BRIGHTNESS PROPERTIES

This application is a continuation-in- part of U.S. Patent Application No. 07/513,350, filed April 20, 1990.

Technical Field

The present invention relates to bacterial cellulose having enhanced brightness properties and methods of producing bacterial cellulose having enhanced brightness in commercial scale quantities.

Background of the Invention It is well known that cellulose can be synthesized by certain bacteria, particularly those of the genus Acetobacter. Bacterial cellulose has been produced, for example, under static cell culture conditions and has been used in a variety of applications. Bacterial cellulose produced employing agitated cell culture techniques has also been developed and is useful for many purposes. Cellulose fibers produced by micro¬ organisms, although chemically similar to cellulose produced from wood pulp, differ from such plant-derived cellulose in a number of important respects. An important difference is that cellulose fibers produced by capable strains of Acetobacter under agitated cell culture conditions are about two

orders of magnitude narrower than the cellulose fibers typically produced by pulping birch or pine wood. The small cross-sectional size of this bacterial cellulose and the concomitantly greater cross-sectional surface area provide important advantages in a variety of applications. Microbial cellulose produced under agitated cell culture conditions exhibits a highly reticulated "network" or "web" structure. The properties of highly reticulated bacterial cellulose produced using agitated cell culture conditions, as well as suitable bacterial strains and cell culture conditions are disclosed in European Patent Application 0 228 779, published July 15, 1987, which is incorporated herein by reference in its entirety. Moreover, sheets may be formed from reticulated bacterial cellulose as described in United States Patent No. 4,863,565, entitled "Sheeted Products Formed From Reticulated Microbial Cellulose".

Discoloration of bacterial cellulose may be problematic when the bacterial cellulose is produced by processes such as those discussed above in commercial scale quantities. The quality of bacterial cellulose and products incorporating bacterial cellulose, as measured principally by consumer desirability, is directly impacted by the color characteristics of the bacterial cellulose. Bacterial cellulose produced in laboratory scale quantities typically does not suffer from problems associated with color impurities, because relatively expensive cell culture media components having low levels of colored impurities can be selected and used in relatively low volume laboratory scale cultures. The economics of industrial scale fermentation, however, can be significantly affected

by the cost of cell culture media components and it is not always economically feasible to use laboratory scale media.

Discoloration results principally from the use of certain culture media components during industrial scale fermentation processes. After fermentation is complete, the fermentation broth is treated with alkali at elevated temperatures (about 60°C) to digest the bacterial cells and permit harvesting of the cellulose. The alkali interacts with certain components of the fermentation media to produce colored impurities. Extensive washing procedures employed to separate alkali from the cellulose typically do not remove colored impurities to an extent sufficient for many commercial applications of the cellulose.

Unfortunately for commercial scale-up purposes, the culture media components which interact with the hot caustic digestion agent to produce colored impurities in the bacterial cellulose are those which are the most economically advantageous. It would be desirable to employ these cost-effective cell culture media components, such as corn steep liquor and yeast extract, and yet produce bacterial cellulose which is free from or has a substantially reduced level of colored impurities and exhibits a satisfactory level of brightness.

Discoloration concerns sometimes arise with regard to cellulose products made from wood pulp, and bleaching techniques have been employed to reduce the level of colored impurities in wood pulp. Colored impurities in wood pulp fibers arise from lignin and lignin-derived materials and carbohydrate degradation products generated during the pulping process. Removal of colored impurities from wood

pulp requires separation of lignin, resins and other extraneous constituents of wood from the cellulose. The degradation of cellulose during pulp bleaching procedures is well documented. Removal of colored impurities from wood pulp and bacterial cellulose would be expected to require entirely different methodologies because the source and nature of the colored impurities are different. Moreover, the physical properties of wood cellulose and bacterial cellulose are different in a number of important respects. Degradation of microbial cellulose during the course of many conventional bleaching procedures would be expected to be substantial and result in an unacceptable reduction in fiber integrity and strength.

