CA1106786A - Method for the production of a bio-mass having a high proteinic content - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
A bio-mass having a high protein content.
is produced by culturing a yeast strain of the genus Candida, having the International Deposit Number NRRL-Y 11062, in a saline solution containing methanol as the only source of carbon and energy, at a temperature of from 20° to 35°C and at pH of from 2.5 to 6.5. The bio-mass obtained can be used as such as proteinic additive for foods and animal feeds, or may be further processed to extract therefrom proteins, aminoacids and nucleic acids.

Description

ri~

CI~S~

This invention relate to th~ production of a microbial bio-mass by culturing a novel yeast ;train which is capable of èxploiting methanol as the sino-le sc~urce of carbon and energyO
I On account o~ the high velocity of reproduction or the ¦micro-organ sms and of their proteinic contents~ the production of microbial bio-masses is a very quick protein-producing mcthodO
For the production of bio~masse3~ there have been exploited in the past scrap carbohydrates such as molasses from sugar ~orks or spent sulfite cooking liqucrs from paper mills.
In more recent times~ on the basis of the considerable availability and the low price of petroleum7 bio-mass pro~uc-tion method3 have been adjusted which use as their substrate either the raw fractions of the petroleum or highly purified mixture of normal paraffins.
The use of such petroleum-based substrates lnvolves a few shortcomin~s from a technological stalldpoint~ which are due to their insolubility on water~ the considerable amount of oxygen required for their assimilation by he micro-organisms and the large heat-build-up during fermental;ion. In addition~ the running costs in the production of -khe bio-masses are boosted by the necessity of purifying the substrate thoroughly and/or careful-ly washing the as-produced bio-mass in order to remove the potential~y harmful petroleum componentsO
; Such difficulties are not experi~nced if the production of bio-masses is carried out by using as the substrates the lower alcohols such as metXanol or ethanol~. As a matter of fact~ their complete solubility in water~ the volatility and the fact of being available at a high degree of purity make it possible to obtain a bio-mass which is exempt from undesirable residues.
Their miscibility with water offset~ the mixing problems which are encountered with the petroleum fractions~ whereas~ on account of the fact that they already contain oxygen in their molecules~ the ~ .

oxygen demand for their assimilatlon is thereby reduced: for this fact a further advantage stems, -that is, that the produc-tion of the bio-mass is accompanied by a reduced heat build-up, the result being a reduction of the cooling costs.
Ethanol is utilized by a large number of micro-organisms and would be the ideal substrate for the production ofbio-masses, but its price is comparatively high. Conversely, methanol can be produced cheaply and at a high degree of purity. This fact gives a reason for the endeavors which have been made in order to find micro-organisms which are capable of efficiently exploiting methanol. With the technical and patent literatures disclosed may bacteria which fulfil these requirements, whereas the efficiency of the yeasts described heretofore is rather low. By enriching the culturing medium and isolation in continuous culture, the present applicants have succeeded in isolating a novel yeast strain (SP M 180 cc) which is the subject-matter of the present invention and is capable of exploiting methanol as a single source of carbon and energy.
The novelty of this strain will become apparent from the specification of its characteristics.
The strain SP M 180 cc is reproduced by multipolar germination and forms discrete ovoidal cells, or tufts made up by a number of elongate cells (pseudomycelium). The novel strain was deposited January 6, 1977 at the Northern Regional Research Laboratory of Peoria, Illinois, and bears deposit number NRRL-Y
11062.
In a liquid culturing medium, it forms a sediment, where-as, in a solid medium there are either smooth and glossy colonies, or opaque and wrinkled colonies. By subculturing the strain on a solid medium, the strain takes more and more a smooth and glossy appearance, which corresponds, in a liquid medium, to the discrete cells, whereas, by cul-turing the strain in a liquid medium under certain conditions, by pseudomyceliar form predominates, which _ 3 _ corresponds to the wrinkled and opaque cells. No spores, of any type, have ever been observed. On the basis of such morphological characteristics, it is thought that the strain belongs to the genus Candida, according to the classification suggested by J. Lodder, ed.: The Yeasts : a taxonomic Study, 1970.

