Cellulose-producing bacteria

Details Cellulose-producing bacteria Cellulose-producing bacteria

2002-06-05

2004-11-16

Lilling, Herbert J. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Bacteria or actinomycetales; media therefor

C435S101000, C435S252100, C435S252300, C536S056000

Reexamination Certificate

active

06818434

ABSTRACT:

TECHNICAL FIELD
This invention relates to novel cellulose-producing bacteria including one producing cellulose having high degrees of polymerization, one producing a Bingham polysaccharide as a by-product, and one producing a small amount of water-soluble polysaccharide; a method for the production of cellulosic material (bacterial cellulose: “BC”), which comprises culturing these cellulose-producing bacteria; and bacterial cellulose thus obtained.
BACKGROUND ART
Since the bacterial cellulose is edible as well as tasteless and odorless, it is utilized in the food industry. The homogenized bacterial cellulose's high dispersibility in water further provides it with many industrial applications, such as to maintain particle sizes of food, cosmetics or coating agents, to strengthen food materials, to maintain moisture, to improve stability of food, and to be used as low-calorie additives and an emulsion stabilizer.
The bacterial cellulose is characterized by a sectional width of its fibrils which is smaller by two orders of magnitude than that of other kinds of cellulose such as those derived from wood pulp.
Due to such structural and physical feature of microfibril, a homogenized bacterial cellulose has plenty of industrial applications as a strengthening agent for polymers, especially hydrophilic polymers. Products prepared by solidification of the homogenized bacterial cellulose in the form of a lump or paper show a high elastic modulus in tension due to the above feature, and are therefore expected to have excellent mechanical properties for use in various kinds of industrial materials.
The methods for the production of BC are disclosed in Japanese Patent Laid-Open Application Sho 62(1987)-265990, Japanese Laid-Open Application Sho 63(1988)-202394 and Japanese Patent Application Publication Hei 6(1994)-43443.
Schramm/Hestrin medium is well known as a nutrient medium suitable for the cultivation of cellulose-producing bacteria, which comprises carbon sources, peptone, yeast-extract, sodium phosphate and citric acid (Schramm et al., J. General Biology, 11, pp.123-129, 1954).
It is also possible to optionally supply accelerators for the cellulose production such as inositol, phytic acid, pyrroloquinoline quinone (PQQ) (Japanese Patent Publication Hei 5(1993)-1718; Mitsuo TAKAI, Japan TAPPI Journal, Vol.42, No.3, pp.237-244), carboxylic acid or their salts (Japanese Patent Laid-Open Application Hei 7(1995)-39386, laid open Feb. 10, 1995), invertase (Japanese Patent Laid-Open Application Hei 7(1995)-184677, laid open Jul. 25, 1995) and methionine (Japanese Patent Laid-Open Application Hei 7(1995)-184675, laid open Sep. 25, 1995) into the culture media. Further, the BC production method with a cultivating apparatus having a specific oxygen-transfer coefficient (KLa) is disclosed in Japanese Patent Application Hei 7(1995)-31787.
Conventionally used culture conditions include static culture, shaken culture, and aerobic agitated culture, and conventionally used culture operation methods include batch fermentation, fed batch fermentation, repeated batch fermentation and continuous fermentation.
Means for agitation include impellers, air-lift fermenters, pump-driven recirculation of the fermenter broth and any combination of these means.
It is well known that the degrees of polymerization of BC are higher than wood pulp (e.g. LBKP and NBKP) and cotton linter used as industrial materials, but lower than a specific cellulose such as that derived from an ascidian and valonia. Polymeric materials including cellulose having higher degrees of polymerization will generally have more excellent mechanical properties such as strength and elasticity. Accordingly, it is expected that the cellulose derived from the ascidian and valonia having high degrees of polymerization is superior in the mechanical properties to the cellulose with low degrees of polymerization.
However, the above specific cellulose such as that derived from the ascidian or valonia has disadvantages that it exists little as resources and can not be efficiently collected, so that it will take much time to produce industrial materials from them. It has been therefore desired to industrially produce the BC having high degrees of polymerization.
Furthermore, it has been found that the BC produced by an industrially advantageous method such as those in an aerobic agitated culture has a lower weight-average degree of polymerization (DPw) than that produced in a static culture. Since the BC with lower degrees of polymerization has been deprived of the excellent mechanical properties, it has been desired to produce the BC having high degrees of polymerization by the aerobic agitated culture method.
It is also known that cellulose-producing bacteria producing no water-soluble polysaccharide as a by-product will show a high BC productivity in the static culture (Journal of General Microbiology (1988), 134, 1731-1736).
On the other hand, it is known that the by-product of water-soluble polysaccharide functions as dispersant in the agitated culture of the cellulose-producing bacteria. Adhesion or catching of BC in the parts of cultivation apparatuses such as impellers and stuffing of these parts with BC may be prevented by its functions so as to form the suspended BC into small clusters. As a result, the BC productivity will be increased (Japanese Patent Laid-Open Application Hei 5(1993)- 284988).
The present inventors have now found the bacteria capable of producing BC having high degrees of polymerization or BC containing only a small amount of the fraction with low degrees of polymerization even under the aerobic agitated culture condition, and the bacteria capable of producing only a small amount of water-soluble polysaccharide as the by-product so as to improve in their BC productivity and yield while maintaining the above function of the water-soluble polysaccharide. The present invention is based on these findings.
DISCLOSURE OF INVENTION
The present invention is related to cellulose-producing bacteria capable of producing of a bacterial cellulose having a weight-average degree of polymerization (in terms of polystyrene) of 1.6×10
4
or above, preferably of 1.7×10
4
or above, more preferably of 2.0×10
4
or above.
The weight-average degree of polymerization of a variety kinds of cellulose such as BC of this invention may be determined by the method using a GPC system (Tosoh HLC-8020) equipped with an RI detector as follows:
A cellulose sample is nitrated with a fuming nitric acid-phosphorous pentaoxide solution according to the method of W. J. Alexander, R. L. Mitchell, Analytical Chemistry 21, 12, 1497-1500 (1949).
Nitrated cotton linter is used as a control.
Nitrated cellulose is then dissolved in THF (Wako Pure Chemical Industries Ltd., the first grade) to a final concentration of 0.05%, and filtered through a 1.0 &mgr;m pore-size filter. THF is also used for an elution solvent.
The flow rate, pressure, and sample-injection volume are adjusted to be 0.5 ml/min., 10-13 kgf/cm
2
and 100 &mgr;l, respectively.
The column system consists of two TSKgel GMH-HR (S) columns (7.5 ID×300 mm) and a guard column (Tosoh Co., Ltd.). The analysis is carried out at a temperature of 35° C.
A relative molecular weight in terms of polystyrene is calculated by using polystyrene standards (Tosoh).
The polystyrene standards having a molecular weight in the range of 2.0×10
7
to 2630 are used and a standard curve is prepared based on the following three-dimension approximate equation:
logM=At
3
+Bt
2
+Ct+D
wherein “t” is an elution time and “M” is a molecular weight.
The weight-average molecular weight and number-average molecular weight are calculated by a program (ver. 3, 10) equipped in a data processor (SC-8020).
The weight-average degree of polymerization and number-average degree of polymerization of the original cellulose samples are finally calculated based on the above data, taking substitution degrees after the nitration into consideration.
The present invention is related to cellulose-p

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