Note: Descriptions are shown in the official language in which they were submitted.
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METHOD FOR PRODUCING LIPID
The invention relates to a method of producing a lipid or a lipid mixture from
organic raw materials according to the preamble of Claim 1. The invention also
relates to the use of the lipid or the lipid mixture produced by the method as
a
biofuel according to Claim 17, as well as to a biofuel according to Claim 18.
The
invention also relates to a method of purifying municipal sewage according to
Claim 19.
Background
It is commonly known that the use of traffic fuels manufactured from fossil
raw
materials is extremely large-scale and that the consumption is continuing to
grow.
Thus, the adequacy, the environmental effects, and the aspect of sustainable
development concerning fossil energy resources have quite rightly emerged as
essential global challenges. In this frame of reference, the renewable
alternative raw
materials of traffic fuels have emerged as an object of increasing interest.
One step towards fuel production based on renewable natural resources is to
attempt, at least partly, to replace the fossil raw materials with organic
materials.
Even with this approach, one can envision problems that are fairly difficult
to solve.
In proportion to the present consumption of fossil raw materials, the amount
of
organic raw material required is extremely large, even if only a portion of
the fossil
raw materials are to be replaced. In several connections, it has already been
observed that a unilateral and large-scale consumption of organic natural
resources
or the reclaiming of areas under cultivation for this purpose may result in
consequences to the biodiversity of nature and to the balance of the primary
production of foodstuffs, which will be difficult to solve. The conversion of
the
organic material into a form that can be used for the production of traffic
fuel in an
energy-effective manner also presents a technological challenge.
A particularly favourable raw material source for traffic fuels would be
organic fat,
particularly triacyl glycerol as its energy content is considerably higher
than that of
corresponding carbohydrates or alcohols, for example. Furthermore, it is the
best-
known and can be converted into components of traffic fuel, such as diesel
fuel,
biodiesel or renewable diesel, through relatively effective chemical
processes.
However, the scarcity of the reserves of natural fatty raw materials presents
a
limiting factor. Based on present fatty resources, no more than a marginal
industrial
production of biofuel is feasible, at the most. Thus, increasing the fat
reserves
U6STITUTE SHEET (Rule 26)
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requires quite a considerable increase in the cultivation of fat plants. Such
a large
change in the production sector of cultivation towards fat plants, in turn,
has a
strong influence on the balance of the food economy on the global market. This
need, still at a speculative level, is already manifesting itself as a heavy
rise in the
prices of food and forage raw materials.
The total amount of naturally renewable organic masses is quite large;
calculated as
an amount of carbon, considerably larger than the annual use of fossil carbon
as
traffic fuel. However, the major part of these renewable masses, about 60%,
consists of compounds, which still contain a substantial amount of oxygen and
whose fuel value is thus quite low.
On the basis of prior known technology, it is known that the utilization of
the
relatively low energy content of carbohydrates into higher-energy compounds
containing more reduced hydrocarbon chains is already in theory, and
particularly
as known chemical applications, such as the gasification technique, an
extremely
energy-intensive process (R. Agrawal, N. R. Singh, F. H. Ribeiro and W. N.
Delgass 2007. "Sustainable fuel for the transportation sector", PNAS 104: 4828
-
4833, and WO 2006/117317), wherein the total efficiency between the feed and
the
yield remains low. A corresponding basic problem is also associated with
biotechnological processes according to the known technology, which are used
to
convert the hexose sugars contained in carbohydrates into higher-energy
compounds. An example of this is the production of alcohols, particularly
ethanol,
which is described, among others, in the specifications US 2002/0185447, US
5637502, and WO 03/038067.
The patent specification US 2004/0231661 also describes the treatment of a
material
containing lignocellulose by means of water and acid extractions and by means
of
hydrolysis, so that xylose and glucose are formed, which can be used in the
preparation of ethanol. The patent specification US 5,221,357 describes the
treatment of a material containing hemicellulose and cellulose by means of
acid
hydrolysis, and the treatment of the solid phase mechanically and by means of
acid
hydrolysis to produce monosaccharides, such as pentose and hexose sugars,
which
could be used in the preparation of ethanol. The patent specification US
4,752,579
describes the treatment of the husks of corn seeds to separate monosaccharides
by
means of acid and/or alkali, and enzymatic hydrolysis.
Some specifications describe the production of lipids by means of microbial
fermentation from different organic materials. The patent specification US
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4,368,056 suggests the use of carbohydrates, which exist in low contents in
industrial waste materials, in butanol fermentation, and the use of microbial
glycerides, which are generated in the fermentation, in the production of
biodiesel.
The publication Dai et al. (Dai, C. Tao, J., Xie, F., Dai, Y. and Zhao, M.
Biodiesel
generation from oleagenous yeast Rhodotorula glutinis with xylose assimiating
capacity. Afr. J. Biotechnol. Vol 6 (18), pp. 2130-2134, 19 September 2007)
describes the extraction of carbohydrates that are present in the straw of
plant
material, such as corn stems, tree leaves and rice, by means of grinding and
an acid
treatment, and the use of the filtrates and washing waters, obtained from the
treatments, as raw material for the production of lipid by the Rhodotorula
yeast.
The lipid obtained from the microbe was used in the production of biodiesel.
Angerbauer et al. (Angerbauser, C., Sierbenhofer, M. Mittelbach, M. and
Guebtz,
G.M. Conversion of sludge into lipids by Lipomyces starkeyi for biodiesel
production. Bioresource Technology 99(2008) 3051-3056), in turn, describes the
treatment of waste water sludge by alkali and acid, as well as its utilization
in lipid
production by the Lipomyces yeast, into a raw material for biodiesel.
