CA1124131A - Bioconversion of cellulosic materials to protein-enriched product - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A cellulosic carbohydrate-containing material, such as, an agricultural of forestry cellulosic residue, is converted into a protein-enriched product having significant amounts of microbial biomass in the form of the fungus, Chaetomium cellulolyticum.

Description

F:IELD OF INVENTION
The present invention relates to the conversion of I cellulosic carbohydrate-containing materials by a fermenta-¦ tion process into products which contain microbial biomass, such products being useful as animal feed for their protein and other nutritive values. In the following description concentrations of substances are expressed as W/V ~for weight per unit volume of total mixture), as V/V ~for volume per unit volume of total mixture), as W/W (for weight per unit weight of total mixture), or as DM (for weight per unit weight of total mixture on a dry matter basis).
BACKGROUND TO THE: INVENTION
Vast quantities of cellulosic materials occur universally as surplus and waste residues of agriculture, forestry and other operations. They occur in native forms, such as, straws of cereal grains, for example, wheat, barley, rice, oats and rye; cornstover; sugarcane bagasse; cotton;
and tree bark; as well as in processed forms,such as, wood sawdust, paper pulp and pulp mill sludges. Basically, these materials contain cellulose, usually in combination with significant amounts of hemicellulose and smaller amounts of lignin. Because of their carbohydrate content, these mater~
ials represent potentially valuable biorenewable resources for animal feed.
Various known processes have been proposed or used to convert cellulosic materials into products which are puryort~d to be sui-tab~, as substitute for ha~ and similar forages, for metabolisable carbohydrate energy in ruminant feeds. These processes use physical and/or chemical changes of the materials merely to enhance thei~r digestibility; none of the original cellulosic material is converted into protein.
Various known processes have also been proposed or used to convert cellulosic materials into products which are purported to be sui-table, as substitutes for soymeal and similar protein-rich substances, for animal feed protein rations. Of these, t~o basic types of fermentation processes are known. In the first method, yeasts are cultivated on liquid sugar solutions which are produced by complete chemi-cal hydrolysis of the hemicellulose and/or cellulose components of the cellulosic material. In the second method, cellulolytic bacteria are cultivated on the solid cellulose and/or hemicellulose components of the cellulosic material.
In the above prior art processes, one or more of the following undesirable features are found~ the processing costs are high, because conversion rates are low and/or ex~reme operating conditions of temperature and/or pressure are required, (2) the product is not suitable as animal feed, because it is too toxic and/or too indigestible, (3) the product is not suitable as animal feed protein ration, because its protein content is too low and/or its protein quality is too poor.
SUMMARY OF INVENTION
~ he present invention obviates the prior art problems by providing a novel fermentation process for treating, under relatively mild operating conditions, a cellulosic carbohydrate-containing material, such as, an agricultural or forestry cellulosic resi~ue. The cellulosic material, supplemented with additives, is con~erted into a protein-enriched product containing significant amounts of microbial biomass in the form of the fungus, Chaetomium cellulolyticum (ATCC 3231g), the product being suitably safe, digestible and nutritious for use as animal feed or as animal feed supplement.

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GE.NERAI. DES~RIT'TION OF IN~NTION
Raw aterial The present invention is concerned with -the conversion of a-t least one cellulosic carbohydrate material 5 into a microbial biomass product. The term "cellulosic material" as used herein signifies a product containing cellulosic carbohydrate having the following chemical composition:
about 35 to about 95% DM cellulose 0 to about 30% DM hemicelluloses 0 to about 25~ DM lignin The cellulosic material may be in relatively pure form, i.e., substantially free from other non-cellulosic carbohydrate materials. Such pure materials may be in native form and processed form. Native form materials include agricultural crop residue, for example, cereal grain straw, particularly wheat straw, sugarcane bagasse, and cornstover.
Processed form materials include forestry residue, for example, paper pulp, pulp-mill sludge, such as, from a 20 Kraft-type pulp mill, and wood sawdust, such as, from Aspen --wood.
While the invention is particularly concerned with utilization of such pure cellulosic carbohydrates, the invention is also concerned with the conversion of cellulosic carbohydrate materials which are associated with substantial quantities of non-cellulosic carbohydrates, such as, starches, into the microbial hiomass product. Cellulosic carbohydrate materials of thls type include root crops, for example, potato, yam and cassava.
The ability to use normally waste materials to ~ form nutritionally useful protein-enriched products is a -~ significant environmental pollution control factor.
The Process The process of the present invention consists 35 essentially of threesteps: (1) pretreatment of the cellulosic material~ (2) fermentation of a mixture of the pretreated cellulosic material and a nutrient supplement solution, and (3) separation of the solids from the fermented mixture.

