Note: Descriptions are shown in the official language in which they were submitted.
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
-5-
'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.
EXAMPLE 1
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
'' '' '' "' ""'. '
. . ' ~, .
'. ~ ' , ~ ,