The problems encountered with the methods described above include the fact
that
the yields of sugars that can be utilized by the micro-organisms remain low,
or the
methods apply to the use of biomaterials as source material that are of minor
importance to the needs of large-scale production. It is obvious that the
quantitative
goals set for the manufacture of biofuel cannot be achieved by means of the
described methods and the source materials used therein.
Thus, there is still a great need for new technological solutions,
particularly
solutions that could be used, more comprehensively than before, to convert the
renewable organic carbohydrate reserve of the globe into compounds having a
higher energy content, irrespective of its chemical or structural composition.
It
would be of particular importance to solve how carbohydrates existing in
various
organic materials, could be effectively converted into compounds having a
higher
energy content, such as fats, which would be more adaptable to traffic fuel
use or to
the raw material of this use.
Summary
The purpose of the present invention is to provide a new solution to the
problem of
how to convert organic biomaterial into compounds having a higher energy
content.
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In particular, the purpose of the present invention is to provide a solution
to the
problem of how to convert the carbohydrate components obtainable from organic
biomaterial into a lipid suitable for the production of biodiesel.
To be more precise, the method according to the present invention is
characterized
by what is stated in the characterizing parts of Claim 1.
The use according to the invention, in turn, is characterized by what is
stated in
Claim 17, and the biofuel by what is stated in Claim 18.
The method for purifying municipal sewage according to the invention is
characterized by what is stated in Claim 19.
The present invention is based on the observation that, when handling biomass
in
different ways for the recovery of cellulose and hemicellulose fractions,
micro-
organism populations appeared quicker in said fractions the further the
cellulose and
hemicellulose fractions were degraded. Surprisingly, it was discovered that in
the
carbohydrate fractions described above, micro-organisms were also growing that
had the ATP citrate lyase enzyme (EC 2.3.3.8, previously EC 4.1.3.8), by means
of
which the organisms collected lipids, particularly triacyl glycerol, into
their cells.
Based on these observations, the invention developed for treating biomaterial
so
that the cellulose and hemicellulose contained in it are separated from the
rest of the
biomaterial and then hydrolyzed so that the hydrolysis products are suitable
for
growing lipid-collecting micro-organisms, and for the production of lipid, and
to
use the lipid thus formed as a raw material in the manufacture of biodiesel.
According to the description of the invention, carbohydrates suitable to be
utilized
by the lipid-synthesizing micro-organisms can be produced from organic raw
material originating from various sources, and/or carbohydrate fractions can
be
produced, from which lipid can be produced by means of micro-organisms.
According to the present invention, such carbohydrates can be produced
particularly
from biomaterials that contain hemicellulose, cellulose, starch or non-starch
polysaccharides. The carbohydrates that are suitable for utilization by micro-
organisms are particularly mono and oligosaccharides, which comprise both
hexose
and pentose sugars. The carbohydrates can also be in polymeric forms, if the
lipid-
producing micro-organism has been selected so that it is capable of using
carbohydrate polymers.
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Wood, in its various forms, comprises the biggest reserve of renewable biomass
that
can be recovered at present. The use of wood, particularly its mechanical or
thermo-
mechanical treatment or other manufacture or production of mechanical mass
from
wood, is large-scale and, as a process, produces a lot of carbohydrate-bearing
minor
5 flows. Very little economic added value has been discovered for these side
cuts of
the wood-refining industry; and in many cases, they incur costs as the
environmental load caused by them must be eliminated. As a technological
challenge, side flows like these are particularly problematic. The side flows
are
large in volume, but their carbohydrate content is typically low. As dilute
water-
based carbohydrate solutions, they are poorly suitable for processes that aim
at
utilizing the carbohydrates in solution by chemical means.
Accordingly, the purpose of the present invention is also to provide a
solution to the
problem of how to utilize large-scales industrial side flows containing
biomaterial,
which presently require costly purification procedures or remain completely
unutilized by the present processes, but which still have potential as a
source of
energy.
In the method according to the present invention, organic source material is
treated,
which contains cellulose, hemicellulose, starch, all of these, some mixture
thereof or
the degradation products thereof or, alternatively, starch or non-starch
carbohydrate
as such or linked with the cellulose or hemicellulose materials. The source
material
can be pre-treated mechanically, thermo-mechanically, physically, chemically,
biologically or by combinations of these treatments, or it may be suitable to
be used
as such. When containing carbohydrates in polymeric form, it is preferably
treated
with water or acid or alkali, or combinations thereof. After these preliminary
treatments according to the present description, the mixture is divided into a
filtrate
and solid matter, i.e. a precipitate (Fig. 1), and the filtrate or both
fractions are
recovered. It is preferable to renew the treatment of the source material that
is
carried out with water or acid or alkali, or their combinations, and to
combine the
filtrates with each other after the separation of the precipitate. The
filtrate obtained
in the treatment containing alkali, either as such or after the recycling
described
above, is preferably conveyed to a mixture, wherein an acid treatment of the
source
material is carried out to increase the amount of soluble monosaccharide. Any
of the
filtrates or precipitates generated in these treatments or the source material
as such,
or the combinations of the source material and the filtrate or precipitate or
filtrates
or precipitates are used to produce single-cell lipid after possible pre-
treatments,
such as neutralization, decolourization and filtering. The filtrates can also
be
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combined, diluted or concentrated to achieve a suitable monosaccharide content
and
composition for the micro-organisms that produce single-cell lipid.