Optionally, the separated solid product may be clried.
The pretreatment opera-tion is effected in a pretreatment medium at a concentration of about 5 to about 30% W/W solids in order to sterilize and sof-ten and/or swell the cellulosic material. When alkali i5 used in the pretreatment, partial delignification and solubilization of hemicelluloses and other ingredients are promoted. The pretreàtment procedure may also result in partial hydrolysis of the cellulosic carbohydrate material to an oligomeric saccharide-containing material.
The pretreatment renders the cellulosic material susceptible to use by the fungus. The conditions used are relatively mild compared to those which are conventionally used when preparing raw materials for use as the carbon nutrient source in the fermentation of common microorganisms, such as, Cellulonomas sp..
The pretreatment is effected under conditions such that the fungus is able to produce from the pretreated starting material a fermented product containing at least 5% DM of biomass. The pretreatment is effected under conditions which do not significantly exceed those which enable a fermented product containing 100% DM of biomass to be produced from the starting material.
The manner of effecting the pretreatment to achieve 25 this result and subsequent utilization of the pretreatment liquor varies depending on -the nature of the raw cellulosic material to be treated. If the cellulosic material is not significantly recalcitrant to the fungus, for ex~mple, some paper pulps, pulp-mill sludges and root crops, then the pretreatment may be effected by heating as a slurry in water, for example, heating for one hour at atmospheric pressure using live steam. The concentration of the slurry may be any convenient value, for example, about 10 to 20 %
W/W solids.
If the cellulosic material is significantly recalcitrant to the fungus and contains significant amounts of both hemicellulose and cellulose, and either no ingredient significantly inhibitory to the fungus, for example, some crop residues, such as, bagasse, cornstover and wheat straw, or an ingredien-t signi~icantly inhibitory to the fungus t for example, some types of wood and tree bark, then the pretreatment may be effected using an a~ueous alkali solutlon, for example, using aqueous sodium hydroxide solution, for example, l~ W/V NaQH, or aqueous ammonium hydroxide solution.
The cellulosic material generally is particulated prior to commencement of the pretreatment. If used in granu-lar form, the particles may have an average particle size in 10 the range oE about 8 to about 40 mesh. If used in fibrous forms, fibre sizes up to 2 cm in length may be used.
If the cellulosic material contains ingredients significantly inhibitory to the fungus, then the spent pretreatment li~uor must be discarded~ Mowever, where that condition does not exist, the spent pretreatment liquor may be retained with the pretreated cellulosic material during fermentation. The ability to utilize the spent pretreatment liquor in this way is important, since the potential polluting capability of the liquor, which contains materials extracted from the cellulosic material, is eliminated.
The pretreated cellulosic material, which may aiso include the spent pretreatment liquor, is mixed with nutrient supplements to provide the essential non-carbon nutrients required in an overall composition of mixture which conforms to conventional guidelines for fermentation medium. Typic~
ally, the ma~or elements as carbont nitrogen, phosphorus and potassium follow the ratios of C:N:P:K - lO0:10;1:1 by weight .
The nutrient supplement solution may be pre sterilized prior to mixing with the cellulosic material or may be sterilized after mixing with the cellulosic material.
The mixing with the nutrient supplement solution usually decreases the consistency of the slurry to a value suitable for submerged fermentation, such as, about 1 to about 3 W/V solids. The fermen-tation may, however, be effected at any desired concentration up to about 30% W/W, for example, by solid state fermentation, at an overall solids concentration of generally about 15 to about 30~ W/W.