Summary of the Invention The present invention is directed to bacterial cellulose having enhanced brightness properties, i.e.. reduced levels of colored impurities, and methods of treating bacterial cellulose to reduce the level of colored impurities. Bacterial cellulose is derived from microbial fermentation processes and subsequently treated to increase its brightness. Preferred embodiments of the present invention contemplate improving the color characteristics of bacterial cellulose produced under agitated culture conditions and characterized by a substantially continuous, reticulated network of fine fiber strands. Treatment according to methods of the present invention includes processing with a brightening agent to reduce color impurities. Sodium hypochlorite and hydrogen peroxide are especially preferred brightening agents. Shear mixing before or after treatment with a brightening agent facilitates removal of colored impurities and

results in a substantial improvement in brightness. Treatment according to methods of the present invention may also reduce the level of nitrogen impurities in bacterial cellulose products. Improvements in brightness of the microbially produced cellulose are achieved utilizing methods of the present invention without impairing important physical characteristics of the bacterial cellulose. For example, properties such as the tensile strength and structural integrity of microbial cellulose are not substantially altered during treatment with the brightening agent and may, in fact, be improved utilizing methods of the present invention. Methods of the present invention moreover may be conveniently adapted in commercial scale bacterial cellulose production processes. Detailed Description of the Invention The present invention provides microbially produced cellulose having reduced levels of colored impurities that is suitable for use in a variety of applications. Bacterial cellulose produced by microorganisms capable of producing cellulose, such as those of the genus Acetobacter. Pseudomonas. Aσrobacterium. and the like may be treated according to methods of the present invention. The term

"bacterial cellulose," as used in the specification and claims herein, refers to cellulose produced by such microorganisms using fermentation techniques. Bacterial cellulose is typically separated from spent fermentation medium and bacterial cells by extraction with a basic solution. Bacterial cellulose is treated with a brightening agent, according to methods of the present invention, to reduce the level of colored impurities and thereby increase its brightness.

Colored impurities in bacterial cellulose generally result from utilization of certain cell culture media components, such as corn steep liquor, yeast extract and similar sources of nitrogen, amino acids, minerals and vitamins. Such culture media components are especially preferred for use in industrial scale cell culture processes due to their relatively low cost, good growth characteristics, and ready availability within the industry. Chemical interactions between the alkali processing agent added to the media to lyse the bacterial cells and components of the growth media such as glucose, corn steep liquor, yeast extract, and similar constituents, generate colored impurities in bacterial cellulose. Even after repeated washing procedures to remove the alkali from the microbial cellulose, colored impurities may render the cellulose unsuitable for many applications. Additionally, nitrogen impurities may be present in bacterially produced cellulose and may or may not be directly associated with colored impurities. Nitrogen impurities typically include amino compounds that are sensitive to slow air oxidation, resulting in the production of undesirable odors and colors. The bacterial cells and the cell culture media constitute sources of nitrogen impurities.

In purifying bacterial cellulose for a variety of end uses, it is desirable to reduce the level of both color impurities and nitrogen impurities. Treatment using the brightening agents of the present invention reduces the level of nitrogen as well as color impurities, without altering important physical properties of the microbial cellulose. Processing protocols employing

a shear mixing step following or preceding treatment of bacterial cellulose with a brightening agent resulted in further improvements in brightness and reduction of nitrogen-containing impurities. Growth and harvesting of bacterial cellulose produced under static conditions may be accomplished as described in Methods in Carbohydrate Chemistry. Volume III - Cellulose. R.L. Whistler, Ed., Chapter 2, Academic Press New York (1963). In preferred embodiments of the methods of the present invention, microbial cell growth is conducted under agitated culture conditions and, as a result, the microbially produced cellulose is characterized by a substantially continuous, reticulated network of fiber strands. Suitable bacterial strains and cell culture conditions are disclosed in European Patent Application No. 0 228 779.

Bacterial cellulose according to a preferred embodiment of the present invention may be produced from a strain of Acetobacter aceti var. xylinum grown as a subculture of ATCC Accession No. 53524, deposited July 25, 1986 under the terms of the Budapest Treaty. The bacteria may be cultured under conditions similar to those described below. The base medium used for cell culture is referred to as R 70-3 medium. Suitable R 70-3 medium comprises:

Carbon source As later specified (usually glucose 2% or 4%, w/v)

Corn steep liquor As later specified (supernatant (usually 1% to 4%, fraction after v/v) centrifugation)

Buffer

3,3 Dimethylglutaric 25 mM acid (DMG)

The final pH of the medium is 5.0, ± 0.2.

