">Sustainable living filtration membranes.</a> Environmental Science and Technology Letters. 7 (3), 213-218 (2020).</li><li>Schr&#246;pfer, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradation+evaluation+of+bacterial+cellulose,+vegetable+cellulose+and+poly+(3-hydroxybutyrate)+in+soil.">Biodegradation evaluation of bacterial cellulose, vegetable cellulose and poly (3-hydroxybutyrate) in soil.</a> Polimeros. 25 (2), 154-160 (2015).</li><li>Orts, W. J., Glenn, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+soil+erosion+losses+with+small+applications+of+biopolymers.">Reducing soil erosion losses with small applications of biopolymers.</a> ACS Symposium Series. 723, 235-247 (1999).</li><li>Mohite, B. V., Patil, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+biomaterial:+Bacterial+cellulose+and+its+new+era+applications.">A novel biomaterial: Bacterial cellulose and its new era applications.</a> Biotechnology and Applied Biochemistry. 61 (2), 101-110 (2014).</li><li>Mikkelsen, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+hydrophilic+polymers+to+control+nutrient+release.">Using hydrophilic polymers to control nutrient release.</a> Fertilizer Research. 38 (1), 53-59 (1994).</li><li>Du, C. W., Zhou, J. M., Shaviv, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+characteristics+of+nutrients+from+polymer-coated+compound+controlled+release+fertilizers.">Release characteristics of nutrients from polymer-coated compound controlled release fertilizers.</a> Journal of Polymers and the Environment. 14 (3), 223-230 (2006).</li><li>Serafica, G., Mormino, R., Bungay, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inclusion+of+solid+particles+in+bacterial+cellulose.">Inclusion of solid particles in bacterial cellulose.</a> Applied Microbiology and Biotechnology. 58 (6), 756-760 (2002).</li><li>Tomaszewska, M., Jarosiewicz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+polysulfone+in+controlled-release+NPK+fertilizer+formulations.">Use of polysulfone in controlled-release NPK fertilizer formulations.</a> Journal of Agricultural and Food Chemistry. 50 (16), 4634-4639 (2002).</li><li>Gonz&#225;lez, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+biodegradable+polymers+as+encapsulating+agents+for+the+development+of+a+urea+controlled-release+fertilizer+using+biochar+as+support+material.">Evaluation of biodegradable polymers as encapsulating agents for the development of a urea controlled-release fertilizer using biochar as support material.</a> Science of the Total Environment. 505, 446-453 (2015).</li><li>Shavit, U., Shaviv, A., Shalit, G., Zaslavsky, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+characteristics+of+a+new+controlled+release+fertilizer.">Release characteristics of a new controlled release fertilizer.</a> Journal of Controlled Release. 43 (2-3), 131-138 (1997).</li><li>Kolakovic, R., Laaksonen, T., Peltonen, L., Laukkanen, A., Hirvonen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spray-dried+nanofibrillar+cellulose+microparticles+for+sustained+drug+release.">Spray-dried nanofibrillar cellulose microparticles for sustained drug release.</a> International Journal of Pharmaceutics. 430 (1-2), 47-55 (2012).</li><li>Zaharia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+cellulose-poly(acrylic+acid-:+Co-N,+N+&#8242;-methylene-bis-acrylamide)+interpenetrated+networks+for+the+controlled+release+of+fertilizers.">Bacterial cellulose-poly(acrylic acid-: Co-N, N &#8242;-methylene-bis-acrylamide) interpenetrated networks for the controlled release of fertilizers.</a> RSC Advances. 8 (32), 17635-17644 (2018).</li><li>Peterson, J. D., Vyazovkin, S., Wight, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+the+thermal+and+thermo-oxidative+degradation+of+polystyrene,+polyethylene+and+poly(propylene).">Kinetics of the thermal and thermo-oxidative degradation of polystyrene, polyethylene and poly(propylene).</a> Macromolecular Chemistry and Physics. 202 (6), 775-784 (2001).</li><li>Goh, W. N., Rosma, A., Kaur, B., Fazilah, A., Karim, A. A., Bhat, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fermentation+of+black+tea+broth+(kombucha):+I.+effects+of+sucrose+concentration+and+fermentation+time+on+the+yield+of+microbial+cellulose.">Fermentation of black tea broth (kombucha): I. effects of sucrose concentration and fermentation time on the yield of microbial cellulose.</a> International Food Research Journal. 19 (1), 109-117 (2012).</li><li>Zhu, H., Jia, S., Yang, H., Jia, Y., Yan, L., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+application+of+bacterial+cellulose+sphere:+A+novel+biomaterial.">Preparation and application of bacterial cellulose sphere: A novel biomaterial.</a> Biotechnology and Biotechnological Equipment. 25 (1), 2233-2236 (2011).</li><li>Nguyen, V. T., Flanagan, B., Gidley, M. J., Dykes, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cellulose+production+by+a+Gluconacetobacter+xylinus+strain+from+Kombucha.">Characterization of cellulose production by a Gluconacetobacter xylinus strain from Kombucha.</a> Current Microbiology. 57 (5), 449-453 (2008).</li><li>Costa, A. F. S., Almeida, F. C. G., Vinhas, G. M., Sarubbo, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+bacterial+cellulose+by+Gluconacetobacter+hansenii+using+corn+steep+liquor+as+nutrient+sources.">Production of bacterial cellulose by Gluconacetobacter hansenii using corn steep liquor as nutrient sources.</a> Frontiers in Microbiology. 8, 1-12 (2017).</li><li>Watanabe, K., Tabuchi, M., Morinaga, Y., Yoshinaga, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+features+and+properties+of+bacterial+cellulose+produced+in+agitated+culture.">Structural features and properties of bacterial cellulose produced in agitated culture.</a> Cellulose. 5 (3), 187-200 (1998).</li></ol>