- 3a -~L~2 67~ :i The physiological character:;.stics of tho str~ SP M 180 cc are the follo~ing 1. FE~ENT~l'IVE UTILIZ~TI0N 0E A Fe~ c~Rr~ol~ sn-uRcEs :
1 D-glucose ¦ D-galactose Saccharose Ma].tose Trealose ..
. Lactose

2. GR0WTH :
D-glucose -~
D-galactose l-sorbose Saccharose ~
Maltose Cellobiose Trealose ~-Lactose Melibiose Raffinose ._ Melezitose Inulin Starch D-xylose -~
l-Arabinose ~-D-Arabinose D Ribose -~ (weak) l-Rhamnose , -Ethanol ~- +
Glycerol Eryt,hritol -~ ~
Ribitol -~
Galacticol .;
D-~Iannitol -~

D-Glucitol +
Lactic acid Succinic acid Citric acid Inositol Nitrate No vitamine at 37 C + (weak~
The comparison of the physiological characteristics of the SP M 180 cc strain with those reported in the study by Lodder quoted above, as well as with those reported in the book "A new Key to the Yea~ts", by J. A. Barrett and R. J. Pankhurst, 1974, has shown that the strain SP M 180 cc differs from all of the yeast species described in tho~e books.
The technical and the patent literatures have described hitherto many yeasts capable of utilizing methanol (cf. C. L.
Cooney and D.W. Levine in "Single-Cell Protein II-MIT Press, 1975), but the characteristics of the strain SP M 180 cc differ from those of all the strains the present applicants have acquired knowledge of.
A "5ui ~eneris" characteristic o~ this strain is its capacity of assimilating methanol more effeciently than ethanol.
The strain can be cultured both in discontinuous and continuous cultures, but its properties are better exploited in continuous cultures.
In addition, by virtue of its ability to form pseudomycelium, a culture can be obtained which set~les very ea~ily: ~uch a property can be exploited for conducting the continuous culture with a partial bio-mass feed back or recycle, a fact which enables a higher hourly output to be obtained. The ease of settling, in addition, make~ the collection '6~

of the bio-mass more convenient.
In accordance with the present invention, there i9 thus provided a method for the production of a bio-mass having a high protein content, which comprise~ culturing a yeast strain of the genus Candida, having the International Deposit Number NRRL-Y 11062, in a saline solution containing methanol as the only source of carbon and energy,at a temperature of from 20 to 35 C and at a pH of from 2.5 to 6~5.
The culturing medium which is inoculated with the yeast strain contains be~ides methanol the usual nutrient elements (N, P, K, Mg, Fe, Ca), growth-factors (yeast extracts and biotin) and mineral trace-elementQ. The broth is incubated with stirring at a temperature preferably between 30 and 33 C.
The preferred pH is between 4 and 4.5.
In a preferred embodiment, the fermentation is effected under a continuous ~upply of a gas mixture which contains oxygen, such as air.
The yeast cells which multiply at the expense of the nutrients which are supplied, are collected by sedimentation and filtration, washed with water and dried by heating.
The bio-mass thus obtained can be used as such as proteinic integrator for food~ and animal feeds, or nobler products can be extracted therefrom, such a~ proteins, aminoaeids, - and nucleic acids.
The fo:Llowing non-limiting examples illustrate the invention.

500-ml Erlenmeyer flasks were prepared, each cont~ining 50 mls of a culturing medium having the following composition :

; ~ i ~ i -6- .

7~
KH2 PO4 2.0 grams per liter NaH2P04 .H20 2.0 do (NH4)2S04 5.0 do MgSO4 7~2 0.2 do FeS04 .?H2o 2.0 milligrams per liter CaC12 2.0 do ZnS04 7~2 2.0 do Yeast extract200,0 do Biotin 25.0 microgram~ per liter Trace elements (soln.) 1 milliliters per liter The solution of trace-elements, prepared in diluted HCl (1 milliliter of conc~ HCl in one liter of water) had the following composition :
CuS04 ,5H20200 milligrams per ~iter H3B03 50C do i MnSO4 .H20 500 do KI 10 . do ... . .... . .. . ~