From each alternative treatment of the source material, a precipitate of a
varying
composition is generated, depending on whether the treatment is carried out
with
water or in the presence of acid or alkali. To increase the sugar yield, the
precipitates are preferably conveyed to mechanical grinding, either as such or
in the
presence of water acid or alkali, from which treatment a filtrate and a
precipitate are
obtained again. The filtrate or the precipitate or some combination thereof is
used
for producing single-cell lipid, or the precipitate is treated preferably with
a strong
acid, when so desired. As a result of the acid treatment, a filtrate and a
precipitate
are generated again, from which the filtrate or the precipitate or the
combination of
these can be conveyed to the production of single-cell lipid. The precipitate
can be
treated in even higher acid concentrations by combining grinding with the
treatment. The filtrate or the precipitate or the combination thereof,
generated as a
result of this treatment, is used for the production of single-cell lipid. The
precipitate can also be removed and burned or used for the production of
biofuel or
a precursor thereof by other methods. Each filtrate fraction or precipitate or
source
material can be used as such or as various combinations for the production of
single-cell lipid. The precipitates obtained from the process steps described
above
can be retreated by means of the earlier or later process steps presented in
the
present description. To increase the sugar yield, it is thus preferable to
treat the
precipitate with an acid or alkali that is stronger than in the previous
treatment.
Further, enzyme treatments or microbial fermentations can be carried out
between
the different process steps.
The invention also relates to a method, wherein the used alkalis and acids are
recycled again in the process.
The invention also relates to a method, wherein the single-cell biomass used
in the
method is recycled as the biomaterial intended by the invention, when the
produced
lipids have been recovered.
Economically exploitable components can be recovered from the precipitate
obtained from the treatments that are carried out according to the method. The
filtrates, in turn, can be used also in other microbial processes than the
production
of lipid.
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The-method according to the description provides a solution to the problem of
how
the hexose and pentose monosaccharides contained in the carbohydrates of the
biomass can be gradually brought into smaller fractions containing a larger
amount
of monomeric sugar units, which fractions the lipid-synthesizing micro-
organisms
can utilize more effectively to produce lipid. In connection with any process
step, in
which an insoluble substance is separated from a soluble substance, a
precipitate,
and in which a filtrate is thus generated, the hexose and pentose sugars
contained in
the filtrate can be used for producing single-cell lipid as such or, after
necessary
pre-treatments, as mixtures of filtrates for the production of single-cell
lipid. In
addition to the filtrates, also precipitates or combinations of filtrates and
precipitates
can be used for the production of single-cell lipid.
A comprehensive advantage of the invention is that it can be used to apply
simple
processes and unit operations, which are already in industrial use, in an
energy-
effective and environmental manner to produce an energy-rich chemical
compound,
a lipid, from compounds of biological origin containing less energy, such as
hexose
and pentose monosaccharides or the oligomers formed by these, as well as the
mixtures of these.
In the method according to the invention, it is particularly preferable that,
by means
of mechanical or thermo-mechanical grinding, a new surface is always exposed
in
the organic material, which can again be subjected to treatments with water,
or acid
or alkali with a different strength, and thus a solution and precipitate
distribution
can be provided, from which, particularly with the solution, more sugars
usable in
the microbial lipid production can be obtained.
When combining several separations of the solution and the precipitate, a
considerably enhanced yield of hexose and pentose sugars is obtained for the
mechanical or thermo-mechanical grinding, compared with single-phase
hydrolyzing methods.
In the method according to the invention, by combining the filtrates obtained
from
the acid and alkali treatments or by combining the filtrates and the
precipitates, the
flows obtained from the various treatments neutralize each other. Thus, the
filtrates
or the precipitates or the combinations thereof are usable as such, without
adjusting
the acidity, or at least the need to adjust the acidity is smaller. The
possibility to
combine several different biomasses and biomasses from different origins with
each
other is also advantageous.
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The exploitability of the method becomes particularly emphasized so that the
sugars
formed therein, which are usable in lipid production, can naturally be
microbiologically used, either as such or partly, to produce also other
compounds,
such as alcohols.
A special advantage of the invention is that, in addition to the utilization
of the side
or main flows of the mechanical and thermo-mechanical treatment of wood, it is
also suitable for the utilization of other biomaterials that release
carbohydrates for
producing lipid.
The invention further relates to a method for forming lipid or a lipid mixture
from a
mixture that, according to the method, was generated by recycling once again
the
fibre, which was generated in the thereto-mechanical treatment, in the tr
eatments
with alkali or acid.
Other organic raw materials that are difficult to utilize on a large scale,
which
according to the present description can be processed into hexose and pentose
sugars or into oligomers formed by these and formed from these into lipid by
means
of micro-organisms, are recycled fibre that is obtained, for example, from the
recycling of newspaper, beet pulp that is obtained from sugar beets, and the
chaffs
and straws of grains, such as oat; and other similar devalued part of field-
cultivated
plants, sawdust, refined mechanical pulp, straw and peat, particularly
slightly
decomposed peat. Other organic materials that so far have hardly been utilized
at all
are swampy or submerged biomass, biomass from the catchment areas of cellulose
mills, and the biomass that goes to activated sludge plants from municipal
sewage,
or other organic municipal waste that goes to dumping areas or incineration.
These
organic materials can also be treated by alternating the various embodiments
of the
method so that the carbohydrates contained in these materials are rendered
useful
for the micro-organisms to produce single-cell biomass and lipid. For example,
municipal wastewater could be treated by means of the present method, whereby
there would, among others, be the advantage of harmful microbes dying in the
treatment.
An essential point of the present invention is that the carbohydrate-
containing
biomaterial, regardless of which organic material it originates from, is
treated so
that monomeric hexose and/or pentose sugars or their oligomers suitable for
the
production of single-cell lipid are obtained from the carbohydrates. It is
preferred to
carry out the treatment by a combination of two or more treatments.
