DD201694A5 - METHOD FOR PRODUCING FEED LEAD AND / OR AETHYL ALCOHOL FROM PLANT WASTE - Google Patents

Abstract

The invention relates to a process for the production of feed yeast and / or ethyl alcohol from vegetable waste materials, cellulose-containing materials of plant origin (for example also paper) and agricultural wastes. According to the invention, the wastes are pretreated with dilute mineral acid and / or diluted caustic in the heat and then with the deposited on September 23, 1980 under the number CU 28 00199 at the Hungarian National Collection (OKI) strain Candida utilis var. Cellulolytica in per se Fermented known manner. If the fermentation is carried out under aerobic conditions, feed yeast is produced, which can be dried after being filtered off or directly by atomization drying. Fermentation under anaerobic conditions produces ethyl alcohol, while fermentation under moderate ventilation (up to 1 mg Otief 2/1) leads to the simultaneous production of feed yeast and ethyl alcohol.

Description

2359 84 2

METHOD FOR PRODUCING FEED LEAD AND / OR ETHYL ALCOHOL FROM PLANT WASTE

Field of application of the invention; ,

The invention relates to a method for producing.

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feed and / or ethyl alcohol from vegetable waste.

Characteristic of the known technical solutions:

The plants, more precisely: 25-40 % of the dry matter

They also contain lignin in an amount of about 20-40 % and lesser amounts of pentose polymers, hexose polymers and other inorganic and organic substances.

A 2240-3916 To - '-

For the utilization of plants and vegetable waste by fermentation, two ways are known «. One of them is: the direct fermentation of cellulose with. Help of celiulose-degrading (cellulolytic) microorganisms. The other possibility is to break down the cellulose into glucose, acid or enzymatically, and. to ferment the obtained glucose.

There are only a few microorganisms capable of direct fermentation of cellulose, and these organisms are only very slowly able to decompose cellulose. Cellulosezersetzende. Microorganisms are for example of the bacteria the Cellulomonas and Cellovibrio kinds, of the yeasts for example Myrrothecium verrucaria, Trichoderma viride etc ...

For the production of microbial protein by using cellulose degrading microorganisms, Ch.E »Dunlap: (a * Chem., Soc. Symp., .97θ) with the strains Cellulomonas and. Alcaligenes experiments: F., Kargi and M..L * Schuler (-Biotechn .. and: Bioeng * Vol. XXII, 1567-1600 ,. Ι98θ) experimented with a mutation,.,,, Of Pseudomonas , fluorescens *

The attempts to produce microbial protein (Single Cell: Protein, .SCP) with the aid of bacteria and molds are gradually becoming less important, because the use of these microorganisms as feed protein is objectionable from a veterinary point of view.

The possibilities for the utilization of cellulose by fermentation are much wider, if the cellulose is first hydrolysed to glucose, because the glucose is assimilated by almost all micro-organisms ...

At the turn of the century, Sholler and Thornes were the first to develop a process for the total hydrolysis of cellulose ... In this process, mineral acids were used

medium concentration and temperatures above 100 C applied. The process was therefore very energy-consuming, and acid and pressure resistant devices were needed to carry it out. Although the process was later modernized (acid concentration and pressure could be reduced), new factories operating on this process are no longer being built.

Since 1950, attempts have been made to hydrolyze the cellulose using Enzynis cellulase produced by fungi (B. Hagerdal et al .: Biotechn. And Bioeng XXII, 1515-1526 and 1527-1542, 1980). For hydrolyzing the cellulose, the enzyme system of Trichoderma viride is generally used (D. Sternberg, Biotechn. And Bioeng., Symp., No. ', 35-53, 1976).

The enzymatic hydrolysis of cellulose is an expensive process. It pays ..im general only of _K when the glucose solution obtained by .the hydrolysis can be weiterve'rarbeitet .to an expensive product. ,., ... ί.

In the preparation of the process of this invention, it has been understood that, for the production of SCP, only microorganisms which are acceptable for the purposes of animal feed and can utilize a wide variety of vegetable components other than lignin can be used.

