Bacterial cellulose : atomized nutrient delivery and gelatin for morphological control

Abstract

Bacterial cellulose (BC) is a naturally derived three-dimensional mesoporous matrix, having high crystallinity, high mechanical strength, high water holding capacity, low density, low electrical conductivity, and is biocompatible. These traits are highly desirable for industrial applications in textiles, air-water filtration, acoustic diaphragms, external wound healing bandages, and tissue scaffolds. The potential benefits of BC use in a wide variety of industrial applications are challenged by long growth times, low cellulose productivity, random fiber distribution, and small average pore size. One way to combat these challenges is to use intermittent top-feeding as a strategy to increase the amount of available nutrients to the bacteria along with assisting in delaying the drop in pH of the growth media from cellulose production, thereby increasing overall BC production. A second way to combat these challenges is the use of non-nutritional additives. These force the bacteria to grow cellulose around areas of high stiffness or template like structures within the media, forming a more organized and larger pore sized matrix. Here, we set out to develop an alternative intermittent top-feeding strategy and study the use of non-nutritional additives on pore formation. First, we developed a method of an intermittent top-feeding strategy utilizing an atomization nozzle apparatus on the growth of BC pellicles. In this method, BC pellicles were fed at 12-hour intervals over a 90-hour cultivations. Here, we found that, by implementing this low-impact top-feeding method, there was a 50% increase in the overall cellulose production by the bacteria compared to normally grown static cultures where all nutrients were provided in the cultivation medium. Pellicles formed by top feeding of atomized nutrient droplets have higher specific modulus and specific tensile strengths then those grown statically. In addition, atomized pellicles had a 250% increase in the water holding capacity along with a higher average porosity. Next, we set out to understand how non-nutritional, naturally derived additives affect the static growth and average pore size development of bacterial cellulose. The additive chosen was gelatin in concentrations of 0.1 wt%, 2.5 wt%, 5 wt%, and 7.5 wt%; as it is a biologically produced material that forms a three-dimensional semi-ordered gel with good thermal stability. We found that, with the addition of gelatin, there is an almost four times increase in cellulose productivity and an almost ten times increase in the average pore size found within the BC matrix. Additionally, there are improved specific modulus and specific tensile strengths seen the gelatin based samples along with a maximum increase of 300% in water holding capacity. In response to these findings, a method was devised to combine the effects of gelatin with the intermittent top-feeding atomization strategy. This was investigated with interval feeding times of 6 hours, 12 hours, and 24 hours over a 7-day period. Additionally, the same total gelatin concentrations used in the static work was implemented in the gelatin media intermittent top-feeding atomization strategy allowing direct comparison of these cultivation strategies. This work revealed that a unique layering morphology was developed within the BC matrix, in which two distinct structural bands were created. The primary structure band resembled that seen in non-gelatin atomized cultures, with larger than natural BC pore sizes and low interstitial fiber counts. The secondary structure band more closely resembled the morphology of the static gelatin pellicles, with maximum average pore sizes of over 20 µm and increasingly thick distinct layer thickness. Overall, the result of this work examined the ability of BC to be fine-tuned for individual needs in industrial applications including the average pore size, productivity, water holding capacity, and mechanical properties. Additionally, our findings informs our understanding of interactions between intermittent top-feeding strategies and non-nutritional viscous additives.