The nutrien-t supplement solution may be provided ~rom any convenient source, such as, a synthetic mixture of chemicals, Eor example, a fertili~er blend. The nutrient supplemen-t solu-tion may also be provided by an animal manure, such as, cattle or swine manure. The animal manure may be anaerobically predigested, if digested, to co-produce methane as a va~uable fuel by-product.
The fermentation medium is adjusted, if necessary, to a pH in the range of about 5 to about 8, particularly about 5 to about 7, and innoculated with the fungus, Chaetomium cellulolyticum. Fermentation is effected at a temperature of about 25 to about 40~C~ typically around 37C, using sterile air, typically supplied at a flow rate of 1 to 2 volumes of air per unit volume of medium per minute~
During the fermentation, the fungus uses the raw materials to reproduce itself and generate cellulase enzymes, which tend to soften and thereby improve the diges-tibility of any solid cellulosic material which remains unutiliæed. The fermentation is continued until the desired fungus growth has been effected, for example, for about 12 to about 24 hours.
Since cellulase enzymes are produced duriny the fermentation, the fermentation may be used to soften non-pretreated cellulosic material to render the same moredigestible in animal feed. Such a procedure ~orms part o~
this invention and produces a product containing up to 10%
DM of biomass.
Followiny completion of the fermentation, the solid ~hase is separated from the liquid phase. The separated solid phase may be used as such, or the separated solid phase may be dried to a low moisture content, generally below about - 10% W/W, typically about 8% W/WO The liquid phase may be reused, i~ desired.
The operation of -the multistage process may ~e effected batchwise or on a continuous basis. For batch operations, the pretreatment and fe~mentation steps may be -h~
effected in the same vessel.
The solid phase product contains _haetomium cellulolyticum in variable quanti-ties, depending on the extent and conditions of the pretreatment and the fermenta-5 tion. The product contains at least about 5~ DM of biomass,usually from about 20 to about 80~ ~M, and up to 100% ~M, i~ desired. Products having a biomass content greater than about 55P6 DM may be used as ruminant and non-ruminant feed while products having a biomass content less than this value may be used only as ruminant feed. The remainder of the solid phase is unfermented cellulosic material.
Chaetomium cellulolyticum is a newly isolated fungus freely available from the American Type Culture Collection (ATCC No. 32319) and has the following capabilities: (l) utilization of a variety of cellulosic as well as non-cellulosic carbohydrate materials as carbon nutrient for growth, (2) utilization of a variety of synthetic as well as non-synthetic mixtures as non-carbon nutrient supplement for growth, (3) grow-th over a range of pH of about 5 to 8, the optimal being about pH 5 for insoluble cellulose and about pH 7 for solubilized hemicellulose, and (4) growth over a range of temperature of about 30C to 45C, the optimal being about 37C.
The average composition of the fungus is as follows (%DM basis): 45% crude protein, 40% carbohydrates, 10% fats, 5% vitamins, minerals, etc. The following Table shows that the amino acid profi]e of the protein component of the fungus is nutritionally sound and is comparable with soymeal protein and the UN-FAO reference protein for human nutrition.

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T2~B LE
Amino Acid _ C cellulolyticum _ Soymeal F~O reference Threonine 6.1 4.0 2.8 Valine 5.8 5.0 4.2 Cys-tine 0.3 1.4 2.0 Methionine 2.3 1.4 2.2 Isoleucine 4.7 5.4 4.2 Leucine 7.5 7.7 4.8 Tyrosine 3.3 2.7 2.8 10 Phenylalamine 3.8 5.1 2.8 Lysine 6.8 6.5 4.2 The product of the process of the invention has been found by in-vivo and in-vitro feeding trials to be suitable as animal feed or animal feed supplement for both non-ruminants and ruminants.
EXAMPLES
The examples given below further illustrate the present invention. It should be unders~ood that the invention is not limited to these particular examples.
Unless otherwise specified, the source of nutrient supplement referred to in the examples below are as follows:
(1) Solution A. A synthetic mixture containing in one litre of aqueous solution 2 g KH2PO4, 1.4 g (NH4)~ SO4, 0.3 g urea, 0.3 g MgSO4.7H2O, 0.3 g CaC12, 5 mg FeSO4.7H2O, 25 1.6 mg MnSO4.H2O, 1.4 mg ZnSO4.7H2O, 2 mg CoC12.

