Acetobacter xylinum: An Inquiry into Cellulose Biosynthesis

Cellulose is the most abundant natural polymer on Earth. It is the major constituent of higher plants, and cellulose fibers have important industrial applications including the clothing that we wear and even the paper upon which this article is written. Cellulose forms the cell wall of eukaryotic plants, algae and fungi, and is synthesized by a variety of bacteria, including Acetobacter xylinum. A. xylinum, a Gramnegative bacterium found in the soil, can frequently be isolated from decaying fruit such as apples that have fallen to the ground. In addition to serving as a model organism for the study of cellulose synthesis, A. xylinum can serve as an interesting and instructive organism in the classroom to help students understand the process of science. The major focus of this article will be upon how students can experiment with A. xylinum using an inquiry approach to their learning. Acetobacter xylinum is an aerobic soil bacterium in the family of bacteria that ferments carbohydrates to vinegar (A. aceti). A. xylinum is an unusual member of this family because it synthesizes and extrudes fibrils of cellulose as part of the metabolism of glucose. The glucose subunits that form the cellulose microfibril are extruded through pores in the cell wall of the bacteria. In standing laboratory culture of the bacteria, the cellulose fibrils bundle together to form a mat or pellicle within which the bacteria are held. The pellicle floats on the surface of the medium allowing the bacteria to obtain plenty of oxygen, which they require for growth, multiplication, and more cellulose synthesis. Cellulose biosynthesis by A. xylinum may serve the bacterium in a number of ways in their microenvironment. These may include holding the bacteria at the air interface to ensure adequate oxygen, protection from ultraviolet radiation, protection from desiccation by holding water, acting as a biofilm to hold the organisms on substrates that they may be decomposing, and/or possibly providing protection from predators or competitors. Cellulose synthesis occurs as a multi-step series of chemical reactions beginning with glucose that is catalyzed by enzymes. The pathway is believed to be as shown in Figure 1. Cellulose is considered to be a secondary metabolite, which the organism synthesizes when major nutritional needs are met. There is genetic and molecular evidence that cellulose synthesis in A. xylinum is under operon control. Some practical applications of bacterial cellulose include: sensitive diaphragms for stereo headphones, thickeners for foods, paint, ink and adhesives. It has also been used as short term protection for burned skin prior to skin grafting. Most commercial applications of cellulose produced by Acetobacter have employed static batch culture to produce sheets of cellulose that can be sterilized to kill the bacteria and washed to remove microbial waste product contaminants. One large scale commercial application is the production of bacterial cellulose in a nonaseptic medium of sugar-enriched coconut milk in the Philippines, yielding an edible product called "nata de coco." This food product has become popular in Japan as a high-fiber, low-calorie food. Recently, Japanese investigators have developed a technique to harvest Acetobacter cellulose from shallow culture pans with a windup roller device to pull the cellulose slowly from the surface of the culture medium. High yield commercial production of bacterial cellulose will require agitated (shaking) culture techniques. There has been some limited success in the laboratory studies producing cellulose in this way, but, at present, there are no commercial applications of this process.