The utilis species belonging to the species Candida and Torulopsis have been used as feed yeasts for over ten decades. These known yeasts can not decompose either the lignin or the cellulose. Also in the literature so far no Hsfeart has been described -worden., Which is able to decompose cellulose.

Surprisingly, it has now been found that the

Isolated in 1978 using genetic and strain selection methods: strain of Candida utilia able to grow the hydrate and partially degraded cellulose _a . For the hydration and degradation of the cellulose, a simple pretreatment process has been developed, with the aid of which a nutrient solution suitable for the yeast strain mentioned can be prepared from the cellulose-containing material.

Explanation of the essence of the invention:

The invention accordingly provides a method

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for the production of: feed yeast and / or ethyl alcohol from vegetable waste «is characteristic of the process. that the pretreated with dilute mineral acid and / or diluted lye vegetable waste with the on the 23. September 1980 under the number CU 28 00199 at the Hungarian National. .Stammsammlung-. (0Kl) _: deposited strain Candida utilis var. Ce.llulo.lyti.ca. under aerobic or anaerobic conditions; in: itself, known manner · fermented *

The 'Stamm.CU 28 was isolated from a Feldstamra. _; l, -g rotted wood: was homogenized with 9 ml of sterile physiological saline solution, - became from the solution; a, decimal serial dilution: prepares, was inoculated with the CMC-containing .Sabouraud agar * (The Carboxymethylcellulose-containing .. Sabouraud agar corresponds in composition to the nutrient medium Qxoid CM 41 with the difference that it contains carboxymethylcellulose instead of dextrose. ") The plates were incubated at 25, 30 and 37 C 'for 72 hours *. Using cell morphological methods, the yeast cultures grown on the plates were selected and inoculated onto a nutrient medium containing glucose and carboxymethylcellulose of the following composition.

L ό D a ö k I

Beef extract (Difko) 5.0 g _: '. ''"

Glucose 7.5 g

Carboxymethylcellulose. 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 0.15 g

Sodium dihydrogen phosphate 2.0 g

Disodium hydro gen phosphate 1.5 g

Magnesium sulfate 0.3 g;

Zinc sulfate. 0.1 g

Ammonium sulfate 3.0 g

Iron (III) chloride. _u . 0.1 mg

Copper sulfate ., ... 0.6 mg

Manganese chloride. 0.15 mg.

Potassium iodide .... 0.05 mg

Boric acid '0.1 mg

Agar-agar _: 15 g water ad 1 liter

The usual purification methods were cultivated from single-cell cultures and maintained on glucose and carboxymethylcellulose-containing nutrient medium.Of these isolates, the one culture which best degraded the carboxymethylcellulose was selected.

The synchronous culture of this strain was mutagenized in 1978 using an N-methylnitroso-N'-nitroguanidine concentration which reduced the number of living germs by at least three orders of magnitude. The surviving cells were segregated in a liquid diet containing carboxymethylcellulose and inoculated onto solid, CMC-containing Sabouraud broth. The cellulase

Activity (d "h, - the ability to decompose cellulose). of the resulting mutant colonies was determined with dinitro-salicylic acid, and the strain with the greatest cellulase activity (Candida, utilis var. cellulolytica) was selected, and No. ₀ 00199 at the National Microorganism Collection of the State Institute for Health Care (Orszagos Közegeszsegügyi Intezet Mikroorganizmusok Nemzeti Gyüjtemenye) deposited *

The cells of the said strain have ellipsoidal shape, the small diameter is between 2 ", 5-4.5, um, the large diameter between 4, .5-7.5, um, the stem with white, convex colonies shiny surface and smooth edges *. -

For hydration and partial degradation, the cellulose is treated with dilute mineral acid and / or diluted leach at temperatures between 80 and 100 ° C: treated by the nature of the vegetable waste for 0.5-5 hours. "Acid and lye have a concentration from 0.5-10% by weight,

The optimum concentration as well as the temperature and duration of the pretreatment are determined individually for each case, depending on the chemical composition and the structure of the vegetable waste.