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Using the method according to the invention, suited to the dimensions of the
needs
for large-scale production of biofuel, source materials can be simultaneously
produced from several different biomasses, which source materials are usable
in the
production of the celimass of lipid-producing micro-organisms and in the
production of lipid. The components suitable for the lipid production of
microorganisms can be produced irrespective of the composition, availability
and
structure of the biomass. A considerable advantage of the present invention is
also
that, by the method according to the invention, usable sugars can be produced
with
a high yield and the consumption of chemicals needed for the adjustment of
acidity
can be reduced.
Compared to the existing state of the prior art, the invention described
herein
provides a breakthrough technique, which combines the conversion of
biomaterials
that contain both cellulose and hemicellulose into usable hexose and pentose
sugars.
By the same embodiments, the invention also enables the utilization of the
monosaccharide units of the starch and the non-starch polysaccharides
contained in
these biomaterials into usable sugars for the production of single-cell
lipids. The
invention is particularly implementable so that it can be applied on an
industrial
scale to materials, which originate from renewable natural resources or their
devalued side flows, which are generated by the industry or communities. The
method according to the invention can be used to treat material containing
cellulose
and hemicellulose in a controlled manner, so that precursors of single-cell
lipids are
formed therefrom, which can be used by safe microorganisms in the production
of
single-cell lipids.
In the following, the invention is described more closely by means of the
appended
drawings and a detailed description.
Brief description of the drawings
Fig. 1 shows the main steps of performance of the method according to the
invention.
Fig. 2 shows the use of hexose and pentose sugars as such or in combinations
for
the production of cellular mass and lipid.
Fig. 3 shows the growth of yeasts and the production of lipid in a culture
medium,
to which a mixture has been added as a source of carbon and for the production
of
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lipid, which mixture was generated from chaff by an alkaline treatment and by
the
10% acid hydrolysis of the filtrate obtained therefrom.
Fig. 4 shows the growth of yeasts in a culture medium, to which commercially
available pentose sugar was added as a source of carbon.
5 Detailed description of the method
"Carbohydrates" refer to organic molecules that include an aldehyde, acid or
keto
group and, in addition, several hydroxyl groups. The sphere of carbohydrates
thus
includes compounds that are described by the terms monosaccharide,
oligosaccharide, polysaccharides, sugar, cellulose, hemicellulose, starch and
non-
10 starch carbohydrate.
"Cellulose" is a long-chain polysaccharide, whose primary structure consists
of a
polymer formed by the (3-1-4 bonds of glucose.
"Starch" is a long-chain polysaccharide that mainly consists of a-1-4 and a-1-
6
glucose units.
"Usable sugar" herein refers to sugars, using which the microorganisms are
able to
multiply, and from which the lipid- and alcohol-producing microorganisms are
capable of producing lipid or alcohols.
"Hemicellulose" refers to a group of compounds consisting of several different
hexose and pentose sugars, such as galactose, mannose, glucose, xylose and
arabinose.
"Monosaccharide" is a monomer unit of carbohydrates, (C-H20)n, which typically
consists of 3 - 9 carbon atoms and which has stereochemical differences in one
or
more carbon atoms. These include hexoses, such as glucose, galactose, mannose,
fructose, which have 6 carbon atoms, and pentoses, such as xylose, ribose and
arabinose, which have 5 carbon atoms.
"Oligosaccharide" refers to a carbohydrate, which has been formed from two or
more monosaccharides by 0-glycosidic bonds.
"Pentose sugar" refers to a monosaccharide containing five carbon atoms.
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"Hexose sugar" refers to a monosaccharide containing six carbon atoms.
"Hydrolysis" refers to carbon-carbon, carbon-oxygen, carbon-nitrogen, or
carbon-
sulphur bonds breaking under the influence of either water, acid or alkali,
irrespective of whether the water participates in the reaction. In an
enzymatic
hydrolysis, the corresponding reactions are catalyzed by enzymes. Hydrolysis
is, for
example, a reaction, where the 0-glycosidic bond between the monosaccharides
of
the carbohydrates or the peptide bond between the amino acids of proteins
breaks.
"Treatment with water- acid or alkali" in this connection means that an
organic
material either as such, or a product derived from it is extracted, treated
mechanically or thermomechanically, or combinations of these treatments are
carried out in the presence of water, acid or alkali. Acid refers to a
chemical
substance, molecule or ion, which is capable of donating a hydrogen ion (a
proton),
and alkali refers to a substance, molecule or ion, which is capable of
accepting the
hydrogen ion (the proton), according to the Brt nsted-Lowry acid alkali
theory. Acid
also refers to the so called Lewis acid, which is capable of accepting an
electron
pair, and the Lewis alkali refers to the so called Lewis alkali, which is
capable of
donating a base pair. According to the definitions, the activity of the
substances as
acids or alkalis is not limited to aqueous solutions. In the present
description, the
terms "acid" and "alkali", according to the definitions, also refer to acid
and alkali
catalysts. In the present description, acid also refers to any acid phase,
wherein it
can function as acid, such as in the form of gas, solid matter or liquid; for
example,
as an aqueous solution. Correspondingly, alkali refers to any alkali phase,
wherein it
can function as an alkali, such as in the form of gas, solid matter or liquid;
for
example, as an aqueous solution.
"Organic source material" in the present description refers to any organic
matter
that is produced by a living organism. The organic source material in the
present
description is also called biomaterial.