(2) Solution B. A synthetic mixture similar to Solution A but enriched to contain,in one litre of aqueous solution~2.5 g KH2PO4, 2.36 g (~H4)2 S 4, 4 2 146 mg ZnSO4.7H2O, 1.92 mg FeC13~ 0-114 mg H3BO3, 0-48 mg 30 (NH4~6 M7O24 4H2O~ 0.78 mg CuSO4.5H2O, 0.144 mg ~nC12.4H2O.

(3) Solution C. A non-synthetic mixture of swine manure (feces plus urine) diluted with water to contain about 0.05% W/V inherent nitrogen, the other naturally-occurring ingredients being reduced accordingly, and enriched with 0.05% W/V added nitrogen as (NH4)2SO4.

(4) So]u-tion D. A non-synthetic mixture of cattle manure (feces plus urine) diluted with water to con-tain about 0.05% W/V inherent nitrogen, the other naturally-occurring ingredients bei~g reduced accordingly, and erriched with 0.05O W/V added nitroyen as (NH4)2SO4.
(5j Solution E. A non-synthetic mi~ture of a stabilized anaerobically-digested cattle manure, pre-diluted with water, contalning about 0.1% W/V inherent nitrogen.
The inoculum for the fermentations referred to in the examples below was prepared as follows. The fungus Chaetomium cellu]olyticum is grown in serial transfers from __ a glucose-based fermentation medium to the actual cellulosic-based medium, using well-known standard techniques~ Visible microbial growth was allowed to develop for one day after which it was removed and disrupted under aseptic conditions in a blender resulting in a suspension of microbial pieces, typically containing about 5~ W/V solidsO A small amount of this suspension, typically 5~ V/V of the medium to be fermented, constitutes the inoculum.
~xample 1 A fermentation medium was prepared by b~iling for one hour with live steam at atmospher-ic pressure, a solid-liqu;d slurry consisting oE 1% W/V solid -particles of a dry sample of a paper pulp (a chemically delig-nified spruce wood pulp having average fibre lengths of about 1 cm) in a solution containing 50% V/V tap water and 50% ~/~
Solution A. After cooling the prepared medium to 37C and adjusting the pH to 5 with 2N H2SO4 solution, it was seeded with a 5~ V/V inoculum and fermented at 37C with sterile air using standard submerged fermentation techniques.
~ fter a growth period of 20 hours, the fermented solids were removed by filtration and dried in an oven overnight at about 80C to a moisture content of about 80 % W/W . It was found that the microbial biomass content - of the solids in the original fermentation medium increased from ~ero to 91% DM, corresponding to a crude protein content of about 41% DM, the balance being unfermented cellulosic material. In standard in-vivo feéding trials using rats, the product was found to be favourably comparable to casein for up to 40~ DM protein replacements of casein which was used in the "control" dietsl with respect to non-toxicity, non-teratogenecity, digestibility and protein nutri-tional value.
Example 2 The experiment of Example 1 was repeated, except that Solution C was used as the nutrient supplement in place of Solution A.
Following a growth period of 20 hours and dryiny of separated solid material, the microbial biomass content of the dried product was 49% DM, the balance being admixed unfermented cellulosic material. In standard in-vivo feeding trials with rats, the product was comparable to casein for up to 20% DM protein replacements in the "control" dietsO
Example 3 The experiment of Example 1 was repeated using a ground sample of wheat straw of average particle size of about 40 mesh as the cellulosic material in place of the paper pulp, and the 50% V/V tap water was replaced with 1%
W/~ NaOH solution, the pretreatment medium again being retained as part of the fermentation medium. The fermentation is conducted at an initial pH of 6.5.
After a growth period of 20 hGurs and drying of separated solid material, the dried product contained 44%
DM of the microbial biomass with the remainder being admixed unfermented cellulosic material. In standard in-vivo feeding trials with rats, the product was comparable to casein for up to 20% DM protein replacements in the "control" diets.
Example 4 A fermentation medium was prepared and fermented as described in Example 1 for a growth period of 18 hours using a ground sample of Kraft-type pulp-mill sludge having an average particle size of about ~0 mesh as the cellulosic material.
The dried product contained 60% DM of the microbial biomass and 40% DM unfermented cellulosic material and had an in-vitro digestibility, as determined by standard in-vitro feeding trials using cattle rumen fluid, of 45% DM. By way of comparison, the value for norma] forage-quality hay is about 55% DM.

