copyright by R. Malcolm Brown, Jr. Department of Botany, The University of Texas at Austin, Austin, Texas 78713-7640
Cellulose is the earth's major biopolymer and is of tremendous economic importance globally. Cellulose is the major constituent of cotton (over 94%) and wood (over 50%). Together, cotton and wood are the major resources for all cellulose products such as paper, textiles, construction materials, cardboard, as well as such cellulose derivatives as cellophane, rayon, and cellulose acetate.
Traditionally, cellulose is harvested from plant resources. Bolls from cotton plants are collected, and the cotton fibers are detached from the seeds and processed into bales (see Proceedings, 1992 Cotton Fiber Cellulose: Structure, Function, & Utilization Conference). In Texas alone, the 1992 cotton crop produced $1.0 billion in cash receipts and had an economic impact of $4.0 billion (from the Crop Biotechnology Center Datasheet on Cotton, Texas A & M University). The 1985 global production of cotton was approximately 17,540,000 tons (see Textile Organon 57: 107, June 1986)
Wood timber is cut from the forest and sent to the sawmill for cutting and drying. Trees also are transported to the paper mill where the wood is shredded into chips which are then processed into a thick, watery pulp. This process requires energy and chemicals often harmful to the environment since unwanted lignin must be removed and the cellulose must be bleached. Pulp is made into paper and cardboard. In the United States alone, more than two million tons of newsprint and writing paper are produced each year from pulp (Moore, et al, 1995). The forest products industry in the United states is a $70. billion/year industry, not insignificant in relation to other major industries (from the Crop Biotechnology Center Datasheet on the Pine Biotechnology Program, Texas A & M University). In 1985, the world production of pulp reached 140 million metric tons (see Pulp and Paper, August, 1986, p 43)
With our increased population explosion and the quest to continue using cellulose crops from wood and cotton, more land is required to meet the global demand. This has a direct impact on the earth's carbon cycle. In this context, we need to understand cellulose in terms of the global carbon cycle as well as its use by humans. The carbon cycle on earth is a vast interplay between the carbon dioxide of the atmosphere and its sequestration or "fixation" via photosynthesis into organic products, among which cellulose is the most abundant macromolecule on earth. Over 10 11 tons are estimated to be synthesized and destroyed annually on earth (Brown, 1979). Cellulose can be thought of as a giant carbon "sink" because carbon incorporation into cellulose remains in the product for a rather lengthy time, sometimes for thousands of years. The global warming cycle affected by the emergence of the industrial age is based, in part, by the release of carbon dioxide into the atmosphere through the burning of wood and hydrocarbons. This emerging problem could have immense consequences if the carbon dioxide content continues to rise and trap heat from the sun's irradiation. Thus, one long term alternative is to reverse this conversion by increasing photosynthesis which results in the trapping of more carbon dioxide. Harvesting huge acreage of trees is neither contributing to the ecological management of the photosynthetic potential, nor maintaining a global sink for carbon dioxide.
SOURCES OF CELLULOSE
Cellulose from such major land plants as forest trees and cotton is assembled from glucose which is produced in the living plant cell from photosynthesis. These are macroscopic, multicellular photosynthetic plants with which we are all familiar. In the oceans, however, most cellulose is produced by unicellular plankton or algae using the same type of carbon dioxide fixation found in photosynthesis of land plants. In fact, it is believed that these organisms, the first in the vast food chain, represent Nature's largest resource for cellulose production. Without photosynthetic microbes, all animal life in the oceans would cease to exist.
Several animals, fungi, and bacteria can assemble cellulose
(Brown, 1979); however, these organisms are devoid of photosynthetic capacity
and usually require glucose or some organic substrate synthesized by a
photosynthetic organism to assemble their cellulose. Some bacteria can
utilize methane or sulfur substrates to produce glucose and other organic
substrates for cellulose.