This protocol outlines BC sphere production and encapsulation methods that are easy to conduct and cost effective. Through various adjustments to the original protocol, an adequate process has been identified. Critical steps must be followed to ensure viable spheres. All the ingredients involved in BC formation play a key role in the health and durability of the spheres. The sucrose feeds organisms, the tea provides nitrogen, and the vinegar lowers the pH to optimal conditions to prevent undesired contaminants<sup class=...

Bacterial cellulose (BC) has been noted for its potential industry use due to its mechanical strength, high purity and crystallinity, water retention abilities, and intricate fiber structure1,2,3,4. These characteristics make BC a favorable biomaterial for a variety of applications, including biomedical, food processing, and environmental remediation uses1. Formation of a BC film can be done with single organism cultures or mixed cultures like those used for kombucha5, a fermented tea beverage. Brewing kombucha relies on a &#34;Symbiotic Culture Of Bacteria and Yeast&#34;, commonly known as a SCOBY. Using this symbiotic culture of organisms, a similar technique is employed to create BC spheres. This biomaterial may be employed to help isolate environmental contaminants and anchor agricultural amendments like biochar to achieve more efficient crop production.
Previous literature has discussed how the characteristics of BC produced in agitated conditions compare to BC produced in a stationary culture. A stationary culture results in a film that forms at the liquid-air interface, while a shaken culture results in varying BC particles, strands, and spheres suspended within the liquid6. Many studies have referenced the claim that commercial production of BC is more feasible in the dynamic conditions6,7, providing rationale for applying this paper&#39;s method. Additionally, various investigations on the structure and properties of BC spheres have been conducted. Toyosaki et al.6 compared baffled and smooth-walled Erlenmeyer flasks in their agitated BC production. A study by Hu and Catchmark4 determined conditions for BC spheres that were used as guidelines for the current BC sphere production process, and their results indicate that the sphere size does not continue to increase after 60 hours. A review of BC production by Mohammad et al.1 indicates that shaking the BC culture ensures even oxygen supply and distribution, which is necessary for successful BC growth. Holland et al.8 have studied the crystallinity and chemical structure of BC using X-ray diffraction and Fourier transform infrared spectroscopy. It is assumed BC capsules will exhibit similar characteristics and future research will investigate structural properties. Studies have also explored the beneficial effects of using BC to produce improved biocomposites. Using epoxy-resin as a base, researchers have showed that the addition of BC improves material characteristics like fatigue life, fracture toughness, and tensile and flexural strength9,10. As shown by past and current research, many are interested in commercializing BC use.
Many researchers have investigated bacterial cellulose in controlled release systems, and the method described here generates capsules that could be utilized as controlled release systems. Much of this research focuses on controlled release in the biomedical field, as well as some exploration in controlled release fertilizer (CRF) administration. Based on the success of BC&#39;s controlled release of amoxicillin11, lidocaine12, and ibuprofen13, BC may exhibit similar delivery characteristics with other substances, such as a pelletized fertilizer. An overview of CRF&#39;s by Shaviv and Mikkelsen14 acknowledges that CRF&#39;s are more efficient, save labor, and generally cause less environmental degradation than conventional fertilizer application. Bacterial cellulose may work as a favorable encapsulating material for CRF&#39;s. Fertilizers may leach out of BC membranes or discharge as BC biodegrades15,16. BC&#39;s high water swelling capacity can also act