CA1178221A

CA1178221A - Process for the preparation of fodder yeast and/or ethanol from wastes of plant origin

Info

Publication number: CA1178221A
Application number: CA000392973A
Authority: CA
Inventors: Gyula Nagy; Robert Kerekes; Boldizser Vajda; Pel Somogyi; Judit Rezessy
Original assignee: AGRICULTURAL CO-OPERATIVE "JOZEF ATTILA"
Current assignee: AGRICULTURAL CO-OPERATIVE "JOZEF ATTILA"
Priority date: 1980-12-23
Filing date: 1981-12-22
Publication date: 1984-11-20
Also published as: FR2496690B1; CS974881A2; HU181771B; CU21613A3; SU1218927A3; FR2496690A1; PL129919B1; GB2090514B; CS241494B2; PL234381A1; GB2090514A; DE3151176A1; DD201694A5

Abstract

ABSTRACT OF THE DISCLOSURE

The invention relates to a process for the preparation of fodder yeast and/or ethanol from plants or cellulose-containing wastes of plant origin. Accor-ding to the invention the 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 Candide utilis var. cellulolytica strain deposited at the Hungarian National Collection of Microorganism 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. By use of a yeast strain which is both suitable for animal fodder and capable of decomposing hydrated and partially degraded cellulose, the process provides a way of producing animal fodder directly from cellulose.

Description

1 ~78~2~

PROCESS FOR ~H~ P~PARhTION OF ~ODDE~ Y~A~ AND/OR E~HANOL
FROM WAST~S OF PIA~ o~IGIN

~ he inventio~ ralate~ ~o a proce3~ for th~ prepara--t~on of ~oddsr ysast and/or ethanol ~rom waste~ of pla~t origin~
Pl~nb~, more particularly~ 25-~ % o~ bh0 dry ~ub-stanc~ con~nS o~ wasto~ o~ plant origin con~ist o~ c~llulo~.
Be~ide thi~ sub~tance, th~y al~o co~ lig~in in a~ amount o~ abou~ 20-40 %, furthermor~ small~r ~mountY of penbose pol~-: mer~ he~ose polymers and additlon~l inoxg~ic ~nd orga~ic su~tanca~O ~hera are two known w~ or th~ utilization o~ 10 plant~ and wasbes of plant origin b~ ~ermentation. One of themi~ the direcb ~ermentation of cellulo~e utiliz ing callulolytic (i~,e. ~elluloss-decomposing) microorganisms~ whereas the other po~ibility i~ to convert ~;ellulose f irst iuto glucose by acidic or enzymatic degrad3tlo~ and than to subject the result-ing glucose to fermentation~
The choice oX microorga~isms capable of tha direct fsrm~ntation of cellulosa is rathar restricted, and even they permit only a very 910w decompo~ition of callulose. 0~ the cellulolytic microorganism~ bacteria, such as those balonging to the Cellulomonas and Callovibrio species~ furthermore r ~ ~

_ 2 - l ~ 782;~ 1 ~

yeas~s, e~g. Myrrothacium verrucaria, Trichodsrma virids, etc.
are to be mentio~ed.
In orde:r to prep~re single cell protein ~ SCP) b;sT
applylrlg cellulolytic microorganisms e~periments were performed 5 by Ch. i~. Dunlap (Am. Chem. Soc. Symp. 1970) with Cellulomo~s and ~lcaliga~as strains. ~. Kargi and M.L. Schuler ~iotechn., a~d Bioeng. Vol~ ~XII~ 1567-1600 (lg8o27 utilized a mutan~ o~
Pseudomonas ~luoresce~ in their ~esSsr A~tempts to prepara ~ingle cell protains by appl~ing bacteria and mould ~ungi ar~ getting 18ss and less important, ~ince kh~ u~a o~ these microorga~isms as Yodder prote~n i9 objectioDable Yrom veterinary aspect3~
~ he pos~ibil~ties ~or ukilizing celluloso by ~erme~ta-bion ara ~ar broader wh~n callulose is hydrolys~d firs~ into glucose 9 ~inc/~ alMo9~ 811 microorganisms are cap9ble o~
assimilating glucose~
Sholler and ~horne~ wera the ~irst to elaborate a~
indu~trial process at the turn o~ th~ ce~tury ~or the to~al hydxol~9is of cellulose~ In thi~ process mi~eral acids o~ medium conce~tration and temperatures abovs 100C were applied. ~hi~
proce~s wa 9 ~ however, vsry energy-intensive and req~lired e~pensive acid- and pressure-rssi~tant apparatuses. Although the proce~s had later been moderni7.ed (acid concentration and tsmperature were reducad~, no new plants based on this process ars built.
~ ests have been per~or~ed since 1950 to hydrolyse cellulose with cellulase enzyme preparations obtained ~ro~
~ungi ~ . Hagerdal et al.: Biotechn. and Bioeng. XXII, 1515 1526 and 1527 1542 ~1980 ~ . ~ha enzyme systam o~ ~richoderma viride is applied generally to hydrolyss cellulosa ~ ~ Stern-1 178~21 berg: Biotechn. and Bioeng. Symp. No. 6, 35-53 (1976)].
The enzymatic hydrolysis of cellulose is an expensive process, profita-ble generally only when the glucose solution obtained by hydrolysis can then be converted into valuable products.
When elabora-ting the process according to the invention, the major asp-ect considered was that the microorganisms applied for the preparation of SCP are acceptable from the aspects of animal foddering, and are capable of the utiliza-tion 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 have been reported in the literature, either.
Now i-t has been Eound, unexpectedly, that a Candida utilis strain iso-lated by us in 1978 by genetic and strain selection methods i5 capable of decom-posing 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.
According to the present invention there is provided 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 pre-treated with a dilute mineral acid and/or a dilute base, and then fermen-ted under aerobic or anaerobic conditions in the presence of a Candida utilis var. cellulolytica strain deposited at the Hungarian National Collection of Mic-roorganisms of the National Institute for Public Health (OKI), Budapest, on 23rd September, 1980 under No. CU 28 00199.
The starting substance may be, for example, agricultural cultivated 1 1~8~.2~