ll E~ample 5 The experiment of Example 3 was repeated using a sample of corn~tover of average fibre lengths of about 1 cm as the cellulosic material and Solution s as the nutrient supplement. The fermentation was conducted for a growth period of 1~ hours.
The dried product contained 73~ DM of the micro-bial biomass and its in-vitro digestibility was 68% DM.
Example 6 A fermentation medium was prepared and fermented as described in Example 3 using a sample of sugar-cane bagasse of average fibre lengths of about 1 cm as the cellulosic material and Solution B as the nutrient supplement. After a growth period of 12 hours, the dried product contained 57% DM of the microbial biomass~
_xample 7 The experiment of Example 6 was repeated using a 3%
W/V slurry of a ground sample of cornstover of average particle size of about 20 mesh as the cellulosic material 2a and Solution D as the nutrient supplement. After a growth period of 45 hours, the dried product was found to contain 23% DM of the microbial biomass.
Example 8 A ~rmentation medium was prepared and fermented as described in Example 7 using Solution E as the nutrient supplement. After a growth period of 20 hours, the dried product was found to contain 66% DM of the microbial biomass.
- Example 9 A fermentation medium was prepared and fermented as described in Example 3 using a ground sample of Aspen wood sawdust of average particle size of about 40 mesh as the cellulosic material, except that the spent alkali liquor was removed before fermentation and replaced with sterile water.
After a growth period of 34 hours, the dried product was found to contain 24% DM of the microbial biomass.
Example ].0 A fermentation medium, having a moisture content of about 80~ W/W, was prepared containing Maple wood sawdust, preground to pass 8 mesh and pretreated as described in Example 9, and a double strength Solution A as ~utrien-t supplement. The medium was ferrnented under the pH and temperature conditions outlined in Examp]e 1 with a 4% W/V
inGculum, according to standard solid state fermentation operations.
After a growth period of 10 days, the dried product contained 22% DM of microbial biomass.

Example _ A fermentation medium, having a moisture content of about 85% W/W, was prepared and fermented as described in Example 10, except that Solution B was used as the nutrient supplement and a sample of wheat straw was used, precut to fibres up to 2 cm in length and pretreated by soaking in a 15 3% W/V NH40H solution at 25C for about 30 days.
After 4 days growth period, the dried product contained 34% DM of microbial biomass.
SUMMARY
In summary of this disclosure, the present invention, therefore, provides a procedure for converting cellulosic materials into products which contain a fungal microbial biomass, and useful as protein-enriched animal feedstuff. Modifications are possible within the scope of the invention-

Claims (26)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A process for increasing the protein content of a solid cellulosic carbohydrate-containing material in which the cellulosic carbohydrate material has the chemical composition about 35 to about 95% DM cellulose, 0 to about 30% hemicelluloses and 0 to about 18% DM lignin by fermen-tation, which comprises:
pretreating a slurry of said cellulosic-containing material in particulate form in a pre-treatment medium at a concentration of about 5 to about 30% W/W solids to render the cellulosic-containing material susceptible to fermentation by the hereinafter-named fungus at least to the extent sufficient to enable a fermented product containing at least about 5% DM of biomass to be produced therefrom and not significantly exceeding the extent sufficient to enable a fermented product containing 100% DM of biomass to be produced therefrom, fermenting said pretreated cellulosic-containing material under aerobic conditions in a culture of the fungus Chaetomium cellulolyticum in the presence of nutrient supplement sufficient to provide essential non-carbon nutrients at a solids concentration of up to about 30%
W/W, said fermentation being effected at a pH of about 5 to about 8 and at a temperature of about 25° to about 40°C
for a time sufficient to grow said fungus to provide at least about 5% DM of said fungus in the final product, and separating the resulting solid mass consisting essentially of said fungus and unfermented cellulosic material from liquid phase.

2. The process of claim 1, including drying said separated solid mass to a moisture content of less than about 10% by weight to form a dried product.