In particular, the organic source material refers to an organic material that
comprises polysaccharide. "Polysaccharide" refers to a carbohydrate polymer
formed from monosaccharides, which can also contain compounds other than
monosaccharide. Polysaccharides are, for example, cellulose, hemicellulose and
starch. Polysaccharides also include, among others, alginate, glucane, inulin
and
arabic gum. Out of other polysaccharides, mannane should also be mentioned.
The
source material can comprise polysaccharides as such or as a mixture, or it
may
comprise their degradation products.
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"Non-starch polysaccharide" refers to a carbohydrate, whose molecular
structure
lacks the a-1-4 bonds typical of starch, or they are scarce. Non-starch
polysaccharides are, for example, glucane, alginate, inulin and arabic gum.
The
non-starch polysaccharides also include hemicellulose and cellulose. Other non-
starch polysaccharides are, for example, the carbohydrate polymers that occur
in
algae.
"Cultivated plant" refers to a plant, which is planted or seeded for
beneficial
purposes in the soil that is prepared for it.
The term "lipid" refers to a fatty substance, whose molecule generally
contains, as a
part, an aliphatic hydrocarbon chain, which dissolves in organic solvents but
is
poorly soluble in water.
In the present invention, the lipids that are formed in the micro-organisms
are
mainly tri-, di- or mono-acylglycerols, or sterol esters, but other lipids,
such as
phospholipids, free fatty acids, sterols, polyprenols, sfingolipids,
glycolipids and
diphosphatidyl glycerol can also be formed in the cells.
The present invention can be used in the manufacture of biodiesel or renewable
diesel.
According to the EU directive 2003/30/EY, "biodiesel" refers to a methyl-ester
produced from vegetable or animal oil, of diesel quality to be used as
biofuel.
Renewable diesel refers to a hydrogen treated lipid of animal, vegetable or
microbial origin, whereby the microbial lipid can originate from a bacterium,
a
yeast, a mould, an alga or another microorganism.
The source material of the method according to the invention can be cellulose,
hemicellulose and biomass, preferably wood pulp, possibly containing binders,
which has been generated by mechanical or thereto-mechanical methods or other
physical methods, or chemically, enzymatically or microbiologically or by
combinations of these methods. Without modifying the method, plant materials
that
contain starch, such as potato, its parts, the seeds of cultivable crops,
maize and rise,
respectively, sugar beet as well as, in addition, sugar beat pulp including
the non-
starch polysaccharides contained in the same, can also be the source material
of the
method according to the invention. Parts of plants that contain non-starch
polysaccharides, such as P-glucan, are also suitable as source material. The
method
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is also suited for the use of carbohydrates, such as alginate, which originate
from
single-cell organisms, as source material. The composition of the source
materials
described above can also include varying amounts of protein and lipid, which
can
also act as source materials for the growth of lipid-synthesizing
microorganisms and
for the lipid production.
The source material of the method according to the invention can also be, for
example, recycled fibre obtained from newspaper recycling, sugar beet pulp and
the
chaffs of grains, such as oat, sawdust, refined mechanical pulp, peat and
straw.
Other source materials useful in the method according to the invention are,
for
example, microbial mass, such as single-cell biomass, swampy or submerged
biomass, including algae and micro-algae, biomass from the catchment areas of
cellulose mills, and the biomass that goes to activated sludge plants from
municipal
sewage, or other municipal waste that contains a biological component and that
is
presently used in incineration, composting or in some other method, which
results
in the comprehensive release of the carbon contained in the waste as carbon
dioxide.
The method according to a preferred embodiment of the invention comprises at
least one step, wherein the filtrate or the combination of filtrates, the
precipitate or
the combination of precipitates, which is obtained from the organic material
according to the method, the organic material as such or a combination of any
of
these, is conveyed to the mixture, where the lipid production takes place. The
alternative embodiment of the method can be selected on the basis of what kind
of a
monosaccharide composition is preferred in the filtrate or the combinations of
filtrates, or in the precipitates, the combinations of precipitates, or in the
combinations of filtrates or precipitates for growing the microorganism and
producing the lipid. Thus, the filtrates or precipitates are selected from a
group that
is generated by treating the biomaterial preferably with a substance that is
selected
from the group comprising:
i) water,
ii) acid, and
iii) alkali, and
by thereafter separating the fibre-containing precipitate and the fibre-
free filtrate.
Optionally, the precipitate is once or more times again subjected to
treatment(s) of
any of the items (i), (ii) or (iii), and the precipitate obtained is
preferably subjected
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to mechanical or thermo-mechanical grinding, and the precipitate and the
filtrate are
separated.
Depending on which biomaterial is used and which monosaccharides are desired,
the biomaterial is treated with water, acid or alkali, preferably acid or
alkali;
typically, by means of an aqueous solutions of acid or alkali. When needed,
the
treatment can also be carried out several times. The same biomaterial can also
be
treated sequentially with several different solutions, and compounds that
enhance
the separation and hydrolyzation of the carbohydrates can be added to the
water.
The source materials that are listed in the following list in Group I can be
treated
with water in the first step or, if the treatment result is to be enhanced,
with a
mixture of water and acid.
The biomaterials of Group II are treated in the first step, preferably with
acid.
The biomaterials of Group III are treated in the first step, preferably with
alkali.
When the precipitate and the filtrate have been recovered, the filtrate can be
re-
treated with acid.
Group I
Biomaterial originating from mechanical, thermomechanical, enzymatic or
microbiological treatment of wood or from combinations of these treatments, or
from cultivated plants.
