GB2090514A

GB2090514A - A process for the preparation of fodder yeast and/or ethanol from plants or cellulose-containing wastes of plant origin.

Info

Publication number: GB2090514A
Application number: GB8137322A
Authority: GB
Original assignee: ORSZAGOS KOZEGESZSEGUGYI INTEZ
Current assignee: ORSZAGOS KOZEGESZSEGUGYI INTEZ
Priority date: 1980-12-23
Filing date: 1981-12-10
Publication date: 1982-07-14
Also published as: CU21613A3; FR2496690B1; DD201694A5; DE3151176A1; PL234381A1; GB2090514B; CS974881A2; HU181771B; CS241494B2; CA1178221A; PL129919B1; SU1218927A3; FR2496690A1

Abstract

A cellulose-containing starting substance is pre-treated with a dilute mineral acid and/or a dilute base, and then fermented under aerobic or anaerobic conditions in the presence of a Candida utilis var. cellulolytica strain deposited at the Hungarian National Collection of Microorganisms of the National Institute for Public Health on 23rd September, 1980 under No. CU 28 00199. When fermentation is performed under aerobic conditions, fodder yeast or a mixture of fodder yeast and ethanol is obtained, depending on the aeration rate, whereas when fermentation is performed under anaerobic conditions, ethanol is obtained.

Description

SPECIFICATION A process for the preparation of foder yeast and/or ethanol from wastes of plant origin The invention relates to a process for the preparation of fodder yeast and/or ethanol from wastes of plant origin.

Plants, more particularly, 25-40% of the dry substance content of wastes of plant origin consist of cellulose. Beside this substance, they also contain lignin in an amount of about 20-40%, furthermore smaller amounts of pentose polymers, hexose polymers and additional inorganic and organic substances. There are two known ways for the utilization of plants and wastes of plant origin by fermentation. One of them is the direct fermentation of cellulose utilizing cellulolytic (i.e. cellulose-decomposing) microorganisms, whereas the other possibility is to convert cellulose first into glucose by acidic or enzymatic degradation and then to subject the resulting glucose to fermentation.

The choice of microorganisms capable of the direct fermentation of cellulose is rather restricted, and even they permit only a very slow decomposition of cellulose. Of the cellulolytic microorganisms bacteria, such as those belonging to the Cellulomonas and Cellovibrio species, furthermore yeasts, e.g. Myrrothecium verrucaria, Trichoderma viride, etc. are to be mentioned.

In order to prepare single cell protein (SCP) by applying cellulolytic microorganisms experiments were performed by Ch. E. Dunlap (Am. Chem. Soc. Symp. 1970) with Cellulomonas and Alcaligenes strains. F. Kargi and M.L. Schuler [Biotechn. and Bioeng. Vol. XXI I, 1 567-1 600 (1 980)]utilized a mutant of Pseudomonas fluorescens in their tests.

Attempts to prepare single cell proteins by applying bacteria and mould fungi are getting less and less important, since the use of these microorganisms as fodder protein is objectionable from veterinary aspects.

The possibilities for utilizing cellulose by fermentation are far broader when cellulose is hydrolysed first into glucose, since almost all microorganisms are capable of assimilating glucose.

Sholler and Thornes were the first to elaborate an industrial process at the turn of the century for the total hydrolysis of cellulose. In this process mineral acids of medium concentration and temperatures above 1 OOoC were applied. This process was, however, very energy-intensive and required expensive acid- and pressure-resistant apparatuses. Although the process had later been modernized (acid concentration and temperature were reduced), no new plants based on this process are built.

Tests have been performed since 1 950 to hydrolyse cellulose with cellulase enzyme preparations obtained from fungi [B. Hagerdal et al.: Biotechn. and Bioeng. XXII, 1515-1526 and 1 527-1 542 (1980)]. The enzyme system of Trichoderma viride is applied generally to hydrolyse cellulose [D. Sternberg: Biotechn. and Bioeng. Symp. No. 6, 35-53 (1976)].

The enzymatic hydrolysis of cellulose is an expensive process, profitable generally only when the glucose solution obtained by hydrolysis can then be converted into valuable products.

When elaborating the process according to the invention, the major aspect considered was that the microorganisms applied for the preparation of SCP are acceptable from the aspects of animal foddering, and are capable of the utilization of a wide range of plant components over lignin.