.
'' , ~ , ' ' ~

CoC~.2~6l~0 10 milligr~ms per liter MoO3 10 do The pll of the meclium was brout~ t to 500 anc; the f:Lask were ¦
then sterili~d at 116C -for 20 minures~ To two flafiks -t;hcre ~rere addecl 0.75 mls (1~9% volume 1~ olume ratio) of metlllnol, ¦and the flasks were -then inocu:latcd w`th a slant of the strain SP M 180 cc. Thc flasl~s were incubate.d fol 72 hours on a rotary sti.rring de~rice (220 rpm~ the diame-ter o the ~isplacement being

3.5 centimeters)~ a -temperature of 32.5C being thermos-tatically controlledO
Other flasks containing the same medium ancl to which there had been added 1% (volwlle/volllmc) o methanol~ 2% (v/v) of ethanol and ~.% (weigl~jvolume) of g'.ucose~ were inoculated with 5 mls each of the pre-cùlture spec.if`ied above~ After a 24-hour incubation~ an additional 1% volumeJvolume of methanol was added to each fla3k. After a total of 48 hours of incubation~ the contents of bio-mass in the flask was measured~ the results having been tlle following :
Subst;ra-te Optical clenc:ity (1:10) Dry bio~mass . at 660 nm grams/ Proteins Iiter Glucose oO34o 3.99 54.5 Methanol 00270 3.68 51.0 Ethanol 0.095 1.18 5401 The protein contents has been determined with -the biuret method.
E~AMPLE 2 A fermenter having an effective volume of abou-t 8 liters and con~aining the culturing medium of Example 1 ~as inoculated with a suspension of the strain SP M 180 cc~ To the fermenter~
which was thermostatically controlled at 32.5C there was added methanol~ making sure that the residual concentration in the broth never exceeded 1% on a volw~e by ~dlume basis. As the culture had satisfclctorily grown~ the continuous addition of the sterile broth to the fermenter was started~ the ~-terile broth 67~6~
¦~containing 290h gr.ln;s o~.` methallol p'`I' li-ler~ wh:ile a quant.ity of Otll-CULtUre Wa5 si.multaneously witlldra~.rn~ :idcIlt:ica:L t;o tha~ of the added culturiIIg mediwnO The qu..antity of added mcdium and of withdra~n brotll-culture were increasecll~ltil at-taining a dilut:ion .~elocity ( D = incoming rate of 10~/-~-ol.un-.e of the culture) o 0.166 h O Un&er these conditions t;he outgoing s-trcam contained lQo28 grams per l.iter of dry bio-mas6 and 146 parts per mill:ion of residual methanol~ ~ith a yield oi-` 35% ancl an hourly pro-duction o 1.72 grams per liter of b:;.o-mass~ The as-obta:i.ned bio-mass contained 55o6~ of proteins ~biuret test).
~ IPLE 3 To a fermenl;er of the kind descri.bed in Example 2 ~as applied a settling tank for the outgoing stream and a portlon of the broth-culture enricned with bio--}~ass was regularly fed bacl. to the fermenter~ By aclding fresh culturing medi~n to the fermenter~ the medium conta:ining 24 grams o methanol per liter~ at such a rate o flow as -to reach a dilution velocity o 0.267 h ~ there was obtained in the nonrecycled portion emerging from the settling tank a brot.h-culture coIltai.ning 7 D 80 grams per liter of bio-mass and 120 parts per million of rasidual methanol7 ¦
with a yield of 32.5% and a hourly OUtpllt of 2008 grams per liter~l The bio-mass thus obtained contained 5300% of proteins (biuret tes- ;) D

'' '' '' "' ""'. ' . . ' ~, .
'. ~ ' , ~ ,

Claims (5)

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

1. A method for the production of a bio-mass having a high protein content, which comprises culturing a yeast strain of the genus Candida, having the International Deposit Number NRRL-Y 11062, in a saline solution containing methanol as the only source of carbon and energy, at a temperature of from 20° to 35°C and at a pH of from 2.5 to 6.5.

2. A method according to claim 1, wherein the temperature is between 30° and 33°C.

3. A method according to claims 1 or 2, wherein the pH is between 4 and 4.5.

4. A method according to claim 1, wherein the fermentation is effected under a continuous supply of a gas mixture containing oxygen.

5. A method according to claim 4, wherein said gas mixture is air.