Depending on the type of plant waste, at least 25-60 % can be converted by bioconversion to feed yeast or Athylalkoh 1. The following is a comparison between the usual for the production of forage yeast strain Torulopsis utilis and the strain Candida utilis var. Cellulolytica drawn. In the table, the amount of dried feed yeast obtainable from 100 kg dry raw material is in kg and the quantity of 100 kg

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On dry raw material recoverable ethyl alcohol in hl (expressed in liters of absolute alcohol yield) ange- 'given.

Starting material and product. yield

Candida utilis Torulopsis var. Cellulo-utilis lytica

Feed yeast from straw 9,6-11,2 6,4-7,8

Straw alcohol 12.0-13.0 8.2-9.4

Feed yeast

Newsprint 13.2-14.0. 8.6-9.2 '

Ethyl alcohol

Newsprint 13,6-14,0 8,6-9,2

Feed yeast from Kenaf 16., 0-18.0 9.2-10.0

Ethylene alcohol from kenaf _: 20.0-22.0 11.4-12.2

Feed yeast and ethanol from Kenaf 9,0-10,0 4,0-5,0

8.0-10.0 3.5-4.5

Feed yeast from the prepared in the ratio 75:25. 18.0-19.0 9.8-10.6 Mixture of sugar cane waste and bagas

Ethyl alcohol from the im

Ratio 75:25 ready 22,0-23,6 12,2-13,6

teten mixture of, sugar:. Rohrabfall and Baqas

- Kensf is a fiber plant, Bagas the remaining after pressing the sugarcane rest » embodiments:

The invention will be apparent from the following examples

explained in more detail, but is not limited to these 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 3.2% sulfuric acid are added to the material, and the mixture is heat treated in an acid-proof container at 96 ° C. for 120 minutes. Then, 7.2 parts by weight of superphosphate and 4.0 parts by weight of added technical ammonium sulfate and dissolved. The solution is centrifuged, its pH adjusted to 6.5 with technical sodium hydroxide solution. The nutrient solution prepared in this way is cooled to 37 C and inoculated in a ratio of 1: 100 with a preculture of Candida utilis var. Cellulolytica containing per milliliter of cells.

The preculture is prepared as follows. , Glucose 7.5 g

Carboxymethyl cellulose 7.5 g

(water soluble)

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 sulphate heptahydrate 0,3g

Zinc sulfate. 0.1 g

Ammonium sulfate 3.0 g

Beef Extract (Oifco) 5.0 g

Bacto peptone 4.0 g

Yeast Extract (Oxoid L 21), -. ,,,. ,,, -;. ^ ,;.,., · .. 1,0. g iron (III) 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

are dissolved in 1 liter of distilled water. The solution is sterilized in a flask sealed with cotton plaques at 105 C for one hour. The Nährlösung.wird at 37 ⁰ C.mit 10 ml of a 24 hour old.

235 9

Inoculated suspension of Candida utilis var, celulolytica and then incubated at 37 C .. With the suspension containing per ml ml cells, the nutrient solution of the main culture is inoculated.

The fermentation is carried out under aeration with an oxygen excess of 1-2 mg Op / 1, In the case of discontinuous fermentation, the fermentation takes 36-40 hours, At the end of the fermentation (after reaching the maximum number of bacteria), the yeast can be filtered on a vacuum drum filter or a filter press or dried directly by spray drying.

From 100 kg kenaf can be obtained in the manner described 16-18 kg of dried feed yeast,

Example 2 .

The procedure described in Example 1 with the difference that fermented under anaerobic conditions, without aeration .. The fermentation takes 46-50 hours, The distillable from the fermentation broth amount of alcohol is - converted into absolute alcohol - 20.0-22 , 0 liters.

Example 3

It works in the manner described in Example 1, but uses as raw material instead of kenaf straw. Pre-treated with 3.7% sulfuric acid at 98 ⁰ C for 38 minutes. After filtration, the residue is treated with 2.8% sodium hydroxide solution at 98 ° C for 42 minutes. Both acid and alkali were used in an amount of 4500 parts by weight, and fermentation was carried out at pH 6.3 for 46 hours , From 100 kg of dry straw, obtain 9.6-11.2 kg of dry forage yeast.