A suitable vitamin mix may be formulated as follows:

Ingredient Cone. (Mg/L)

Inositol 200

Niacin 40 Pyridoxine HC1 40

Thiamine HC1 40

Ca Pantothenate 40

Riboflavin 20 p-Aminobenzoic acid 20 Folic acid 0.2

Biotin 0.2

The carbon source generally comprises monosaccharides or mixtures thereof, such as glucose and fructose, disaccharides such as sucrose, and mixtures of mono- and disaccharides. The carbon source, typically glucose, is generally provided in concentrations of about 0.5% to about 7.0% (w/v) , and preferably about 2.0%-4.0% (w/v).

Corn steep liquor, yeast extract, casein hydrolysate, ammonium salts or other nitrogen-rich substances may be used as a general source of nitrogen, amino acids, minerals and vitamins. Corn steep liquor is preferred, and suitable concentra¬ tions thereof range from about 0.1% to about 10% (v/v) . Cell culture media comprising about 5% (v/v) corn steep liquor is preferred for shaking flask cultures. In fermenters, an initial concentration of corn steep liquor may be supplemented during the fermentation run with additional aliquots of corn steep liquor. Yeast extract may be employed in place of corn steep liquor as an additive to the culture medium. Yeast extract in a quantity of about 1% (v/v) is suitable and may be obtained from Universal Foods, Milwaukee, WI, under the tradename Amberex 1003.

Corn steep liquor varies in composition, depending upon the supplier and mode of treatment.

A product obtained as Lot E804 from Corn Products

Unit, CPC North America, Stockton, California, may be considered typical and has a pH of about 4.5 and the following composition:

Major Component

Solids

Crude protein Fat

Crude fiber

Ash

Calcium

Phosphorous Nitrogen-free extract

Non-protein nitrogen

NaCl

Potassium

Reducing sugars (as dextrose)

Starch 1.6

Bacteria were first multiplied as a pre-seed culture using R 70-3 medium with 4% (w/v) glucose as the carbon source and 5% (w/v) corn steep liquor. Cultures were grown in 100 L of the medium in a 750 mL Falcon No. 3028 tissue culture flask at 30°C for 48 hours. The entire contents of the culture flask were blended and used to make a 5% (v/v) inoculum of the seed culture. Preseeds were streaked on culture plates to monitor for homogeneity and contamination.

Seed cultures were grown in 400 mL of the above-described culture medium in 2 L baffled flasks in a reciprocal shaker at 125 rpm at 30 ^β C for two days. Seed cultures were blended and streaked as

before to check for contamination before further use.

Bacterial cellulose was initially made in a continuously stirred 14 L Chemap fermenter using a 12 L culture volume inoculated with 5% (v/v) of the seed cultures. An initial glucose concentration of 32 g/L in the medium was supplemented during the 72-hour fermenter run with an additional 143 g/L added intermittently during the run. In similar fashion, the initial 2% (v/v) corn steep liquor concentration was augmented by the addition of an amount equivalent to 2% by volume of the initial volume at 32 hours and 59 hours. Cellulose concentration reached about 12.7 g/L during the fermentation. Throughout the fermentation, dissolved oxygen concentration was maintained at about 30% air saturation.

Following fermentation, cellulose was allowed to settle, and the supernatant liquid was poured off. The remaining cellulose was washed with deionized water and then extracted with 0.5 M NaOH solution at 60°C for two hours. After extraction, the cellulose was again washed with deionized water to remove residual alkali and bacterial cells. More recent experimental studies have shown that a 0.1 M NaOH solution is entirely adequate for the extraction step. The purified microbially produced cellulose was maintained in wet condition for further use. This material was readily dispersible in water to form a uniform slurry. Bacterial cellulose for the later samples was made in 250 L and 6000 L fermenters.