The utilis species belonging to the Candida and Torulopsis genera have been applied as fodder yeasts since several decades. These known yeast species are unable to decompose, however, either cellulose or lignin. No yeast species capable of decomposing cellulose has been reported in the literature, either.

Now it has been found, unexpectedly, that a Candida utilis strain isolated by us in 1 978 by genetic and strain selection methods is capable of decomposing hydrated and partially degraded cellulose. A simple pre-treatment method has also been elaborated for the hydration and partial degradation of cellulose, which enables one to prepare an appropriate culture medium for the propagation of this yeast strain from cellulose-containing substances.

Accordingly, the invention relates to a process for the preparation of fodder yeast and/or ethanol from wastes of plant origin. According to the invention one proceeds so that a waste of plant origin, pre-treated with a dilute mineral acid and/or a dilute base, is fermented under aerobic or anaerobic conditions in the presence of Candida utilis var. cellulolytica deposited at the Hungarian National Collection of Microorganisms of the National Institute for Public Health (OKI), Budapest, on 23rd September, 1 980 under No. CU 28 00199.

The CU 2800199 strain was isolated from a field strain as follows: About 1 g of punk wood was homogenized with 9 ml of sterile physiological saline solution. A decimal dilution series was prepared from the solution, and samples of the diluted solutions were inoculated onto carboxymethyl cellulose (CMC) containing Sabouraud agar. (CMC-containing Sabouraud agar corresponds in composition to the Oxoid CM 41 culture medium with the difference that it contains OMO instead of dextrose).The plates were incubated for 72 hours at temperatures of 25, 30 and 37or. The yeast cultures grown on the plates were selected by cell morphological methods, and then trans-inoculated onto a culture medium containing glucose and CMC, having the following composition: beef extract (Difco) 5.0 g glucose 7.5 g carboxymethyl cellulose 7.5 g Bacto peptone (Difco) 4.0 g yeast extract (Oxoid L 21) 1.0 g potassium dihydrogen phosphate 0.2 g dipotassium hydrogen phosphate 0.1 5g sodium dihydrogen phosphate 2.0 g disodium hydrogen phosphate 1.5 g magnesium sulfate 0.3 g zinc sulfate 0.1 g ammonium sulfate 3.0 g ferric chloride 0.1 g copper sulfate 0.6 g manganese chloride 0.1 5g potassium iodide 0.05mg boric acid 0.1 mg agar 15 g water q.s. ad 1 litre The strains developed from single-cell cultures were isolated by conventional purification techniques and maintained on a glucose- and CMC-containing culture medium.

Of the isolated strains the one with the best CMC-decomposing ability on the CMC-containing Sabouraud medium was selected for the purpose of further processing.

A synchronous culture of this strain was subjected to a mutagenic treatment in 1 978 by utilizing N-methyl-N-nitroso-N'-nitroguanidine in a concentration which provoked a decrease in the number of living germs of at least three orders of magnitude. The surviving cells were segregated in a CMC-containing liquid culture medium, and inoculated onto a solid CMCcontaining Sabouraud medium.The cellulase activity (i.e. the capability of decomposing cellulose) of the resulting mutant colonies was determined with dinitrosalicylic acid, and the strain with the highest cellulase activity (Candida utilis var. cellulolytica) was selected and deposited at the National Collection of Microorganisms of the National Institute of Public Health (Orszagos KSzegBszsBgiigyi Intézet, M ikroorganizmusok Nemzeti Gyüiteménye), Budapest, under No. 00199.

The cells of this microorganism strain are ellipsoidal with a short diameter of 2.5-4.5 ,um and a long diameter of 4.5-7.5 ium. The strain forms white, convex colonies with shiny surfaces and smooth rims.

For partial hydration and degradation, cellulose is treated with a dilute mineral acid and/or a dilute base at a temperature of 80 to 100 C for 0.5 to 5 hours, depending on the type of the waste of plant origin. Dilute acids and bases with concentrations of 0.5-10% by weight are applied for this purpose.

The optimum concentration, as well as the optimum temperature and time of re-treatment are determined individually for each of the wastes of plant origin, depending on their chemical compositions and structures.

Depending on the type of waste processed, at least 25-60% of its dry substance content can be converted into fodder yeast and/or ethanol according to the method of the invention. The yields obtained when fermenting various wastes of plant origin with Candida utilis var.

cellulolytica, the strain utilized according to the invention, or with Torulopsis utilis, the strain applied conventionally for the production of fodder yeast, are given in Table 1. The amount of dry fodder yeast obtained from 100 kg of dry raw material is indicated in kilograms, whereas the amount of ethanol obtained from 100 kg of dry raw material is indicated in units of hl" (a yield expressed as litres of absolute ethanol).