as a beneficial soil amendment17,18,19 because both fertilizer nutrients and moisture may release into the ground through application of BC spheres. With these traits, a CRF formed by BC sphere encapsulation may have an advantage over other fertilizer coating materials that could have negative effects during their production and disposal stages. Adapting BC into a fertilizer coating may further improve CRF technologies. By lowering fertilizer release rate, crops will have sufficient time to uptake the fertilizer and prevent excess runoff into bodies of water, thereby reducing eutrophication and unoxygenated zones. Similar slow-release fertilizers have been prepared and piloted using polymer coatings20.
Unlike protocols outlined in previous research, this one focuses on uniform, cohesive sphere production rather than high cellulose yield. Moreover, BC encapsulation of other solids has been studied with cellulose films, but not spheres21. By expanding the research on bacterial cellulose spheres, further steps can be made to produce BC commercially, which is beneficial because of BC&#39;s environmentally safe features. This method of BC sphere fabrication utilizes inexpensive, readily available culinary ingredients. After the initial assembly, BC spheres begin to form within 2 days without interference. Producing BC spheres through this strategy requires little space and has an edible by-product, the fermented tea &#39;kombucha&#39;. Encapsulation techniques mentioned in other studies include coatings formed through the phase inversion technique22,23, matrix formation24, spray drying25, and direct encapsulation during synthesis26. The direct encapsulation method outlined in this manuscript is useful for those who desire an easy, inexpensive process that utilizes readily available materials.
Through this research, a successful protocol for BC sphere production and encapsulation was created. BC spheres can encapsulate solid particles of biochar, mine tailings, and polystyrene microbeads within their individual structures. Although not widely used in industry yet, BC is a practical, sustainably made, and naturally occurring material that could be used for future applications.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mL graduated cylinder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 mL graduated cylinder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Erlenmeyer baffled flask</td>
			<td>Chemglass</td>
			<td>CLS-2040-02</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL beaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Balance</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biochar</td>
			<td></td>
			<td></td>
			<td>Ponderosa pine heat treated under argon gas, heated at 15 &#176;C per minute to 800 &#176;C</td>
		</tr>
		<tr>
			<td>Black tea</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Distilled white vinegar</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Elastic band</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microbial starter culture</td>
			<td>Cultures for Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mine waste</td>
			<td></td>
			<td></td>
			<td>Collected from Butte, MT: 46.001978,-112.582465. Mine waste contains soil and metals originating from past copper mining. Mn, Si, Ca, Al, and Fe were the five most prevalent elements measured in the mine waste through x-ray diffraction.</td>
		</tr>
		<tr>
			<td>Mortar and pestle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td></td>
			<td></td>
			<td>Used various brands</td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microbeads</td>
			<td>Polybead</td>
			<td>17138</td>
			<td>3 micron diameter</td>
		</tr>
		<tr>
			<td>Stir rod</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tea kettle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TGA</td>
			<td>TA Instruments</td>
			<td>TA Q500</td>
			<td>400 &#176;C/min to 800 &#176;C, 100 mL/min N2</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>XRF Analyzer</td>
			<td>ThermoFisher Scientific</td>
			<td>10131166</td>
			<td></td>
		</tr>
	</tbody>
</table>