plants and/or wild-growing plants or was-tes thereof OL a cell~lose-con-taining indusirial w~ste.
The pretreatment is preferably conducted with a 0.5-10% mineral acid and/or with a 0.5-10% base, more preferably a 0.5-4.0% mineral acid and/or a 0.5-4.0% base. The duration of the pretreatment is preferably from 0.5-5 hours, more preferably from 0.5-3 hours. The fermentation is preferably conducted at 33-39C, more preferably at 35-37C and may be continuous or batchwise. If batchwise, then the duration of -the fermentation is preferably 16-72 hours, more preferably 16-50 hours depending on the type of starting substance.
Fermentation may be performed under aerobic conditions, for example so that the free oxygen content of the broth is 1.0-4.0 mg, preferably 0-1 mg, of O2/litre of fermenta-tion broth, or, to produce subs-tantially ethanol, under anae-robic conditions.
The CU 2~ 00199 s-train was isolated from a field strain as follows:
About l g of punk ~ood 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) con-taining Sahouraud* agar. (CMC-containing Sabouraud* agar corresponds in composi-tion to the Oxoid* CM 41 culture medium with the difference that it contains CMC
instead of dextrose). The plates were incubated for 72 hours at temperatures of 25, 30 and 37C. 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 followinq composition:
beef extract (Difco*) 5.0 g glucose 7 5 g carboxymethyl cellulose 7.5 g * Trademark l 17~2~1 Bacto peptone (Difco*) 4.0 g yeast extract (Oxoid* L 21) 1.0 g potassium dihydrogen phosphate 0.2 g dipotassium hydrogen phosphate 0.15 g 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 mg copper sulfa-te 0.6 mg * Trademark - 4a -.~

_ 5 _ ~7~22 mangano~e chloride 0.15 m~
potassium iodide 0005 m~
boric acid 0.1 mg agar 15 g water q.8. ad 1 libra ~ho ~trains d~v~loped ~rom sin~le-cell cultures were isolabed by conv~ntional puri~ication l;echniques and mai~tained o~ a glQcose- and CMC-containing culture medium.
0~ the i~olatad strain~ th~ one with the be3t CMC-de-composi~g ability on tha C~C~contai~ing Sabouraud medium was ~elected ~or th~ purpose of ~ur~her proces~i~g, A ~ynchronous culture o~ this strai~ wa~ subject0d to a mutag~ic treatmenb in 1978 by utilizing N-methyl~ itro~o-~t~ibroguanidi~ in a conce~trabion which pxovokad a decr~ase in the number o~ living germs o~ at lea~t thro~ orders o~
mag~itude. ~he surviving call~ wer~ sagr~ga~ed in a CMC-contain-ing liquid culture mediumt and inoculat~d onto a solid C~C-conta~ning S~bouraud medium~ ~he cellulase activi~ (i.e. the capability of d~composing cellulose) o~ the resulting mutan~
coloni~ was det~rmined with dinitrosalicyLic acid, and ~he strain with the highest cellulase activity (Ca~dida utilis var~ cellulolytica) ~as selected and deposited at ~he ~ational Collection of Microorganisms o~ the ~ational Institute of Public Health ~Orszagos Kozegészségugyi I~tézet~ ~ikroorganiz-musok ~emzeti G~ujteménye), Budapest, undar No. 00199~
The cells of this microorganism strain are ellipsoidal ~ith a short dia~netar of 2.5-4.5 ~um and a lo~ diameter of 4.5_7.5 um. The strain ~orm~ whi~e, convax colo~ics with shi~y surfaces and smoo~h rimsO
For partial hydration and degradation, cellulose is - 6 ~ c~ 2 2 ~ ~