Group II
Recycled fibre, beet pulp, chaffs, straw, bran, grain granule, whole
cultivated plant,
cultivated plant, TMP pulp, MDF pulp or a source material containing starch or
non-starch polysaccharides
Group III
Sawdust, refined mechanical pulp, chaffs, straw and woody plant parts, TMP
pulp,
MDF pulp, beet pulp, cultivated plant, which may contain varying amounts of
starch.
The treatments can be enhanced by adding, for example, one or more enzymes
into
the treatment solution, preferably into a treatment solution that is made
using water.
Enzyme treatments or microbial fermentations can also be added between the
various process steps.
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In the following step, the lipid-producing micro-organism is contacted with
any of
the filtrates or precipitates or their combinations in a culture medium, and
the
microorganism cells are allowed to produce lipid, and the lipids are
recovered.
The fibre-containing precipitate obtained from the above-described biomaterial
5 treatment steps is also preferably treated using a method, wherein the fibre-
containing precipitate is mechanically ground and the fibre-containing
precipitate
and the fibre-free filtrate are separated.
In addition, the fibre-containing precipitate obtained from the mechanical
grinding
can be treated with a strong acid, and the fibre-containing precipitate and
the fibre-
10 free filtrate can be separated. After the acid treatment, the precipitate
can also again
be recycled back to the mechanical grinding or, as stated above, the
precipitate can
be used in lipid production using microbes.
In addition, the biomaterial can be acidified and ground mechanically or
thermo-
mechanically, and the fibre-containing precipitate and the fibre-free filtrate
can be
15 separated.
The filtrate or precipitate or their combination, obtained from any of the
above
described treatments can be added to the culture medium of the lipid-producing
microorganisms.
Typically, the total amount of sugars in the filtrates is 0.5-10% by weight.
Of these,
sugars usable for the production of biomass and lipids typically comprise at
least
0.5% by weight, preferably at least 3% by weight, more preferably 4 - 5% by
weight. For example, by grinding and re-extracting the biomaterial, more
sugars can
be detached from the biomaterial and, in this way, the amount of sugars can be
increased to a more advantageous level. However, the amount of sugars in the
filtrate is preferably less than 30% by weight, more preferably less than 20%
by
weight.
The microorganisms that are capable of producing lipid can be grown so that
they
first produce biomass and then lipid, or simultaneously both biomass and
lipid.
Depending on the origin of the biomaterial and the method of treatment it is
subjected to (treatments with water, acid, alkali), hexose monosaccharides,
pentose
monosaccharides or both in various ratios can be obtained, as shown in Fig. 2.
From
some biomaterials, mainly hexose sugars can be obtained, whereas from others,
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16
mainly pentose sugars. With a suitable selection of the microorganism capable
of
producing lipid, the filtrate or precipitate or their combination that mainly
contains
hexose sugars can be used for the production of cellular mass and, after that,
the
filtrate or precipitate or their combination that mainly contains pentose
sugars can
be used for the production of lipid into the cellular mass. Alternatively,
lipids can be
produced in the cellular mass from hexoses. The cellular mass and the lipids
can be
produced from hexoses. Correspondingly, by a suitable selection of the
microorganism that is capable of producing lipid, the filtrate or precipitate
or their
combination that mainly contains pentose sugars can be used for the production
of
cellular mass and, after that, the filtrate or precipitate or their
combination that
mainly contains hexose sugars can be used for the production of lipid into the
cellular mass. Alternatively, lipids can be produced into the cellular mass
from
pentoses, or the cellular mass and the lipids can be produced from pentoses.
Both
cellular mass and lipids can also be produced from a mixture of pentoses and
hexoses.
Treatment of biomaterial
In the following, the treatment of biomaterial according to the preferred
embodiments of the invention is described. Typically, the treatment is carried
out as
a combination of two or more treatments:
The source material is preferably extracted at a temperature of 90-100 C. An
advantageous embodiment of the acid extraction is to use 5-10% of a mineral
acid,
such as sulphuric acid, or an organic acid, such as citric acid or acetic
acid, and in
the alkali extraction, preferably 0.5-2.OM of NaOH. The treatment time can
range
widely; it is preferably 1-10 hours, typically 2-8 hours, most suitably 2-4
hours.
Other treatments, such as treatments with enzymes, microorganisms, oxidizing
or
reducing chemicals, or combinations of these treatments, can also preferably
be
combined with the water extraction.
According to the method, the precipitate generated in the extraction can be
mechanically ground preferably at a temperature of 100-210 C, typically 150-
200 C, preferably for 2-20 minutes, typically for 5-11 minutes. The pressure
is
preferably 6-8bar. The generated mass is filtered, the filtrate is treated
using the
method described above in order to be suitable for the production of single-
cell
lipid. The precipitate can be conveyed to an acid treatment with a strong
acid, which
preferably is a treatment with 40-72% sulphuric acid, suitably 65-70%
sulphuric
acid. Normally, the treatment time is 2-8 hours, preferably 2-4 hours. The
method
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can be implemented with any acid, by means of which a proton-catalytic
hydrolysis
is provided. Suitable acids are, for example, strong mineral acids, phosphoric
acid,
sulphuric acid, or the oxyacids of sulphur, nitrogen, chlorine, bromine and
iodine.
The result of the hydrolysis is divided into a filtrate, which is treated in
order to be
suitable for the production of single-cell lipid, and the precipitate can be
conveyed
into a dilute acid solution, preferably a solution of 5-10% sulphuric acid,
and
grinding can be carried out at a temperature of 170-200 C and at a pressure of
6-10
bar, for 10-20min. The mixture is divided into a filtrate and a precipitate,
of which
the former is treated in order to be suitable for the production of single-
cell lipid and
the precipitate can be removed.