bacterial cellulose spheres that encapsulate solid materials

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an affordable and simple method for BC sphere production. In addition to producing these spheres, an encapsulation method for solid particles has also been identified. To produce BC spheres, water, black tea, sugar, vinegar, and bacterial culture are combined in a baffled flask and the contents are agitated. After determining the proper culture conditions for BC sphere formation, their ability to encapsulate solid particles was tested using biochar, polymer beads, and mine waste. Spheres were characterized using ImageJ software and thermal gravimetric analysis (TGA). Results indicate that spheres with 7.5 mm diameters can be made in 7 days. Adding various particles increases the average size range of the BC capsules. The spheres encapsulated 10 - 20% of their dry mass. This method shows low-cost sphere production and encapsulation that is possible with easily obtainable materials. BC spheres may be used in the future as a contaminant removal aid, controlled release fertilizer coating, or soil amendment.

BC spheres have the fastest growth rate during the first 48 h of culture (Figure 2). Figure 2 also shows how the spheres tend to reach a maximum average size and then remain constant. In this experiment, the spheres reached an average diameter of 7.5 &#177; 0.2 mm. Although the BC spheres never completely deteriorate within the 10 day growth period, they did start to form tendrils that extend off the main body of the sphere around the eighth day. This can be see...

Watch this Scientific Journal Video about Bacterial Cellulose Spheres that Encapsulate Solid Materials at JoVE.com

Bacterial Cellulose Spheres that Encapsulate Solid Materials

This protocol presents an easy, inexpensive method of forming bacterial cellulose (BC) spheres. This biomaterial can function as an encapsulation medium for solid materials, including biochar, polymer spheres, and mine waste.

Bacterial cellulose (BC) spheres have been increasingly researched since the popularization of BC as a novel material. This protocol presents an ...

Our protocol is significant, as it expands on current methods of producing a biodegradable, naturally-occurring biomaterial. Our method emphasizes production of reproducible bacterial cellulose spheres. The main advantage of this technique is the ease of access to the necessary materials:sugar, water, tea, vinegar, bacterial culture starter, a baffle flask, and an orbital shaker.Someone performing this technique for the first time may not be able to correctly identify the spheres and remove irregular bacterial cellulose masses. We have demonstrated these steps in the video, but do not be discouraged if it does not work the first time. Visual demonstration of this method is usually helpful for researchers to visualize what the sizes, amounts, and shapes of bacterial cellulose masses are preferred and not preferred.Begin by boiling 350 milliliters of deionized water using a tea kettle. Then transfer the hot water to a 500 milliliter beaker. Use heat protection gloves, and make sure that the glass bar can withstand the boiling water temperatures.Completely dissolve 42.5 grams of granulated sucrose into the hot water using a stir rod. Steep a bag with 2.54 grams of black tea in the flask containing sucrose solution for one hour. Remove the tea bag with the stir rod without breaking it open, and dispose of it in the trash.Add 100 milliliters of distilled white vinegar to the beaker, and thoroughly stir the mixture. Transfer 80 milliliters of the prepared acidic tea mixture to a 250 milliliter baffled flask, and allow that to mixture to cool to 20 to 25 degrees Celsius. Add 20 milliliters of microbial starter culture liquid to the baffled flask when the liquid is at room temperature, and cover the flask with Parafilm.Place the baffled flask on an orbital shake table, and allow it to shake at 125 rotations per minute for three days at 20 to 25 degrees Celsius to produce BC spheres. Remove unwanted BC masses of nonspherical shapes with tweezers to prevent further irregular BC masses from forming. Once the BC spheres have formed, gently pour them from the flask for further use.In this protocol, the BC spheres showed a high growth rate for the first 48 hours of culture, and the size remained constant after reaching a maximum. The BC spheres started to form tendrils around the eighth day of culture. The size distribution of spheres after encapsulation of solid contaminants like biochar, polymer beads, and mine waste was tested.It was observed that the addition of solids to the BC spheres does have a consistent effect on sphere size or frequency. The orbital shaking speed, ambient temperature, and formation of irregular particles seem to be the main factors that affect shape, size, and frequency of spherical particles. It was noticed that too high of a room temperature or improper removal of irregular masses changed the shape of an intact BC sphere to stellate particles or stringy clumps.To determine the fraction of encapsulated solids in the BC spheres, a thermal gravimetric analysis was done. Thermal and microscopic evaluation together confirmed the effective encapsulation of solid particles within BC spheres. The differential TGA profile of plane BC appeared in nearly identical magnitudes with the BC with polystyrene beads.However, an additional peak corresponding to thermal decomposition of polystyrene beads was observed. It is most important to add the microbial starter culture when the tea has cooled down to room temperature. Additionally, we must ensure the culture has active and healthy organisms.This method can be used for contaminant removal in environmental remediation. The spheres can also be utilized for controlled release of a substance of interest. This technique enables us to encapsulate environmental materials within bacterial cellulose spheres.This may be useful for a biodegradable controlled release or remediation platforms.