treat0d with a dilute mineral acid and/or a dilute base at a tamperature of 80 to L00C for 0.5 to 5 hours, dependinæ on the type o~ the waste o~ plant originO Diluta acids and bases with concontrations of 0.5-10 ~ by wsight a.r~ applied for this purposeO
The op~imum concentration, as well ~9 the op~imum temperat~re and ~lme o~ rs-trsatment are debermined individualLy for each o~ tha wastes o~ plant origin, depending on thsir chemical compositions and structures.
De`pe~diDg on the t~pe o~ the waste proces~led, ab lea~t 25-60 % o~ it~ dry ~ubstancs content can be con~orted into fodder yea3b and/or ethanol according to the method o~ the invention. The yields obtained when ~ermsnting various waske~
o~ plant origin with Candid~ utilis var. cellulol~tica, ~he ~train ubilized according to the inven~ic)n, or with ~orulopsis utilis, the strain appli~d conventionally for ~hs produation o~ ~odder yeast, axo given in ~able lo ~he amount o~ dry fodder yeast obtained Prom 100 kg of dry raw material is indicated in kilograms~ wheraas the amount o~ ~thanol obtained from 100 kg of dry raw material is indicatsd in units o~ hl (a yield ~xpressed as li5res of absolute ethanol).
Remar~ to Table 1: Ke~a~ is a fibre plant bslonging to the family o~ malvaceae, and bagass is the residue obtained after pressi~g out sugar cane.

1 ~78221 Table 1 __ _ _ ~__ Y i e 1 d Starting substance and Candicla utilis var.Torulop~is 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 Erom kenaf 16.0-18.0 9.2-10.0 Ethanol from kenaf 20.0-22.0 1l.4-12.2 Fodder yeast and c-~thanol 9.0-10.0 4.0- 5.0 from kenaf 8.0-lO.0 3.5- 4.5 Fodder yeast from a 75:25 mlx-ture of sugar cane wastes ~ 18.0-19.0 9.8-10.6 and bagass Ethanol from a 75:25 mixture 22.0-23.6 12.2-13.6 of sugar cane wastes and bagass 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 part-icle 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 96C 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 centri-fuged and the pH of the supernatant is adjusted to 6.5 with soda lye of techni-cal quality. The resulting culture medium is cooled to 37C and inoculated with a preculture of Candida utilis var. cellulolytica, containing lO

,~, cells/ml, in a ratio of l: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 g potassium dihydrogen phosphate 0.2 g dipotassium hydrogen phosphate 0.15 g sodium dihydrogen phosphate monohydrate 2.0 g disodium hydrogen phosphate 1.5 g magnesium sulfate heptahydrate 0.3 g zinc sulfate 0.l g ammonium sulfate 3.0 g beeE extract ~Difco) 5.0 g Bacto peptone ~.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 0.15 mg potassium iodide 0.05 mg boric acid 0.1 mg The solution is filled into a flask, the flask is closed with cotton, and sterilized at 105C 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 10 cells/ml, is applied to inoculate the main culture.
Fermentation is performed Lmder 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 .1 - 9 - ~178221 ~.
be filterod of~ on a vacuum drum ~iltsr or through a filter press, or can be dried directly by spray dryi~g.
I~ this way 16-18 kg of dry foddsr y~ast can be obtained ~rom 100 kg of L~ena~.
Exam ~le 2 One proc0eds as de~cribsd in ~xarnple 1 wi~h tha di~erence that ~ermentation i9 performed under anaerobic condi-tion~, i.c. without aaration. Fermentation lasts for L~ 50 hours.
The amou~t o~ alcohol distillsd o~ from the ~ermentation broth is 20.0~22.0 litre~, expres~ed as absolute ethanol ~a~
O~e proceeds as dascribed in ~xampl~ 1 wlth ~he di~ere~ce that ~traw, pre-treatcd with 3.7 % 8~U~OU9 sul~u~ic acid at 98C ~or 30 minutes~ is appli0d as star~i~g ~ubsta~c~
i~stead o~ ke~af. Aft~r Yiltratio~ th~ residue ls treated with a 2~8 ~ soda lye solution at 98C for 42 minutes. Both the acid and the baso are applied i~ amounts o~ 4500 part~ by weight.
Ferme~tatio~ is performed at pH 6.~ for 46 ho~ ~. In this way 9.6-11.2 kg o~ dry ~oddsr yeast are obtained from 100 kg of dr~ stra~.
~x~ 4 One procaeds as described in ~xample 3 with the difference that fermentation i9 performed u~der anaerobic conditions, i.a. without aaration. ~ter 52 hours o~ fermenta-tion the broth is distilled. 12-13 litres of alcohol, calculat-ed as absolut~ ethanol, ara obtained from 100 kg of straw.

