Chemistry: molecular biology and microbiology – Process of utilizing an enzyme or micro-organism to destroy... – Treating animal or plant material or micro-organism
Reexamination Certificate
2001-10-15
2003-09-30
Witz, Jean C. (Department: 1651)
Chemistry: molecular biology and microbiology
Process of utilizing an enzyme or micro-organism to destroy...
Treating animal or plant material or micro-organism
C435S277000, C435S278000, C424S769000
Reexamination Certificate
active
06627430
ABSTRACT:
TECHNICAL FIELD
The present invention relates to a process for the cultivation of chitin/chitosanrich, filamentous fungi with capacity to assimilate mannose as well as xylose, galactose, and glucose for a further manufacture of a porous structure comprising cell walls having good absorption ability, as well as a black liquor freed from sugars.
BACKGROUND
In traditional biotechnology the aim has been to use an organism to produce one product of commercial value. This might be a substance with high biological activity and value such as an antibiotic or a hormone. Also less valuable material such as single cell protein and single cell oil are produced. The higher the value of the product the less is the importance of the cost of media and recovery of by-products. When the commercial value of the product is low, the economy of the process is improved by development of a system consisting of a low-cost substrate, a non-expensive production process and the use of several products from the process both such that are produced by the biosynthesis and such that remain in the medium used for cultivation, since the cultivation process might have removed undesired components from the cultivation medium.
In the preparation process for piper pulp from wood in the sulfite process ca half of the wood is extracted in the sulfite liquor as lignosulfonate 60%, sugars 20%, and other material 20%. The dominating sugars are mannose, xylose, galactose, and glucose. Xylose in a pentose, the others are hexoses. The hexoses are readily fermented by yeast,
Saccharomyces cerevisiae,
into ethanol, whereas the pentoses are not fermented unless the yeast is genetically modified. Xylose it particularly abundant in hardwood. Furthermore the removal of the yeast by centrifugation and the recovery of ethanol by distillation are expensive processes, such that the economy of the entire process has narrow margins.
Lignosulfonate may be concentrated by evaporation and used for binding in animal feed and concrete. In animal feed part of the sugar may be used as a nutrient by the animal being fed, however, the concentration of sugars to be used is fairly low.
When lignosulfonate is used for binding and dispersion in concrete the presence of sugars will delay hardening. Thus different procedures are being developed in order to remove the sugars.
Moulds in contrast to yeasts grow as filaments, and may be harvested from liquid media by simple sieving. Zygomycetes moulds are known to grow well on moist wood causing respiratory allergy in law-mill workers.
Certain zygomycetes species, particularly
Rhizopus oligosporus
and
R. oryzae
have been used since long for food preparation in south-east Asia especially tempeh which is like a camembert made from soy furthermore zygomycetes are used for the production of a great number of different extracellular and intracellular enzymes. One taxonomic characteristic of zygomycetes is that the hyphae (filaments) are lacking septa which make them into microscopical tubes. Another characteristic is that their cell walls contain very high concentrations of chitin/chitosan in a net-work like a micro-sponge. It has previously been found that these cell walls have very good binding properties for several kinds of biological material being negatively charged (EP 0 494 950 B1) as well as for water (Swedish patent application 9801373-3); the material has also antimicrobial effect. The water binding capacity is in the same range as polyacrylate super-absorbents. The polyacrylates are petrochemical products which are difficult to decompose by composting, whereas the cell wall material is made from renewable sources and is readily decomposed in the environment. As a consequence the cell wall material is being exploited for hygienic and infection-preventive purposes (Swedish patent Application 9801373-3).
Thus there is a need for a process which can transfer the drawbacks of the present sulfite liquor because it is an environmental problem, when it is discarded into the environment as such and it is not optimal for use of the lignosulfonate because of the presence of sugar.
The present invention exploits the growth of zygomycetes with the consumption of sugars. The zygomycetes growth will primarily be used for the preparation of cell wall material. However, the very use of an edible mould will eventually make a number of products useful as well as a number of metabolites, e g lactic acid might be recovered.
SUMMARY OF THE PRESENT INVENTION
The present invention concerns a procedure for cultivation of zygomycetes for the production of a material consisting of a porous structure of cell walls with high capacity to absorb and transport liquid, particularly water, and good binding power for biomacromolecules and cells including micro-organisms. In this process the sulfite liquor from the paper pulp industry is cleared from its sugar content.
An extraction process is employed which removes the cell content (protoplasm) and opens tip the cell walls, such that a microporous net-work is formed; alternatively a large number of finely dispersed filaments may surround the former cell wall. The fungal cell walls mainly exist as microtubes (capillaries), and the large number of microtubes gives the material a filamentous appearance.
The porous cell wall material is obtained from a fungus e g belonging to the division Zygomycota and contains a high proportion of hexosamine. The fungal cell material may be disintegrated and is extracted with chemicals such that a suspension is formed. Alternatively the cell material is extracted directly. The suspension is then dried in such a way that the resulting material gets a porous structure e g by air-drying, spray-drying, or preferably by freeze-drying. The resulting material gets a unique capillary system which is capable of absorbing and transporting large volumes of liquid. It can also adsorb proteins and other macromolecules, and cells as bacteria and yeasts. In this context porous implies that the material contains large quantities of air. Thus it has a low density, 0.1 g/cm
3
, preferably 0.05 g/cm
3
at the most. Since the binding capacity of the material is very much dependent of an acid pH, it is of great importance that the counter-ion (anion) is non-volatile. One very useful anion is lactate, since it is non-volatile and has been used widely in skin applications. It is even produced by Rhizopus under the present conditions.
After freeze-drying the material contains a large proportion of air which can be replaced by liquid, macromolecules, and cells e g microorganisms. Consequently the porous fungal cell-wall structure may be used as an absorbent for liquid as well as molecules, particles and cells. After absorption of water the material retains its original form as set before freeze-drying and does not show any sign of disintegration, neither after a long time (>1 week). Due to the fine capillary system of the material, it has a good capacity to spread the liquid without the addition of other fibres.
The material can be dried as it is or bound to another surface. This surface may be a foam, a film or a fibre e g cellulose or synthetic fibre. If the fibre is absorbing, mainly liquid absorbing capacity is obtained. If the fibre is made from plastics, the capacity to adsorb macromolecules and microorganisms is more conspicuous. The material can also be fastened to a surface after drying. It can also be adsorbed to a surface after disintegration of the cell wall filaments as by freeze-pressing.
Furthermore the material may be doped with polar or negatively charged macromolecules such as certain proteins and polysaccharides. New properties are added to the material with the doping. When the material is doped with an enzyme, the material acquires enzymatic activity.
DETAILED DESCRIPTION OF THE PRESENT INVENTION
In order to find zygomycetes strains with good capacity to use the sugars present in sulfite liquor 30 strains representing 10 species were tested with respect to assimilation of 10 different sugars including mannose, xylose, galactose, and glucose.
Samuels , Gauthier & Stevens, LLP
Witz Jean C.
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