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Research

JoVE Journal

Engineering

Solid-state Graft Copolymer Electrolytes for Lithium Battery Applications

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 &deg;C), flammable, and volatile organic electrolytes. These organic based electrolyte systems are viable at ambient temperatures, but require a cooling system to ensure that temperatures do not exceed 80 &deg;C. These cooling systems tend to increase battery costs and can malfunction which can lead to battery malfunction and explosions, thus endangering human life. Increases in petroleum prices lead to a huge demand for safe, electric hybrid vehicles that are more economically viable to operate as oil prices continue to rise. Existing organic based electrolytes used in lithium ion batteries are not applicable to high temperature automotive applications. A safer alternative to organic electrolytes is solid polymer electrolytes. This work will highlight the synthesis for a graft copolymer electrolyte (GCE) poly(oxyethylene) methacrylate (POEM) to a block with a lower glass transition temperature (Tg) poly(oxyethylene) acrylate (POEA). The conduction mechanism has been discussed and it has been demonstrated the relationship between polymer segmental motion and ionic conductivity indeed has a Vogel-Tammann-Fulcher (VTF) dependence. Batteries containing commercially available LP30 organic (LiPF6 in ethylene carbonate (EC):dimethyl carbonate (DMC) at a 1:1 ratio) and GCE were cycled at ambient temperature. It was found that at ambient temperature, the batteries containing GCE showed a greater overpotential when compared to LP30 electrolyte. However at temperatures greater than 60 &deg;C, the GCE cell exhibited much lower overpotential due to fast polymer electrolyte conductivity and nearly the full theoretical specific capacity of 170 mAh/g was accessed.

Lithium ion batteries employ flammable and volatile organic electrolytes that are suitable for ambient temperature applications. A safer alternative to organic electrolytes are solid polymer batteries. Solid polymer batteries operate safely at high temperatures (&gt;120 &deg;C), thus making them applicable to high temperature applications such as deep oil drilling and hybrid electric vehicles. This paper will discuss (a) the polymer synthesis, (b) the polymer conduction mechanism, and (c) provide temperature cycling for both solid polymer and organic electrolytes.

Battery safety has been a very important research area over the past decade. Commercially available lithium ion batteries employ low flash point (&lt;80 ...