One proceeds as dsscribed i~ ~ample 1 with the difference that newsprin* is applied as starting substance ~0 instead of kena~. ~ho starti~g substance i9 pre-~r~ated with - 10 - 1~782~

a 3.6 % aqueous soda lye at 98C for 68 minutas. Fermentation requires 454 hours. In this wa~ 13.2-14.0 kg o~ dry ~odder yaast are obtained ~rom 100 kg oP naw~pri~t.
~a~
0~9 procoeds a~ described in ~Xample 5 with the di~ference tha~ fermenbatlon is per~ormed under anaerobic conditions9 i.e. without aeration. After 60 hour~ o~ fermenta-tio~ 13.6-14~0 litres o~ alcohol, calculated as ab~olute othanol, ca~ be o~tained ~rom lO0 kg of ~ew~print9 ~Z
One proceeds a~ do~cribed in E~ampl~ l wibh the di~ere~ce that a mi~ture o~ 75 p~rts o~ ~ugar ca~e wa~te and 25 p~rts of baga~s i~ appli~d as starting ~ubsta~lc~ Thi~ mix-ture i~ pre~breat~d with a 2~8 ~ aqueous ~ul~uric aaid ~olution at 96C for 150 minute~0 Fermentation requires 40-44 hours.
In thi~ wa~ 18-l9 kg o~ dry fodder yea~t are obtain~d ~rom 100 kg of tha ~tarting mixture.
ample 8 O~e proceeds a~ described in ~xample 7 with the diff~rsnce that ~ermentation i~ performed under anaerobi¢
conditions 9 i~e. wi~hout aerationO Af~er 50-56 hours of f~rmenta-tio~ 22.0-23.6 litres o~ alcohol, calculated as absolute ethanol, are obtained ~rom lOO kg of the starting substance.

One proceeds as describ~d in ~xample l wi~h tha dif~er~nce that the culture is aerated with an oxygen excess o~ 0.2 mg o~ 02/litre0 In this instance 9-lO kg of dry fodder yeast and 8-lO litres of absolute ethanol are obtained ~rom 100 ~g of ~ena~.

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

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 pre-treated with a dilute mineral acid and/or a dilute base, and then fermented under aerobic or anaerobic conditions in the pre-sence of a Candida utilis var. cellulolytica strain deposited at the Hungarian National Collection of Microorganisms of the National Institute for Public Health (OKI), Budapest, on 23rd September, 1980 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-con-taining industrial waste is applied as starting substance.

4. A process as claimed in claim 1, 2 or 3, characterized in that the plants, agricultural wastes or industrial wastes of plant origin are pre-treated with a 0.5-10% mineral acid and/or with a 0.5-10% base.

5. A process as claimed in claim 1, 2 or 3, characterized in that the plants, agricultural wastes or industrial wastes of plant origin are pre-treated with a 0.5-4.0% mineral acid and/or with a 0.5-4.0% base.

6. A process as claimed in claim 1, 2 or 3, characterized in that pre-treatment is performed at 80-100°C.

7. A process as claimed in claim 1, 2 or 3, characterized in that pre-treatment is performed at 90-98°C.

8. A process as claimed in claim 1, 2 or 3, characterized in that pre-treatment is conducted for 0.5-5 hours.

9. A process as claimed in claim 1, 2 or 3, characterized in that pre-treatment is conducted for 0.5-3 hours.

10. A process as claimed in claim 1, 2 or 3, characterized in that fermen-tation is performed at 33-39°C.

11. A process as claimed in claim 1, 2 or 3, characterized in that fermen-tation is performed at 35-37°C.

12. A process as claimed in claim 1, 2 or 3, characterized in that fermen-tation is performed batchwise.

13. A process as claimed in claim 1, 2 or 3, characterized in that fermen-tation is performed continuously.

14. A process as claimed in claim 1, 2 or 3, characterized in that fermen-tation is performed batchwise for 16-72 hours.

15. A process as claimed in claim 1, 2 or 3, characterized in that fermen-tation is performed batchwise for 16-50 hours.

16. A process as claimed in claim 1, 2 or 3, 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 O2/litre of fermen-tation broth.

17. A process as claimed in claim 1, 2 or 3, 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 1.5-2.5 mg of O2/litre of fermen-tation broth.

18. A process as claimed in claim 1, 2 or 3, for the production of ethanol, characterized in that fermentation is performed under anaerobic conditions.

19. A process as claimed in claim 1, 2 or 3, for the simultaneous product-ion of fodder yeast and ethanol, characterized in that the fermentation broth isaerated so that the free oxygen content of the broth is 0-1 mg of O2/litre.