The bacterial cellulose produced under stirred or agitated conditions, as described above, has a microstructure quite different from that of bacterial cellulose produced in conventional static

cultures. It is a reticulated product formed by a substantially continuous network of branching, interconnected cellulose fibers. The bacterial cellulose prepared as described above by agitated fermentation has filament widths much smaller than softwood pulp fibers or cotton fibers. Typically, these filaments are about 0.1 to 0.2 microns in width with indefinite length due to the continuous network structure. A softwood fiber averages about 30 microns in width and 2 to 5 mm in length, while a cotton fiber is about half this width and about 25 mm long.

According to preferred embodiments of the present invention, cellulose-producing micro- organisms of the genus Acetobacter are cultured under agitated conditions to produce bacterial cellulose characterized by a substantially continuous, reticulated network of fiber strands. Exemplary of such cellulose-producing species are Acetobacter aceti subsp. xylinum and Acetobacter pasteurianus. Characteristics of cellulose- producing bacteria and preferred growth and agitated culture conditions are fully described in U.S. Patent No. 4,863,565, entitled "Sheeted Products Formed From Reticulated Microbial Cellulose", which is herein incorporated by reference in its entirety.

Reducing the level of colored impurities in microbially produced cellulose is accomplished according to methods of the present invention by treatment of the cellulose with a brightening agent. A brightening agent, as contemplated by the present invention, encompasses any substance capable of significantly reducing the level of colored impurities in microbially produced cellulose by contact therewith and thereby imparting a significant brightening effect, without damaging or

substantially altering important properties of the cellulose. A significant brightening effect (i.e. , reduction in the level of colored impurities) is observed as a result of a two to three point brightness increase, as measured by the Technical Association of the Pulp and Paper Industry (TAPPI) testing method designated T525-OM-86.

Brightening agents of the present invention encompass both oxidative and reductive chemical compounds, as well as mixtures thereof. Oxidative brightening agents, such as ozone or chlorine dioxide, increase the brightness of the bacterial cellulose via oxidation of conjugated chromophore functional groups, for example. Reductive brightening agents, such as sodium bisulphite, increase the brightness of the bacterial cellulose via reduction of chromophores containing carbonyl functional groups, for example. Exemplary brightening agents of the present invention include hydrogen peroxide, sulfur dioxide and sulfur dioxide enriched water, sodium hypochlorite, sodium borohydride, sodium dithionite, sodium bisulfite, sodium sulfite, chlorine dioxide, ozone and the like, or combinations thereof. Sulfur dioxide in admixture with water results in the formation of bisulfites, which are also brightening agents useful in accordance with the present invention. Sodium hypochlorite, hydrogen peroxide and sulfur dioxide are preferred brightening agents. Treatment of microbially produced cellulose to increase the brightness thereof requires that the brightening agent be contacted with the microbially produced cellulose. A preferred and commercially viable contacting method in accordance with the present invention is the bubbling of a brightening agent through a dilute

suspension of bacterial cellulose. Sulfur dioxide, for example, is a gaseous brightening agent which may be brought into contact with bacterial cellulose in this manner to allow the brightening agent to brighten the bacterial cellulose. Any other method sufficient to provide contact of a gaseous or non-gaseous brightening agent with the cellulose, i.e. simply admixing the two components with agitation, can be utilized according to methods of the present invention.

An "effective amount" of brightening agent must be contacted with the cellulose to provide a significant brightening effect. Typically, an effective amount may be expected to range from about 0.05% to about 20% (w/w) brightening agent, based upon the dry weight of microbially produced cellulose, with from about 0.25% to about 5% being preferred. Preferred brightening agents such as sodium hypochlorite and hydrogen peroxide are effective at surprisingly low concentrations of about 2.0% or less (w/w, grams brightening agent per 100 grams bacterial cellulose) .

To achieve a significant brightening effect, bacterial cellulose is preferably incubated with a brightening agent or agents for a period of time under appropriate reaction conditions. Appropriate reaction conditions vary depending upon the brightening agent employed and are described more fully in the examples included herein. Typical exposure times of bacterial cellulose to a brightening agent or agents are generally from about 5 minutes to about 5 hours, with about 10 minutes to 3 hours being preferred. For brightening agents of the oxidative type, such as hydrogen peroxide, sodium hypochlorite and chlorine dioxide, the brightening reaction is preferably carried out at

elevated temperatures. Preferred ranges of elevated temperatures are generally from about 40"C to about 75°C. For brightening agents of the reductive type, such as sulfur dioxide enriched water, the brightening reaction is preferably carried out at ambient temperature.