Probing and Mapping Electrode Surfaces in Solid Oxide Fuel Cells

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen 1-7. The performance of SOFCs and the rates of many chemical and energy transformation processes in energy storage and conversion devices in general are limited primarily by charge and mass transfer along electrode surfaces and across interfaces. Unfortunately, the mechanistic understanding of these processes is still lacking, due largely to the difficulty of characterizing these processes under in situ conditions. This knowledge gap is a chief obstacle to SOFC commercialization. The development of tools for probing and mapping surface chemistries relevant to electrode reactions is vital to unraveling the mechanisms of surface processes and to achieving rational design of new electrode materials for more efficient energy storage and conversion2. Among the relatively few in situ surface analysis methods, Raman spectroscopy can be performed even with high temperatures and harsh atmospheres, making it ideal for characterizing chemical processes relevant to SOFC anode performance and degradation8-12. It can also be used alongside electrochemical measurements, potentially allowing direct correlation of electrochemistry to surface chemistry in an operating cell. Proper in situ Raman mapping measurements would be useful for pin-pointing important anode reaction mechanisms because of its sensitivity to the relevant species, including anode performance degradation through carbon deposition8, 10, 13, 14 ("coking") and sulfur poisoning11, 15 and the manner in which surface modifications stave off this degradation16. The current work demonstrates significant progress towards this capability. In addition, the family of scanning probe microscopy (SPM) techniques provides a special approach to interrogate the electrode surface with nanoscale resolution. Besides the surface topography that is routinely collected by AFM and STM, other properties such as local electronic states, ion diffusion coefficient and surface potential can also be investigated17-22. In this work, electrochemical measurements, Raman spectroscopy, and SPM were used in conjunction with a novel test electrode platform that consists of a Ni mesh electrode embedded in an yttria-stabilized zirconia (YSZ) electrolyte. Cell performance testing and impedance spectroscopy under fuel containing H2S was characterized, and Raman mapping was used to further elucidate the nature of sulfur poisoning. In situ Raman monitoring was used to investigate coking behavior. Finally, atomic force microscopy (AFM) and electrostatic force microscopy (EFM) were used to further visualize carbon deposition on the nanoscale. From this research, we desire to produce a more complete picture of the SOFC anode.

We present a unique platform for characterizing electrode surfaces in solid oxide fuel cells (SOFCs) that allows simultaneous performance of multiple characterization techniques (e.g. in situ Raman spectroscopy and scanning probe microscopy alongside electrochemical measurements). Complementary information from these analyses may help to advance toward a more profound understanding of electrode reaction and degradation mechanisms, providing insights into rational design of better materials for SOFCs.

Solid oxide fuel cells (SOFCs) are potentially the most efficient and cost-effective solution to utilization of a wide variety of fuels beyond hydrogen ...

Characterization of Thermal Transport in One-dimensional Solid Materials

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including conductive, semi-conductive or nonconductive one-dimensional structures. This technique broadens the measurement scope of materials (conductive and nonconductive) and improves the accuracy and stability. If the sample (especially biomaterials, such as human head hair, spider silk, and silkworm silk) is not conductive, it will be coated with a gold layer to make it electronically conductive. The effect of parasitic conduction and radiative losses on the thermal diffusivity can be subtracted during data processing. Then the real thermal conductivity can be calculated with the given value of volume-based specific heat (&#961;cp), which can be obtained from calibration, noncontact photo-thermal technique or measuring the density and specific heat separately. In this work, human head hair samples are used to show how to set up the experiment, process the experimental data, and subtract the effect of parasitic conduction and radiative losses.

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials.

The TET (transient electro-thermal) technique is an effective approach developed to measure the thermal diffusivity of solid materials, including ...

Preparation and Reactivity of Gasless Nanostructured Energetic Materials

High-Energy Ball Milling (HEBM) is a ball milling process where a powder mixture placed in the ball mill is subjected to high-energy collisions from the balls. Among other applications, it is a versatile technique that allows for effective preparation of gasless reactive nanostructured materials with high energy density per volume (Ni+Al, Ta+C, Ti+C). The structural transformations of reactive media, which take place during HEBM, define the reaction mechanism in the produced energetic&#160;composites. Varying the processing conditions permits fine tuning of the milling-induced microstructures of the fabricated composite particles. In turn, the reactivity, i.e., self-ignition temperature, ignition delay time, as well as reaction kinetics, of high energy density materials depends on its microstructure. Analysis of the milling-induced microstructures suggests that the formation of fresh oxygen-free intimate high surface area contacts between the reagents is responsible for the enhancement of their reactivity. This manifests itself in a reduction of ignition temperature and delay time, an increased rate of chemical reaction, and an overall decrease of the effective activation energy of the reaction. The protocol provides a detailed description for the preparation of reactive nanocomposites with tailored microstructure using short-term HEBM method. It also describes a high-speed thermal imaging technique to determine the ignition/combustion characteristics of the energetic materials. The protocol can be adapted to preparation and characterization of a variety of nanostructured energetic composites.