ACETOBACTER XYLINUM-
A RICH SOURCE OF PURE
CELLULOSE
Among the bacteria, one of the most advanced types of purple bacteria is the common vinegar bacterium, Acetobacter. This non-photosynthetic organism can procure glucose, sugar, glycerol, or other organic substrates and convert them into pure cellulose (Brown, et al, 1976). Acetobacter xylinum is Nature's most prolific cellulose-producing bacterium. A typical single cell can convert up to 108 glucose molecules per hour into cellulose. Consider that as many as a million cells can be packed into a large liquid droplet, and if each one of these "factories" can convert up to 108 glucose molecules per hour into cellulose, the product should virtually be made before one's eyes. A single cell of Acetobacter has a linear row of pores from which glucan chain polymer aggregates are spun ( Figure 1). As many as one hundred of these pores can produce a composite cable of glucan polymers resulting in a ribbon. Time lapse analysis of individual Acetobacter cells assembling cellulose ribbons reveals a myriad of activities, each cell acting as a nano-spinneret, producing a bundle of sub-microscopic fibrils. Together, the entangled mesh of these fibrils produces a gelatinous membrane known as a pellicle. This membrane of pure cellulose, and cells entrapped within it can be cleaned and dried and the product used for many exciting new applications. One of the unique features of this pure cellulose membrane is that it is very strong in the never dried state, and it can hold hundreds of times its weight in water. This great absorbtivity and strength constitute two of the many novel features of microbial cellulose (Brown, 1989; Brown, 1992; Brown, 1994; White and Brown, 1989).
UNIQUE MICROBIAL CELLULOSE FEATURES AND PRODUCTS
The distinguishing features of microbial cellulose are shown in Table 1. Because the microbial cellulose ribbons are "spun" into the culture medium, membranes and shaped objects can be produced directly during the fermentation process, thus enabling a novel array of non-woven products. Direct dyes can be added during synthesis to alter the cellulose produced. Because of the novel features of microbial cellulose, a variety of product applications of microbial cellulose is possible. These are summarized in Table 2.
CURRENT STATUS OF
INDUSTRIAL DEVELOPMENT OF
MICROBIAL CELLULOSE
Relatively few industries using microbial cellulose are organized; however, as the product becomes more well known and its properties are further exploited, this undoubtedly will change. A few representative product areas and potential products are described below:
Healthfood Industries - Nata de Coco
The Philippine desert, Nata de Coco, has been a cottage industry for the past seventy years in the Philippines (Lapuz, et al, 1969). After WW II, the country began exporting Nata, and in fact, the product can be found in almost any of the thousands of oriental catered stores in the US. The import of Nata from the Philippines into Japan has had a major impact on the global outlook for expansion of microbial cellulose production. In 1992, a fad originated in Japan with the introduction of microbial cellulose into diet drinks enjoyed by young girls. Nata earned the distinction as among the 30 best hit products among Japanese consumers for 1993. Since this time, the export of Nata from the Philippines has risen from approximately $1 million per year to more than $26 million per year as of 1993 (see Philippine Daily Inquirer "Agriculture" Vol. 20. March 3, 1994). In fact, the cottage industry in the Philippines cannot meet the demand for export, so great interest has recently focused on ways and means to increase Nata production.
When visiting Japan in October, 1994, I enjoyed Nata which was part of a fruit cocktail encore for breakfast at a local Japanese Inn. This use suggests that the product is beginning to secure favored recognition by the adult population in Japan. In the US, the general population is not so positive about Nata let alone lacking knowledge in general, about this food product. Thus, in the US, prospects are tremendous for introduction of new food products with microbial cellulose if market niches and advertising are fully exploited. The unique gel-like properties of microbial cellulose, combined with its complete indigestibility in the human intestinal tract, makes this an attractive food base.
Healthfood Industries - Kombucha Elixir or Manchurian Tea
Kombucha is a fermented product which is consumed by a
growing number of individuals for improved health needs. A recent story
on Kombucha aired on CNN News. Acetobacter along with yeasts are
cultured in a medium containing tea extract and sugar. The cellulose is
not consumed in this product. The fermentation extract is utilized. There
is growing interest in using the fermented extract for health improvement;
however, scientific scrutiny of reported health benefits has not been reported
to date. For information on this rather vast subject, consult the following
Internet address:
http://www.webcom.com/~sease/kombucha/roche.html.
Audio Components
Sony Corporation, in conjunction with Ajinomoto developed the first audio speaker diaphragms using microbial cellulose (see US Patent 4,742,164). The first headphones were very expensive, over $3,000 for one pair! The price recently has dropped, and the microbial cellulose headphones are available worldwide as an upper end audio product. The unique dimensional stability of microbial cellulose gives rise to a sound transducing membrane which maintains high sonic velocity over a wide frequency ranges, thus being the best material to meet the rigid requirements for optimal sound transduction. On the horizon, it is expected that larger speaker diaphragms will be made of microbial cellulose.