Mild sodium hypochlorite treatment of bacterial cellulose at concentrations of about 2.0% (w/w) or less sodium hypochlorite in a cellulose/ water slurry is a preferred treatment methodology. Treatment of bacterial cellulose with hydrogen peroxide under alkaline conditions is likewise a preferred treatment protocol. Shearing of the bacterial cellulose prior or subsequent to treatment with a brightening agent results in a substantial improvement in brightness properties and also results in a substantial reduction of nitrogen impurities in the bacterial cellulose product.

The following examples set forth specific brightening agents and methods for their use to enhance the brightness properties (i.e. , reduce the level of color impurities) of microbially produced cellulose, for the purpose of more fully understand¬ ing preferred embodiments of the present invention, and are not intended to limit the invention in any way.

EXAMPLE I Colored Impurity Removal Using Hydrogen Peroxide Five (5) grams of cellulose (o.d., oven dry basis) , produced under agitated culture conditions as described above, was admixed with deionized water in a 2 L conical flask to form a cellulose/water slurry. Hydrogen peroxide was added, and the consistency of the slurry was adjusted to 1.0% (1 gram bacterial cellulose in

100 grams total weight of mixture) . The amount of hydrogen peroxide in the slurry was 2.0% (w/w) hydrogen peroxide/dry weight of bacterial cellulose. Sodium hydroxide was added in an amount equal to 1% of the bacterial cellulose by weight. The conical flask was maintained in a water bath having a temperature of 55°C for 1 hour and agitated by shaking. The treated slurry was then diluted with deionized water to 2 L, and the pH was adjusted to neutral. The cellulose suspension can be filtered at this time, if desired.

Four hundred eighty (480) ml of the neutralized cellulose suspension was processed in a Technical Association of the Pulp and Paper Industry (TAPPI) sheet making apparatus to form a 1.2 gm (o.d. basis - approximately 60 g/m ² ) sheet in accordance with TAPPI procedure number T220 om-83. The sheet was lifted from the apparatus wire using Whatman 541 filter paper backed by a blotter. Excess water was removed by placing the cellulose sheet over another filter paper/blotter assembly and gently hand pressing the cellulose. The hand- pressed cellulose sheet was then placed between two sheets of filter paper and was dried over a drum dryer, such as that available from Noble & Wood.

The filter paper was then removed from the cellulose sheet, and handsheet testing was conducted as described in Example V.

EXAMPLE II

Colored Impurity Removal Using Sodium Hypochlorite Five (5) grams of cellulose (o.d. basis) , produced under agitated culture conditions as described above, was admixed with deionized water in a 2 L conical flask to form a cellulose/water slurry at 1% consistency. Sodium hypochlorite was added at

levels of 2.0%, 1.0%, 0.5% and 0.25% (w/w) (number of grams brightening agent in 100 grams bacterial cellulose) . The conical flask was maintained in a water bath having a temperature of 45°C for 1 hour and agitated by shaking. The treated slurry was then diluted with deionized water to 2 L, and the pH was adjusted to neutral. Cellulose handsheets were prepared in accordance with the protocol set forth in Example I.

EXAMPLE III Colored Impurity Removal Using Chlorine Dioxide Five (5) grams of cellulose (o.d. basis) , produced under agitated culture conditions as described above, was admixed with deionized water in a 2 L conical flask to form a cellulose/water slurry. Chlorine dioxide was added in an amount of 0.3% (w/w) based upon dry weight of bacterial cellulose, and the consistency of the slurry was adjusted to 1% (w/w) . The conical flask was maintained in a water bath having a temperature of 70°C for 2 hours and agitated by shaking. The treated slurry was then diluted with deionized water to 2 L, and the pH was adjusted to neutral. Cellulose handsheets were prepared in accordance with the protocol set forth in Example I.