This protocol describes the preparation of gasless nanostructured energetic materials (Ni+Al, Ta+C, Ti+C) using the short-term high-energy ball milling (HEBM) technique. It also describes a high-speed thermal imaging method to study the reactivity of mechanically fabricated nanocomposites. These protocols can be extended to other reactive nanostructured energetic materials.

High-Energy Ball Milling (HEBM) is a ball milling process where a powder mixture placed in the ball mill is subjected to high-energy collisions from the ...

Advanced Experimental Methods for Low-temperature Magnetotransport Measurement of Novel Materials

Novel electronic materials are often produced for the first time by synthesis processes that yield bulk crystals (in contrast to single crystal thin film synthesis) for the purpose of exploratory materials research. Certain materials pose a challenge wherein the traditional bulk Hall bar device fabrication method is insufficient to produce a measureable device for sample transport measurement, principally because the single crystal size is too small to attach wire leads to the sample in a Hall bar configuration. This can be, for example, because the first batch of a new material synthesized yields very small single crystals or because flakes of samples of one to very few monolayers are desired. In order to enable rapid characterization of materials that may be carried out in parallel with improvements to their growth methodology, a method of device fabrication for very small samples has been devised to permit the characterization of novel materials as soon as a preliminary batch has been produced. A slight variation of this methodology is applicable to producing devices using exfoliated samples of two-dimensional materials such as graphene, hexagonal boron nitride (hBN), and transition metal dichalcogenides (TMDs), as well as multilayer heterostructures of such materials. Here we present detailed protocols for the experimental device fabrication of fragments and flakes of novel materials with micron-sized dimensions onto substrate and subsequent measurement in a commercial superconducting magnet, dry helium close-cycle cryostat magnetotransport system at temperatures down to 0.300 K and magnetic fields up to 12 T.

We describe the methodology of mechanical exfoliation and deposition of flakes of novel materials with micron-sized dimensions onto substrate, fabrication of experimental device structures for transport experimentation, and the magnetotransport measurement in a dry helium close-cycle cryostat at temperatures down to 0.300 K and magnetic fields up to 12 T.

Novel electronic materials are often produced for the first time by synthesis processes that yield bulk crystals (in contrast to single crystal thin film ...

Characterization of Ultra-fine Grained and Nanocrystalline Materials Using Transmission Kikuchi Diffraction

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and nanocrystalline materials. The traditional techniques associated with scanning electron microscopy (SEM), such as electron backscatter diffraction (EBSD), do not possess the required spatial resolution due to the large interaction volume between the electrons from the beam and the atoms of the material. Transmission electron microscopy (TEM) has the required spatial resolution. However, due to a lack of automation in the analysis system, the rate of data acquisition is slow which limits the area of the specimen that can be characterized. This paper presents a new characterization technique, Transmission Kikuchi Diffraction (TKD), which enables the analysis of the microstructure of UFG and nanocrystalline materials using an SEM equipped with a standard EBSD system. The spatial resolution of this technique can reach 2 nm. This technique can be applied to a large range of materials that would be difficult to analyze using traditional EBSD. After presenting the experimental set up and describing the different steps necessary to realize a TKD analysis, examples of its use on metal alloys and minerals are shown to illustrate the resolution of the technique and its flexibility in term of material to be characterized.

This paper provides a detailed method to characterize the microstructure of ultra-fine grained and nanocrystalline materials using a scanning electron microscope equipped with a standard electron backscatter diffraction system. Metal alloys and minerals presenting refined microstructures are analyzed using this technique, showing the diversity of its possible applications.

One of the challenges in microstructure analysis nowadays resides in the reliable and accurate characterization of ultra-fine grained (UFG) and ...

Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of lithium-ion (Li-ion) batteries. However, a number of setbacks prevent their integration into commercial devices. The main limiting factor is due to nanoscale phenomena occurring at the electrode/electrolyte interfaces, ultimately leading to degradation of battery operation. These key problems are highly challenging to observe and characterize as these batteries contain multiple buried interfaces. One approach for direct observation of interfacial phenomena in thin film batteries is through the fabrication of electrochemically active nanobatteries by a focused ion beam (FIB). As such, a reliable technique to fabricate nanobatteries was developed and demonstrated in recent work. Herein, a detailed protocol with a step-by-step process is presented to enable the reproduction of this nanobattery fabrication process. In particular, this technique was applied to a thin film battery consisting of LiCoO2/LiPON/a-Si, and has further been previously demonstrated by in situ cycling within a transmission electron microscope.