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Example 4

The procedure described in Example 3 with the difference that one carries out the fermentation without aeration, under anaerobic conditions, after 52 hours of fermentation, the culture broth is distilled, converted to absolute alcohol from 100 kg straw 12-13 liters of alcohol obtained »

Example 5 ,

It works in the manner described in Example 1, but uses instead of kenaf as starting material newsprint, This is pretreated at 98 ⁰ C with 3.5% sodium hydroxide solution for 68 minutes * The fermentation tion lasts 54 hours .. From 100 kg of newsprint 13 , 2-14.0 kg of dry feed yeast,

Example 6

The procedure described in Example 5 with the difference that one carries out the fermentation without aeration, under anaerobic conditions. After fermentation for 60 hours, from 100 kg of newsprint, alcohol can be obtained in an amount of 13.6-14.0 liters (abs,).

example 7 '

The procedure is as described in Example 1 with the difference that the starting material is a mixture of 75 parts sugar cane crop residues and 25 parts Bagas used., Pretreated at 96 ⁰ C with 2.8% sulfuric acid for 150 minutes. Fermentation takes 40-44 hours »From 100 kg of the mentioned mixture 18-19 kg dry feed yeast can be produced.,.

Example 8

The procedure is as described in Example 7 with the difference that the fermentation

under anaerobic conditions, without ventilation. The fermentation takes 50-56 hours »From 100 kg of the starting material 22,0-23,6 liter of absolute alcohol can be produced. -

Example 9 .

It works in the manner described in Example 1, but aerated the culture with an oxygen excess of 0.2 mg Op / 1 · In this case, from 100 kg of kenaf 9-10 kg of dried feed yeast and 8-10

11 ..

Liter abs. Obtained ethyl alcohol.

Claims (5)

Erfindungsanspr-uch 1 * Method for producing forage yeast and / or Ethyl alcohol from vegetable, agricultural and / or industrial waste, characterized in that the waste is pretreated with dilute mineral acid and / or dilute liquor and then with the on September 23, 1980 under the number CLJ.28 00199 in the Hungarian National Collection ( OKI) deposited strain Candida utilis var _o cellulolytica fermented under aerobic or anaerobic conditions in a conventional manner. 2 "Process according to item 1, characterized in that the starting material used is agricultural crop plants and / or wild plants or their waste" 3 «Pufl ^^ __1 method, characterized in that the starting material used is cellulosic industrial waste * 4, Method according to one of the items 1-3, characterized in that the plants, the agricultural and the cellulosic industrial waste with 0.5-10%, preferably 0.5-4.0% mineral acid and / or with Pretreated 0.5-10%, preferably 0.5-4.0% lye. 5. The method according to any of ' points 1-4, characterized in that one carries out the pretreatment at 80-100 ⁰ C, preferably 90-98 C. 6 ". Method according to one of the items 1-5, characterized in that pretreatment is carried out for 0.5-5 hours, preferably for 0.5-3 hours. _Ί ο A method according to any one of the I - punk te ^ 1-6, characterized in that one carries out the fermentation at 33-39 ⁰ C, -preferably at 35-37 C. 8 · Method according to one of the j-. Points JX-7, characterized in that one carries out the fermentation batchwise or continuously. 9. Method according to one of! Points 1-8, by characterized in that in the case of discontinuous fermentation, the fermentation time depending on the starting material 16-72 ¹ hours, preferably 16-50 hours, towers. ^: 10. Method according to one of! 'Points -51-9, characterized in that for the production of forage yeast during the fermentation of. Content of the ferment broth of free oxygen 1.0-4.0 mg O ₂ /! ,, preferably 1.5-2.5 · mg 0 ₂ / l, is. , , . 11. A method according to any one of the following ^: characterized in that for the production of ethyl alcohol the fermentation is carried out under anaerobic conditions. '. '. · -. 12. The method according to [j ^ jl-jtolc 9, characterized in that is ventilated for the parallel preparation of fodder and ethyl alcohol die.Fermentbrühe so that its ^G ehalt free oxygen 0-1 mg 0 ₂ / l bet rag t.