EXAMPLE IV Colored Impurity Removal Using Sulfur Dioxide Five (5) grams of cellulose (o.d. basis) , produced under agitated culture conditions as described above, was admixed with deionized water in a 2 L conical flask to form a cellulose/water slurry. Sulfur dioxide enriched water was added, and the consistency of the slurry was adjusted to 1% (w/w) . Sulfur dioxide was employed at levels

ranging from 1.18 to 28.4 grams/liter. The conical flask was maintained in a water bath at ambient temperature from 30 minutes to 3 hours. The treated slurry was then diluted with deionized water to 2 L, and the pH was adjusted to neutral. Cellulose handsheets were prepared in accordance with the protocol set forth in Example I.

EXAMPLE V Handsheet Testing - Brightness Measurement

Handsheet testing to measure brightness was conducted, in accordance with standard TAPPI procedures (Method T525-0M-86) , on the handsheets of microbially produced cellulose prepared in Examples I-IV. An untreated cellulose handsheet was additionally prepared in accordance with the protocol set forth in Example I and measured for brightness. The results are shown in Table 1.

Table 1 % Brightness. TAPPI

39.6 45.0 59.4 56.3 51.1 48.9 chlorine dioxide, 0.30% 53.5

S0 ₂ enriched water 52.2

* Percentages indicate the amount of the brightening agent per dry weight of bacterial cellulose.

As can be readily seen, treatment of discolored or

^dull" cellulose with the various brightening agents resulted in significant increases in the brightness level, even at relatively low dosage levels of

brightening agent. Each treatment resulted in at least a five-point brightness improvement.

EXAMPLE VI Handsheet Testing - Brightness Measurement

Handsheet testing was conducted, in accordance with standard TAPPI procedures (Method T525-OM-86) , on handsheets of microbially produced cellulose prepared from two ambient temperature treatments accomplished in the manner described in Example IV. The results are shown in Table 2.

Table 2

S0 ₂ concentration Treatment % Brightness, TAPPI (grams/liter) Time (hr) Batch #1 Batch #2

0.00

1.18

9.45 18.90

28.40

As can be seen, sulfur dioxide was an effective brightening agent at ambient temperatures, low agent concentrations and short treatment times. Such conditions are favorable for use in association with large scale fermentation schemes.

Visual comparisons of handsheet brightness were also conducted to assure that the results observed above were not merely due to the acidity of the solution (S0 ₂ enriched water is acidic, imparting a pH of approximately 2.2). Cellulose was treated with sulfuric and hydrochloric acid to impart a pH of approximately 2.2. No change in the brightness of cellulose treated with sulfuric and hydrochloric acid was observed visually compared to the control cellulose. Thus, sulfuric acid and hydrochloric acid treatments reduced the pH of the slurry, but

did not improve the color characteristics of the cellulose.

EXAMPLE VII Handsheet Testing - Brightness Measurement Handsheet testing was conducted, in accordance with standard TAPPI procedures (Method T525-OM-86) , on thoroughly washed handsheets of microbially produced cellulose. Bacterial cellulose was treated as described in Example II with 0.25% (Trial 1), 0.50% (Trial 2), 1.0% (Trial 3) and 2.0% (Trial 4) sodium hypochlorite brightening agent for 1 hour at 45°C and then washed thoroughly. Various process parameters were measured during and following processing, including the initial and final sodium hypochlorite concentrations, the final pH, and brightness. These treatments produced the following results.

Table 3 Parameter

As can be readily seen, treatment of the discolored or "dull" cellulose with a NaOCl brightening agent resulted in significant brightness increase at relatively low dosage percentages. Moreover, most of the NaOCl brightening agent was consumed during the brightening treatment.

EXAMPLE VIII Handsheet Testing - Brightness Measurement

Handsheet testing was conducted, in accordance with standard TAPPI procedures (Method T525-OM-86) , on thoroughly washed handsheets of microbially produced cellulose. Bacterial cellulose was treated as described in Example IV with sulfur dioxide brightening agent at levels of 0.125 (Trial 1), 0.25 (Trial 2), 0.50 (Trial 3), and 1.0 (Trial 4), all expressed as grams S0 ₂ /5 grams cellulose. Various process parameters were measuring during and following processing. The results are shown below in Table 4.

Table 4 meter rial umbe

* The untreated brightness value was 29.3.

** A blank experiment without the presence of bacterial cellulose in the slurry resulted in an S0 ₂ concentration change from 0.27 g/1 to 0.26 g/1.