Wound Care Products
In the early 1980's Johnson & Johnson pioneered in exploratory investigations on the use of microbial cellulose as a liquid loaded pad for wound care (see US Patent 4,655,758; 4,588,400 ). Since that time, a company in Brazil, Biolfill Industias, has continued to investigate the properties of microbial cellulose and is beginning to market specific microbial cellulose products in the wound care market (Fontana et al, 1990, 1991). In our lab, we have developed a process to produce molded objects of microbial cellulose directly during the fermentation process. This process could provide non-woven, shaped objects in medicine such as artificial arteries, vessels, skin, etc. (see EP 186495). Certainly the unique absorptive properties of microbial cellulose would suggest a large potential market in wound care and drug delivery.
Paper and Paper Products
Microbial cellulose has been investigated as a binder in papers, and because of it consists of extremely small clusters of cellulose microfibrils, this property greatly adds to strength and durability of pulp when integrated into paper. Ajinomoto Co. along with Mitsubishi Paper Mills in Japan are currently active in developing microbial cellulose for paper products (see patent JP 63295793).
THE FUTURE - WHAT DOES IT
HOLD FOR MICROBIAL
CELLULOSE?
It seems clear from this brief presentation of product diversity from microbial cellulose that this source of Nature's abundant biopolymer would have been used much earlier; however, the drawbacks have centered on understanding the biosynthetic process itself, then trying to optimize the fermentation process leading to more cells and cellulose biosynthesis.
Fermentation Optimization
Several routes to fermentation are available. The first is a "deep tank" fermentation which is very similar to large scale industrial fermentor now in operation. In the United States, Weyerhaeuser spent a number of years developing microbial cellulose production in such fermentors; however, their commercial product Cellulon never really obtained commercial success because cost reductions to make this product competitive with other sources of cellulose never were realized. Approximately two years ago, Weyerhaeuser advertised to sell its Cellulon business, this being indicative of the perils and pitfalls for entering into such a business without sufficient fundamental R & D to provide a basis for optimal and economical production. The Cellulon business was sold to Kelco, Inc. a company experienced in large scale fermentation of bacterial products. Kelco is perhaps best known for its commercial production of the bacterial polymer. xanthan gum, used as a food thickener. Recently Kelco, the subsidiary of Merck, was purchased by Monsanto, an agriculturally based company. Thus, with Monsanto's renewed interest in the biopolymer field, an opportunity for expanding microbial cellulose production now exists in the U.S; however, significant gains will be necessary in fermentation upgrading using the deep tank methodology.
The static culture method, on the other hand, is more likely to succeed because microbial cellulose has been produced this way for many years in the Nata industry, and the low shear forces in static culture promote higher productivity. Because microbial cellulose is an extracellular products which is excreted into the culture medium, special care and handling is necessary to maintain optimal production. The cellulose membrane itself can become a barrier for substrates and oxygen necessary for the cells to produce cellulose. Thus, in our laboratory during the past 5 years, novel fermentation approaches have been developed to overcome some of the intrinsic difficulties for mass culture of Acetobacter. Furthermore, a vigorous program of bacterial strain selection from regions all over the world has provided a stock resource of stable, efficient cellulose-producing strains. The recent success with cloning and sequencing the genes for bacterial cellulose synthesis (Saxena and Brown, 1990, 1991) combined with new information on how these genes may function in vivo (Saxena, et al, 1994) gives new input on further optimization.
What is needed at the present is development of intermediate fermentation scale-up so that the conversion to an efficient large scale fermentation technology will truly deliver microbial cellulose to the market at a competitive cost. However, when large scale fermentation does become a reality, the future economies of scale could lead to a major new industry as a new source for an important biopolymer which has been used for centuries.