The above described embodiment of the method aims at the total use of the
carbohydrate present in the source material for the production of single-cell
lipid.
However, starting from the way of extracting the source material, the method
can
also be implemented for selected parts only, for example when the fibre
material of
the precipitate is also to be used for other purposes. The method according to
the
invention is characterized by preferably comprising the steps described above
in
their entirety, but is not limited from carrying out part(s) of the method to
an extent
that deviates from the basic method, or in a unit operation order, and to the
use of
the monosaccharide fractions produced by these operations in the production of
single-cell lipid. A process step can also be attached to the method according
to the
description, wherein the monosaccharide-containing fractions obtained from the
source material are used for the production of single-cell biomass or ethanol,
in
addition to lipid.
In the following description, some methods according to the preferred
embodiments
of the invention are described. The same methods can also be applied to raw
materials other than the ones presented in the description.
1.
According to a preferred embodiment of the invention, wood fibres that
comprise
ground wood, TMP pulp, sawdust or mechanical pulp is treated according to the
following constituent steps:
A. 100 g of wood fibre is extracted in a litre of water at 90-100 C for 2-4
hours, preferably 2 hours. The precipitate is separated from the solution by
filtration, and the solution is recovered. The carbohydrate yield in the
solution ranges from 2% to 5% depending on the manufacturing method of
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18
the wood fibres, typically being 4-5% for TMP fibre of a high treatment
temperature (over 170 C).
B. To increase the carbohydrate yield, the fibre fraction is hydrolyzed in a
litre
of 5-10% acid (preferably 5%), pH about 1 (e.g., mineral acid), at a
temperature of 90-100 C for 2-4 hours, preferably 2 hours. The remaining
precipitate is separated from the solution; the solution is recovered.
C. Excess acid is preferably decanted from the precipitate, which is re-ground
in
a defibrator (e.g. a wing or disc refiner). It is preferred to increase the
temperature of the precipitate by pre-steaming for one minute and to keep the
condensate in the mixture. The temperature is raised 150-200 C, preferably
170 C, the pressure being 6-8bar (wing refiner). The grinding time is
selected according to the wood from the range of 2 to 15 minutes. The
precipitate is separated from the solution by filtering and the solution is
recovered.
D. Strong acid of 40-72% (sulphuric acid) is added to the precipitate
fraction, it
is allowed to absorb for 2-4 hours, preferably 2 hours at room temperature.
Excess acid is decanted off and the precipitate is re-ground in a defibrator
(e.g. a wing or disc refiner). It is preferred to increase the temperature of
the
precipitate by pre-steaming for one minute, without draining the condensate
thereafter. The temperature is raised 150-200 C, preferably 170 C, the
pressure being 6-8bar (wing refiner). The grinding time is selected according
to the wood from the range of 2 to 15 minutes. The mixture is filtered, the
precipitate is separated from the solution. The solution is recovered and the
precipitate is used as fuel.
The solution fraction obtained from any of the constituent steps A - D can be
further processed by decolourization methods, pH adjustments, and other
measures
that promote the growth of microorganism, such as removing water, and the
solutions thus obtained can be used in the culture medium of the
microorganism,
and the microorganism can be allowed to produce lipid. By combining the
constituent steps A - D, 40-65% of the original wood fibre material can be
converted into a soluble form from the fibre that is used as source material.
The
dissolved carbohydrates comprise glucose, galactose, mannose, xylose and
arabinose units, their mutual portions being typical for the wood type used.
II.
According to another preferred embodiment of the invention, 100g of wood
fibre,
ground wood, recycled fibre, TMP pulp, sawdust or mechanical pulp are used,
extracted in one litre of 4-8% alkali solution, preferably 1 M of NaOH at a
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19
temperature of 90-100 C for 2-4 hours, preferably 3 hours. The precipitate is
separated from the solution by filtering at room temperature, and both
fractions are
recovered. Depending on the treatment of the fibre of the source materials,
the
carbohydrate yield in the solution is within a range of 5-8%. This solution is
preferably treated by any of the following constituent steps:
A. The vat liquor is re-used as such in extracting the following fibre batch
in the
way described above, and the solution is recovered.
B. The vat liquor is hydrolyzed with acid according to any of the steps B - D
of
the previous embodiment to treat the lignin and oligo- and polysaccharides
that have dissolved therein.
C. The vat liquor recycled according to step A is used according to step B of
embodiment I.
To increase the monosaccharide yield, the precipitate recovered in the alkali
treatment is preferably treated using any of the following methods or the
combinations thereof:
D. The precipitate is treated according to any of steps B, C, D of the
previous
embodiment I, or according to all of them. The mixture is filtered, the
solution is neutralized, and both the precipitate and the solution are
recovered.
E. The precipitate remaining from step A of embodiment II is re-treated with
alkali, preferably under conditions, where the mixture is ground for 2-8
minutes, preferably 6 minutes, at a pressure of 4-10bar, preferably 8bar, the
temperature being 170 C. Filtration is carried out, the solution is
neutralized
and recovered. By means of this extra grinding, the amount of material that is
dissolved can be increased to 27% of the original amount of fibres in the
source material.
The carbohydrate-containing solution generated in each constituent step A - E,
comprising glucose, galactose, mannose, xylose and arabinose units, is
processed
into a form suitable for lipid production by microorganisms, for example, by
means
of decolourization, pH adjustment, or by removing water from the solution, and
it is
used as a culture medium for the microorganism, or as a part thereof, and the
microorganism is allowed to produce lipid.
III.