Focused Ion Beam Fabrication of LiPON-based Solid-state Lithium-ion Nanobatteries for In Situ Testing

A protocol for the fabrication of electrochemically active LiPON-based solid-state lithium-ion nanobatteries using a focused ion beam is presented.

Solid-state electrolytes are a promising replacement for current organic liquid electrolytes, enabling higher energy densities and improved safety of ...

Electric-field Control of Electronic States in WS2 Nanodevices by Electrolyte Gating

A method of carrier number control by electrolyte gating is demonstrated. We have obtained WS2 thin flakes with atomically flat surface via scotch tape method or individual WS2 nanotubes by dispersing the suspension of WS2 nanotubes. The selected samples have been fabricated into devices by the use of the electron beam lithography and electrolyte is put on the devices. We have characterized the electronic properties of the devices under applying the gate voltage. In the small gate voltage region, ions in the electrolyte are accumulated on the surface of the samples which leads to the large electric potential drop and resultant electrostatic carrier doping at the interface. Ambipolar transfer curve has been observed in this electrostatic doping region. When the gate voltage is further increased, we met another drastic increase of source-drain current which implies that ions are intercalated into layers of WS2 and electrochemical carrier doping is realized. In such electrochemical doping region, superconductivity has been observed. The focused technique provides a powerful strategy for achieving the electric-filed-induced quantum phase transition.

Electric-field Control of Electronic States in WS2 Nanodevices by Electrolyte Gating

Here, we present a protocol to control the carrier number in solids by using the electrolyte.

A method of carrier number control by electrolyte gating is demonstrated. We have obtained WS2 thin flakes with atomically flat surface via scotch tape ...

Experimental Methods for Spin- and Angle-Resolved Photoemission Spectroscopy Combined with Polarization-Variable Laser

The goal of this protocol is to present how to perform spin- and angle-resolved photoemission spectroscopy combined with polarization-variable 7-eV laser (laser-SARPES), and demonstrate a power of this technique for studying solid state physics. Laser-SARPES achieves two great capabilities. Firstly, by examining orbital selection rule of linearly polarized lasers, orbital selective excitation can be carried out in SAPRES experiment. Secondly, the technique can show full information of a variation of the spin quantum axis as a function of the light polarization. To demonstrate the power of the collaboration of these capabilities in laser-SARPES, we apply this technique for the investigations of spin-orbit coupled surface states of Bi2Se3. This technique affords to decompose spin and orbital components from the spin-orbit coupled wavefunctions. Moreover, as a representative advantage of using the direct spin detection collaborated with the polarization-variable laser, the technique unambiguously visualizes the light polarization dependence of the spin quantum axis in three-dimension. Laser-SARPES dramatically increases a capability of photoemission technique.

Here, we combine polarization-variable 7-eV laser with spin- and angle-resolved photoemission technique to visualize the spin-orbital coupling effect in solid states.

The goal of this protocol is to present how to perform spin- and angle-resolved photoemission spectroscopy combined with polarization-variable 7-eV laser ...

Additive Manufacturing of Functionally Graded Ceramic Materials by Stereolithography

An additive manufacturing technology is applied to obtain functionally graded ceramic parts. This technology, based on digital light processing/stereolithography, is developed within the scope of the CerAMfacturing European research project. A three-dimensional (3-D) hemi-maxillary bone-like structure is 3-D printed using custom aluminum oxide polymeric mixtures. The powders and mixtures are fully analyzed in terms of rheological behavior in order to ensure proper material handling during the printing process. The possibility to print functionally graded materials using the Admaflex technology is explained in this document. Field-emission scanning electron microscopy (FESEM) show that the sintered aluminum oxide ceramic part has a porosity lower than 1% and no remainder of the original layered structure is found after analysis.

This manuscript describes the processing of single multifunctional ceramic components (e.g., combinations of dense-porous structures) additively manufactured by stereolithography.