3. The process of claim 1 wherein said cellulosic carbohydrate-containing material is an agricultural crop residue.

4. The process of claim 3 wherein said agricultural crop residue is selected from the group consisting of cereal grain straw, sugarcane bagasse and cornstover.

5. The process of claim 4 wherein said agricultural crop residue is wheat straw.

6. The process of claim 1 wherein said cellulosic carbohydrate-containing material is a forestry residue.

7. The process of claim 6 wherein said forestry residue is selected from the group consisting of a paper pulp, a pulp-mill sludge and wood sawdust,

8. The process of claim 7 wherein said fores-try residue is Kraft-type pulp-mill sludge.

9. The process of claim 7 wherein said forestry residue is Aspen or Maple wood sawdust.

10. The process of claim 1 wherein said cellulosic carbohydrate-containing material is a root crop.

11. The process of claim 10 wherein said root crop is selected from the group consisting of potato, yam and cassava.

12. The process of claim 1 wherein said nutrient supplement is a synthetic mixture of chemicals.

13. The process of claim 1 wherein said nutrient supplement is derived from an animal manure.

14. The process of claim 13 wherein said animal manure is selected from the group consisting of swine manure and cattle manure.

15. The process of claim 13 wherein said animal manure is anaerobically predigested cattle manure.

16. The process of claim 6 or 10 wherein said forestry residue is selected from the group consisting of a paper pulp, a pulp-mill sludge and a root crop not significantly recalcitrant to the fungus and said pretreatment is effected for a period required to permit, but which does not significantly exceed that required to permit, a fermented product containing 100% DM of biomass to be produced using steam in the presence of water as the pretreatment medium.

17. The process of claim 4 wherein said agricultural crop residue is significantly recalcitrant to the fungus, contains significant amounts of both hemicelluloses and cellulose, and contains no ingredient significantly inhibi-tory to the fungus, and wherein said pretreatment is effected for a period required to permit, but which does not significantly exceed that required to permit, a fermented product containing 100% DM of biomass to be produced in the presence of dilute alkali as the pretreatment medium.

18. The process of claim 17 wherein the spent pre-treatment liquor is retained as part of the fermentation medium in said fermentation step.

19. The process of claim 6 wherein said forestry residue is wood sawdust significantly recalcitrant to the fungus and containing significant amounts of hemicelluloses and cellulose and at least one ingredient significantly inhibitory to the fungus, said pretreatment is effected for a period required to permit, but which does not signifi-cantly exceed that required to permit, a fermented product containing 100% DM of biomass to be produced in the presence of dilute alkali as the pretreatment medium and the spent treatment liquor is excluded from the fermentation medium in said fermentation step.

20. The process of claim 17 or 19 wherein said dilute alkali is about 1% W/V aqueous sodium hydroxide solution.

21. The process of claim 17 or 19 wherein said dilute alkali is about 3% W/V aqueous ammonium hydroxide solution.

22. The process of claim l wherein said particulate cellulosic material is in the form of granules having an average diameter of about 8 to about 40 mesh.

23. The process of claim 1 wherein said particulate cellulosic material is in the form of fibres of size up to about 2 cm in length.

24. The process of claim 16, 17 or 19 wherein said fermentation is effected to provide a product containing at least about 20% DM of biomass.

25. A process for increasing the digestibility of a solid cellulosic carbohydrate-containing material in which the cellulosic carbohydrate material has the chemical composition about 35 to about 95% DM cellulose, 0 to 30%
DM hemicelluloses and 0 to 18% DM lignin by fermentation, which comprises fermenting said cellulosic carbohydrate-containing material under aerobic conditions in a culture of the fungus Chaetomium cellulolyticum in the presence of nutrient supplement sufficient to provide essential non-carbon nutrients at a solids concentration of up to about 30% W/W, said fermentation being effected at a pH of about 5 to about 8 and at a temperature of about 25° to about 40°C
for a time sufficient to grow said fungus to provide up to 10% DM of said fungus in the final product, and separating the resulting solid mass consisting essentially of said fungus and unfermented cellulosic material from liquid phase.

26. The process of claim 25 including drying said separated solid mass to a moisture content of less than about 10% by weight to form a dried product.