As the results demonstrate, sulfur dioxide treatment enhanced brightness significantly.

Trial #5 was conducted at low pH without the presence of S0 ₂ to assure that the results observed were not merely due to the acidity of the solution (S0 ₂ enriched water is acidic, imparting a pH of approximately 2.2). No significant change in brightness of the non-S0 ₂ acid treated cellulose (brightness 29.3) was observed over the control cellulose (brightness 29.6). Thus, the non-S0 ₂ acid treatments reduced the pH of the cellulose slurry,

but did not significantly improve the color characteristics of the product cellulose.

EXAMPLE IX Handsheet Testing - Tensile Strength

One advantage of the present invention is that an increase in bacterial cellulose brightness is achieved without harming or substantially altering the structure of the bacterial cellulose. To demonstrate this advantage, tensile indices of bacterial cellulose handsheets prepared in accordance with Examples I and II were measured and compared to that of an untreated control bacterial cellulose handsheet. Tensile indices were computed from tensile measurement data on the microbial cellulose handsheets in accordance with TAPPI Method T494-OM-88 on an Instron Tensile Tester. The experimentation produced the following results.

Table 5 Agent Tensile Index (N-m/g) Control 119 H ₂ 0 ₂ , 2.0% 127 NaOCl. 2.0% 126

As the experimental evidence indicates, processing with brightening agents actually produced a slight increase in the tensile strength of the bacterial cellulose handsheets.

EXAMPLE X Effect of Hydrogen Peroxide Concentration on

Removal of Colored Impurities A series of experiments was run in which different quantities of hydrogen peroxide (w/w, based on bacterial cellulose) were employed to determine the amount of hydrogen peroxide necessary to achieve significant brightness improvement. Ten

(10) grams of bacterial cellulose (o.d. basis) were slurried into deionized water. Hydrogen peroxide was added (for parallel experiments) in the amounts of 0, 2.0, 5.0 and 10.0% by weight to bacterial cellulose fiber. The pH was adjusted to 9.5 by addition of sodium hydroxide, and the final volume was adjusted by dilution to 1000 mL. The consistency was 1% in all cases.

The fiber slurry was heated to 55 ^β C on a hot plate and then transferred to a water bath where the temperature was maintained at 55 ^β C for one hour with occasional stirring. After cooling to room temperature, the suspension was filtered and then reslurried with fresh water and neutralized to pH 7 by addition of aqueous sulfur dioxide. The suspension was then filtered and reslurried with fresh water to wash out salts and impurities.

Handsheets were prepared as in Example 1, except 2.0g fiber were used for each handsheet (basis weight 100g/m ² ) and the sheets were air dried between filter papers mounted within rings in a room controlled at 50% relative humidity. Brightness was measured according to standard TAPPI procedures. The results of the experiments are shown in Table 6. Table 6

Effect of Hydrogen Peroxide Dosage on Handsheet Brightness

H0,. % % Brightness. TAPPI

0 33.7

2.0 49.4 5.0 47.5

10.0 53.3

Nearly all of the attainable brightness improvement is achieved at a hydrogen peroxide

dosage of 2.0% (w/w) to bacterial cellulose fiber. This method, therefore, represents an efficient, economical process to improve the brightness level of bacterial cellulose. Further, it is compatible with the normal purification process wherein the fermentation broth is treated with sodium hydroxide at approximately pH 13 to lyse bacterial cells and separate bacterial cellulose. After separation of the dissolved cell material and washing, the bacterial cellulose product suspension could conveniently be adjusted to pH 9.5 and treated with 2% hydrogen peroxide for further removal of colored impurities. As shown in Table 6, such treatment results in a significant brightness improvement of more than 15 points.