Remark to Table 1: Kenaf is a fibre plant belonging to the family of malvaceae, and bagass is the residue obtained after pressing out sugar cane.

Table 1 Yield Starting substance and Candida utilis var. Torulopsis product cellulolytica utilis Fodder yeast from straw 9.6-11.2 6.4-7.8 Ethanol from straw 12.0-13.0 8.2-9.4 Fodder yeast from newsprint 13.2-14.0 8.6-9.2 Ethanol from newsprint 13.6-14.0 8.6-9.2 Fodder yeast from kenaf 16.0-18.0 9.2-10.0 Ethanol from kenaf 20.0-22.0 11.4-12.2 Fodder yeast and ethanol 9.0-10.0 4.0-5.0 from kenaf 8.0-10.0 3.5-4.5 Fodder yeast from a 75:25 mixture of sugar cane wastes 18.0-19.0 9.8-10.6 and bagass Ethanol from a 75:25 mixture of sugar cane wastes and bagass 22.0-23.6 1 2.2-1 3.6 The invention is elucidated in detail by the aid of the following non-limiting Examples.

Example 1 1000 parts by weight of kenaf are ground in a hammer mill to a particle size of 1-2 mm.

9000 parts by weight of a 3.2% aqueous sulfuric acid are added to the ground material, and the mixture is subjected to heat treatment at 96 C for 120 minutes in an acid-fast container.

Thereafter 7.2 parts by weight of superphosphate and 4.0 parts by weight of ammonium sulfate (technical quality) are introduced and dissolved in the mixture. The mixture is centrifuged and the pH of the supernatant is adjusted to 6.5 with soda lye of technical quality. The resulting culture medium is cooled to 37"C and inoculated with a preculture of Candida utilis var.

cellulolytica, containing 108 cells/ml, in a ratio of 1:100.

The pre-culture is prepared by dissolving the following components in 1 litre of distilled water: glucose 7.5 g carboxymethyl cellulose (water soluble) 7.5 9 potassium dihydrogen phosphate 0.2 9 dipotassium hydrogen phosphate 0.159 sodium dihydrogen phosphate monohydrate 2.0 g disodium hydrogen phosphate 1.5 9 magnesium sulfate heptahydrate 0.3 9 zinc sulfate 0.1 9 ammonium sulfate 3.0 g beef extract (Difco) 5.0 g Bacto peptone 4.0 g yeast extract (Oxoid L 21) 1.0 g ferric chloride hexahydrate 0.1 mg copper sulfate pentahydrate 0.6 mg manganese chloride tetrahydrate 0.15mug potassium iodide 0.05mg boric acid 0.1 mg The solution is filled into a flask, the flask is closed with cotton, and sterilized at 105or for one hour.This culture medium is inoculated at 37 C with 10 ml of a 24 hours old suspension of Candida utilis var. cellulolytica, and then incubated at 37 C. The resulting suspension, containing 108 cells/ml, is applied to inoculate the main culture.

Fermentation is performed under aeration with an oxygen excess of 1-2 mg 02/litre. With batchwise operation fermentation lasts for 36-40 hours. At the end of fermentation, i.e. when the maximum germ number is attained, the yeast can be filtered off on a vacuum drum filter or through a filter press, or can be dried directly by spray drying.

In this way 16.18 kg of dry fodder yeast can be obtained from 100 kg of kenaf.

Example 2 One proceeds as described in Example 1 with the difference that fermentation is performed under anaerobic conditions, i.e. without aeration. Fermentation lasts for 46-50 hours. The amount of alcohol distilled off from the fermentation broth is 20.0-22.0 litres, expressed as absolute ethanol.

Example 3 One proceeds as described in Example 1 with the difference that straw, pre-treated with 3.7% aqueous sulfuric acid at 98 C for 30 minutes, is applied as starting substance instead of kenaf.

After filtration the residue is treated with a 2.8% soda lye solution at 98"C for 42 minutes. Both the acid and the base are applied in amounts of 4500 parts by weight. Fermentation is performed at pH 6.3 for 46 hours. In this way 9.6-11.2 kg of dry fodder yeast are obtained from 100 kg of dry straw.