CONCLUSIONS
In the era of declining forests, global climate changes, continuing expansion of industrialization, it is reasonable to consider the consequences of an alternative source of cellulose. Thus, we return to our original idea of what if's and other questions in the prediction of scenarios for the future of native biopolymers. The process for success depend on education--the public needs to become better educated on all aspects of cellulose, ranging from its fundamental biosynthesis, structural properties, to the biotechnology applications. Education and communication among professionals also is necessary. It takes an international collaboration of scientists from many fields, including botany, microbiology, chemical engineering, forestry, mechanical engineering, polymer science, textiles, food products, marketing, economics, and business to fully realize the merits of a new resource for a widely used product. This is an excellent opportunity to take advantage of new development since the intense interest in Nata and other microbial cellulose products now is fueled by the demand for the product. It is ironical that demand now outpaces the supply for microbial cellulose, largely because of lack of investment in fermentation R & D to optimize microbial cellulose production on a large scale.
Can microbial cellulose compete with traditional cellulose sources? This question remains unanswered until commercial scale up and fermentation development become mature. In the meantime, specialty products could be economically produced from microbial cellulose. Perhaps the evolution of microbial cellulose into mature consumer products would greatly affect present sources of cellulose. Whether this comes to pass will be understood only in terms of supply and demand for the unique properties of the product. Perhaps this brief excursion into microbial cellulose will provide a stimulus for the renewal of interest in combining traditional cellulose technology with biotechnology development to create growth in a new, exciting field.
REFERENCES
To see complete abstracts of papers from the RMB lab click here
Brown, R.M., Jr., J.H.M. Willison, and C.L. Richardson. 1976. Cellulose biosynthesis in Acetobacter xylinum: 1. Visualization of the site of synthesis and direct measurement of the in vivo process. Proc. Nat. Acad. Sci. U.S.A. 73(12):4565-4569.
Brown, Jr. R. M. 1979. Biogenesis of natural polymer systems, with special reference to cellulose assembly and deposition. IN: Proceedings of the Third Phillip Morris U.S.A. Operations Center. Richmond, Virginia, November 1978. pp. 50-123.
Brown, R.M., Jr. 1989. Bacterial cellulose. In: Cellulose:Structural and Functional Aspects, Ed Kennedy, Phillips, & Williams. Ellis Horwood Ltd. pp 145-151.
Brown, Jr. R. M. 1989. Advances in Cellulose Biosynthesis. IN: Assessment of Biobased Materials Ed. H.C. Chum. SERI/TR-234-3610. U.S. Department of Energy. pp 9-1 to 9-7.
Brown, Jr. R. M. 1992. Emerging technologies and future prospects for industrialization of microbially derived cellulose. In: Harnessing Biotechnology for the 21st Century Ed. M.R Ladisch and A Bose. Proceedings of the Ninth International Biotechnology Symposium and Exposition. Crystal City, Virginia. American Chemical Society, Washington, D.C. pp 76-79.
Brown, Jr. R. M. 1994. Understanding nature's preference for cellulose I assembly: Toward a new biotechnology era for cellulose. Proc. Inter.Symp. Fiber Sci. and Tech. p437-239. Publishers. The Society of Fiber Science and Technology, Japan
Cousins, S.K. and R. M. Brown, Jr. 1995. Cellulose I microfibril assembly: computational molecular mechanics energy analysis favors bonding by van der Waals forces as the initial step in crystallization. Polymer. In Press
Fontana, J.D., A. M. deSouza, C.K.Fontana, I.L. Toriani, J.C. Moreschi, B.J. Gallotti, S.J. de Souza, G.P. Narcisco, J.A. Bichara, and L.F.X. Farah. Acetobacter cellulose pellicle as a temporary skin substitute. 1990. Appl. Biochem. & Biotechnology 24/25: 253-264.
Fontana, J.D., V.C. Franco, S.J. deSouza, I.N. Lyra, and A. M de Souza. 1991. Nature of plant stimulators in the production of Acetobacter xylinum ("Tea Fungus") biofilm used in skin therapy. Appl. Biochem. & Biotechnology 24/25: 341-351.
Lapuz, M.M., E.G. Gallardo, and M. A. Palo, 1969. The nata organism-cultural requirements, characteristics, and identity. The Philippine Journal of Science 96: 91-109
Moore, Randy, Clark W. Dennis, and Kingsley R. Stern. 1995Botany, First Edition. Wm. C. Brown Publishers, Dubquie, Iowa. 824pp.
Saxena, I. M., F. C. Lin, and R. M. Brown, Jr. 1990. Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum. Plant Molecular Biology 15:673-683.