EXAMPLE XI Removal of Impurities by Combined Action of Shear Mixing and Hydrogen Peroxide A series of experiments was performed in which the alkaline hydrogen peroxide treatments were identical at a hydrogen peroxide dosage of 2.0% (w/w) to bacterial cellulose fiber at pH 9.5, as described in Example 10. Samples were subjected to shear treatment both before and after treatment with brightening agent to determine the effect of shear treatment on brightness. For "shear, then peroxide" samples, the bacterial cellulose fiber was first slurried at 0.5% consistency and the suspension was sheared using a Cowles Dissolver at 5000 revolutions per minute for periods of 2, 10 and 20 minutes. This was followed by hydrogen peroxide treatment at 1.0% consistency, and ultimately handsheet preparation and brightness measurements as described previously. A similar series of experiments was performed for "peroxide, then shear" samples,

wherein the bacterial cellulose fiber slurry was treated with the hydrogen peroxide brightening agent at 1% consistency. The resulting suspension was filtered, washed and reslurried to 0.5% consistency prior to shearing. The samples were then subjected to the same shear mixing conditions for periods of 2, 10 and 20 minutes.

Samples were also diluted to 1% consistency and sheared at room temperature for comparable times without treatment with peroxide. Handsheets were prepared and brightness levels are included in Table 7 as the "Shear Only" set of samples. Brightness improvements were less than 3%. Another sample, labeled "no H ₂ 0 ₂ ," represents a fiber slurry sheared for 10 minutes at pH 9.5 and 55 ^β C without hydrogen peroxide. This resulted in a small gain (4.7%) in brightness and verified that the combined effects of shear and alkaline hydrogen peroxide results in the maximum brightness improvements. Results are shown below in Table 7.

Table 7

Effect of Shear Mixing and Hydrogen Peroxide Treatment on Brightness

Shear Brightness

Time Shear, then Peroxide, No

Min. Shear Only Peroxide then Shear Peroxide 0 31.5 ^* 41.8

2 31.8 52.6 52.0

37.1

Control sample.

As shown in Table 7, a substantial improvement in brightness is achieved by coupling the action of shear mixing with hydrogen peroxide treatment. The brightness gain due to peroxide alone is about 10%, whereas when shear mixing is employed (for two minutes or longer) preceding or following peroxide treatment, an additional gain of 10-11% in brightness is achieved.

EXAMPLE XII

Removal of Nitrogen Impurities by Com ine Action of Shear Mixing and Hydrogen Peroxide

The combined effect of shear and treatment with hydrogen peroxide also reduces the level of nitrogen-containing impurities in bacterial cellulose. A series of experiments was performed in which alkaline (pH 9.5) hydrogen peroxide treatments were used at a hydrogen peroxide dosage of 2% (w/w) to bacterial cellulose fiber, as described in Examples X and XI. The suspension was sheared using a Cowles Dissolver at 5,000 rpm for 20 minutes. Samples labeled 1-5 were treated as follows: (1) control — no shear and no alkaline peroxide treatment; (2) shear treatment; no alkaline peroxide treatment; (3) no shear treatment; alkaline peroxide treatment; (4) shear treatment then alkaline peroxide treatment; and (5) alkaline H ₂ 0 ₂ treatment, then shear treatment. The experimental protocols were identical to those described in Example XI. Results are shown in Table 8, below.

Table 8

Effect of Shear Mixing and Hydrogen Peroxide Treatment on Nitrogen Impurities

Sample Nitrogen. %

1 0.19 ^*

2 0.19

3 0.16

4 0.14 5 0.13

Control Sample.

The lowest levels of 0.14% and 0.13% nitrogen were achieved when both shear mixing and peroxide treatment were combined. The reduction in nitrogen level (0.13% vs. 0.19%) amounts to a decrease of 32% in nitrogen-containing impurities. It should also be noted that duplicate nitrogen determinations consistently agreed within 0.01%. Thus, the experiments described herein confirm that the improvements in brightness due to alkaline hydrogen peroxide treatment can be increased if shear mixing is applied prior to or following hydrogen peroxide treatment, and such combined use of shear and hydrogen peroxide also results in substantially reduced levels of nitrogen-containing impurities. With the information contained herein, various departures from the precise description of the invention will be readily apparent to those skilled in the art to which the invention pertains, without departing from the spirit and the scope of the invention claimed below. The present invention is not to be considered limited in scope to the products, procedures, properties or components

defined, since the preferred embodiments and other descriptions are intended to be only illustrative of particular aspects of the invention. Any products or procedures for preparing such products which are functionally equivalent to those described herein are considered to be within the scope of the invention.