Example 4 One proceeds as described in Example 3 with the difference that fermentation is performed under anaerobic conditions, i.e. without aeration. After 52 hours of fermentation the broth is distilled. 1 2-1 3 litres of alcohol, calculated as absolute ethanol, are obtained from 100 kg of straw.

Example 5 One proceeds as described in Example 1 with the difference that newsprint is applied as starting substance instead of kenaf. The starting substance is pre-treated with a 3.6% aqueous soda lye at 98 C for 68 minutes. Fermentation requires 454 hours. In this way 13.2-14.0 kg of dry fodder yeast are obtained from 100 kg of newsprint.

Example 6 One proceeds as described in Example 5 with the difference that fermentation is performed under anaerobic conditions, i.e. without aeration. After 60 hours of fermentation 13.6-14.0 litres of alcohol, calculated as absolute ethanol, can be obtained from 100 kg of newsprint.

Example 7 One proceeds as described in Example 1 with the difference that a mixture of 75 parts of sugar cane waste and 25 parts of bagass is applied as starting substance. This mixture is pretreated with a 2.8% aqueous sulfuric acid solution at 96"C for 1 50 minutes. Fermentation requires 40-44 hours. In this way 18-19 kg of dry fodder yeast are obtained from 100 kg of the starting mixture.

Example 8 One prceeds as described in Example 7 with the differences that fermentation is performed under anaerobic conditions, i.e. without aeration. After 50-56 hours of fermentation 22.0-23.6 litres of alcohol, calculated as absolute ethanol, are obtained from 100 kg of the starting substance.

Example 9 One proceeds as described in Example 1 with the difference that the culture is aerated with an oxygen excess of 0.2 mg of 02/litre. In this instance 9-10 kg of dry fodder yeast and 8-10 litres of absolute ethanol are obtained from 100 kg of kenaf.

Claims

1. A process for the preparation of fodder yeast and/or ethanol from plants or agricultural and/or industrial wastes of plant origin, characterized in that the starting substance is pretreated with a dilute mineral acid and/or a dilute base, and then fermented under aerobic or anaerobic conditions in a manner known per se in the presence of a Candida utilis var.

cellulolytica strain deposited at the Hungarian National Collection of Microorganisms of the National Institute for Public Health (OKI) on 23rd September, 1 980 under No. CU 28 00199.

2. A process as claimed in claim 1, characterized in that agricultural cultivated plants and/or wild-growing plants or wastes thereof are applied as starting substances.

3. A process as claimed in claim 1, characterized in that a cellulose-containing industrial waste is applied as starting substance.

4. A process as claimed in any of claims 1 to 3, characterized in that the plants, agricultural wastes or cellulose-containing industrial wastes are pretreated with a 0.5-10%, preferably 0.5-4.0%, mineral acid and/or with a 0.5-10%, preferably 0.5-4.0%, base.

5. A process as claimed in any of claims 1 to 4, characterized in that pre-treatment is performed at 80-100 C, preferably 90-98"C.

6. A process as claimed in any of claims 1 to 5, characterized in that pre-treatment is conducted for 0.5-5 hours, preferably 0.5-3 hours.

7. A process as claimed in any of claims 1 to 6, characterized in that fermentation is performed at 33-39"C, preferably at 35-37 C.

8. A process as claimed in any of claims 1 to 7, characterized in that fermentation is performed batchwise or continuously.

9. A process as claimed in any of claims 1 to 8, characterized in the fermentation is performed batchwise for 16-72 hours, preferably 16-50 hours, depending on the type of the starting substance.

10. A process as claimed in any of claims 1 to 9 for the production of fodder yeast, characterized in that fermentation is performed under aerobic conditions so that the free oxygen content of the broth is 1.0-4.0 mg of 02/litre, preferably 1.5-2.5 mg of O2/litre of fermentation broth.

11. A process as claimed in any of claims 1 to 9 for the production of ethanol, characterized in that fermentation is performed under anaerobic conditions.

1 2. A process as claimed in any of claims 1 to 9 for the simultaneous production of fodder yeast and ethanol, characterized in that the fermentation broth is aerated so that the free oxygen content of the broth is 0-1 mg of 02/litre.

1 3. A process as claimed in claim 1, substantially as hereinbefore described in any one of Examples 1 to 9.

14. Fodder yeast and/or ethanol when prepared by a process as claimed in any preceding claim.