Saxena, I. M. , F. C. Lin, and R. M. Brown, Jr. l99l. Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum. Plant Molecular Biology 169:947-954.
Saxena,. I.M., K.Kudlicka, K. Okuda, and R. M. Brown, Jr. 1994. Characterization of genes in the cellulose synthesizing operon (acs operon) of Acetobacter xylinum: Implications for cellulose crystallization. J. Bacteriology 176: 5735-5752.
White, D.G. and R.M. Brown, Jr. 1989. Prospects for the
commercialization of the biosynthesis of microbial cellulose. In: Cellulose
and Wood - Chemistry and Technology,
Ed. C. Schuerch. John Wiley and Sons, Inc. N.Y., 573-590.
Purity
- cellulose is the only biopolymer synthesized
- absence of lignin or hemicelluloses
- completely biodegradable and recyclable ó a renewable resource
Great Mechanical Strength
- high strength crystalline cellulose I
- consistent dimensional stability
- high tensile strength
- light weight
- remarkable durability
Extraordinary Absorbency in the Hydrated State
- remarkable capacity to hold water
- selective porosity
- high wet strength
- high surface-to-volume carrier capacity
Direct Membrane Assembly During Biosynthesis
- intermediate steps of paper formation from pulp unnecessary
- intermediate steps of textile assembly from yarn unnecessary
- extremely thin, sub-micron, optically clear membranes can be assembled
Cellulose Orientation During Synthesis
- dynamic fiber forming capabilities
- uniaxially strengthened membranes
Direct Modification of Cellulose During Assembly
- delayed crystallization by introduction of dyes into culture medium
- control of physical properties of the cellulose during assembly (molecular weight and crystallinity)
Genetic Modification of Cellulose Product
- direct synthesis of cellulose derivatives (such as cellulose acetate, carboxymethylcellulose, methyl cellulose, etc.)
- control of cellulose crystalline allomorph (cellulose I or cellulose II)
- control of molecular weight of cellulose
Industry Products
Food
Desserts (Nata de Coco, low calorie ice creams chips, snacks, candies)
Thickeners (ice cream and salad dressing)
Weight reduction base
Base for artificial meat
Sausage and meat casings
Serum cholesterol reduction (see US Patent 4,960,763)
Kombucha elixir or Manchurian tea
Healthcare
Wound care dressings (see patent EP 323717)
Drug delivery agents, either oral or dermal
Artificial skin substrate
Cosmetics and Beauty
Skin creams
Astringents
Base for artificial nails
Thickener and strengthener for fingernail polish
Environmental
Oil spill cleanup sponge
Absorptive base for toxic material removal
Petroleum and Mining
Mineral and oil recovery ( see US Patents 5,011,596 and 5,009,797)
Clothing and Shoe
Artificial leather products
One piece textiles
Outdoor Sports
Disposable tents and camping gear
Public Utilities
Water purification via ultrafilters and reverse osmosis membranes (see patent by Nakano Sumise JP 3032726)
Babycare Products
Disposable recyclable diapers
Audio Products
Superior audio speaker diaphragms
Forest Products
Artificial wood strengthener (plywood laminates)
Filler for paper
High strength containers
Specialty Papers
Archival document repair
Paper base for long-lived currency
Automotive and Aircraft
Car bodies
Airplane structural elements
Rocket casings for deep space missions
2% (w/v) glucose
0.5% (w/v) peptone (Difco Bactopeptone)
0.5% (w/v) yeast extract (Difco)
0.27% anhydrous disodium phosphate
0.15% (w/v) citric acid monohydrate
Adjust the pH to around 5.0
using 1 N acetic acid.
Solidify using Difco Bacto Agar (1.5%) and autoclave for 15 min
at 15 lb/square inch pressure. Let the agar cool but
not too much and pour into sterile disposable plastic Petri dishes
(100 mm) while still warm and molten. After
solidifaction, seal the dishes using Parafilm strips and store
upside down to prevent condensation on the agar surface.
Store in the refrigerator
until ready for use. After streaking with the field sample, incubate
at 25-30 degrees C for about 3-4 days until colonies appear. Then pick
individual colonies and transfer into the liquid Schramm and Hestrin medium
in tubes. Dispose of the used plates by autoclaving. Be forewarned to be
careful of any pathogens which may also grow in this medium. We are not
responsible for any misuse of this information.