According to a third preferred embodiment of the invention, wood fibre, ground
wood, TMP pulp, sawdust, mechanical pulp, recycled fibres (or a neutralized
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extraction prepared according to step B described above) are used, hydrolyzed
with
an acid that has a strength of 5-10% (preferably 5%), by adding 1 litre of
said acid
(e.g. a mineral acid) per 100 g of material to be hydrolyzed, at a pH of about
1,
using a temperature of 90-100 C for 2-4 hours, preferably 2 hours. The
precipitate
is separated from the solution by filtration. The solution contained 4-14% of
carbohydrates calculated from the amount of fibres of the source material. The
precipitate can be used as fibres or be further processed in order to increase
the
monosaccharide yield according to steps C or D or both of the first embodiment
described above. The solution is separated from the precipitate, neutralized,
filtered,
and either used as such or in a concentrated form into the culture medium of
the
microorganism or a part thereof, and the microorganism is allowed to produce
lipid.
IV.
According to a fourth preferred embodiment of the invention, IOOg of wood
fibres,
ground wood, TMP pulp, sawdust, mechanical pulp or the precipitate remaining
from steps A-C of embodiment I or step A of embodiment II are used, 5-10% of
acid is added, pH about 1 (e.g. a mineral acid), it is allowed to absorb for 2-
4 hours,
preferably 2 hours. The excess acid is decanted off and the precipitate is re-
ground
in a defibrator (e.g. a wing or disc refiner). The temperature of the
precipitate is
preferably raised by pre-steaming for one minute, and the condensate is not
allowed
to flow out. The temperature is raised 150-200 C, preferably 170 C, the
pressure
being 6-8bar (wing refiner). The grinding time is selected according to the
wood
from the range of 2 to 15 minutes. The precipitate is removed from the
solution by
filtration. The residual precipitate can be used as fuel or be re-ground in
the way
described above to increase the monosaccharide yield in the solution. The
solution
is neutralized, filtered and used as such or in a concentrated form, and is
used in the
manner described in embodiments I-III.
V.
According to a fifth preferred embodiment of the invention, 100g of wood
fibres,
ground wood, TMP pulp, sawdust, mechanical pulp or the residual precipitate
according to any of embodiments I-IV is used, a strong acid of 40-72% (e.g.
sulphuric acid) is added, is allowed to absorb for 2-4 hours, preferably 2
hours at
room temperature. The excess acid is decanted off and the precipitate is re-
ground
in a defibrator (e.g. a wing or disc refiner). The temperature of the
precipitate can be
increased by pre-steaming for one minute, and the condensate is not allowed to
flow
out. The temperature is raised 150-200 C, preferably 170 C, the pressure being
6-
8bar (wing refiner). The grinding time is selected according to the wood from
the
range of 2 to 15 minutes. The carbohydrate mixture is filtered and the
precipitate is
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21
separated from the solution. The portion of matter that is dissolved from the
precipitate used as source material is in the range of 40-65%. The residual
precipitate can be used as fuel or it can be re-ground to increase the
monosaccharide
yield. The solution is neutralized and filtered and treated according to any
of
embodiments I-IV.
Alternatively, strong sulphuric acid of 40-72% is absorbed into the
precipitate at
room temperature for 1-3 hours, preferably 1.5 hours. After this, the acid is
diluted
to 5% and boiled at normal pressure at 100 C for 4 hours. Further processing
as
above.
Lipid production using microorganisms
The present invention allows, through combination of the filtrates generated
in the
various constituent steps, the carbohydrate contained in the source material
as well
as the hexose and pentose monosaccharides to be comprehensively used for lipid
and single-cell biomass by means of microbiological processes. Each recovered
filtrate can, with pre-treatment, such as washing, neutralization,
decolourization or
other after-treatment procedures, be used as such or alternatively in
combination
with various aqueous fractions for the production of single-cell lipid.
Because of the
ways of treatment of the source material, which is part of the invention, the
invention is also applicable to ethanol production.
A filtrate, i.e. an aqueous fraction, or any combination of filtrates, is
added to a
microorganism culture medium which has been or is inoculated with a
microorganism and the microorganism is allowed to produce lipid. The lipid is
recovered in the form of microorganism mass or the lipid is separated from
said
mass and both the lipid and the microorganism mass separated from it are
recovered. Lipids can be recovered using known methods either by removing them
from the cells or by disrupting the cells. The lipid can be extracted from the
disrupted cells using an organic solvent. Methods of lipid recovery applicable
to the
invention are described, for instance, in the publication by Z. Jacob: Yeast
Lipids:
Extraction, Quality Analysis, and Acceptability, Critical Reviews in
Biotechnology,
12(5/6); 463-491 (1992). A preferred method for recovering lipids is phase
separation. The treatment of the lipid formed in the microorganism into fatty
acid
esters can also take place without prior homogenization of the microorganism
cells
and subsequent fat isolation.
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22
A preferred embodiment of the present invention relates to a method of forming
a
lipid or a lipid mixture from a carbohydrate mixture generated in the
processing of
the organic source material, comprising hexose and pentose sugars in monomeric
or
oligomeric forms, according to which method the carbohydrate-containing
mixture
is added to an aqueous culture medium, on which a lipid-producing micro-
organism
is cultured, the medium is supplemented with nutrients required for the
growth,
inoculation of the medium with said organism is carried out, the organism is
cultured and allowed to produce lipid, the cellular mass is recovered, and the
lipid
or the lipid mixture is separated from the cells or the fat-containing cells
or their
constituents are utilized as such.
The method according to the invention provides a particular flexibility for
microbiological lipid production. The fraction containing both hexose and
pentose
sugars is a natural carbon source for many lipid-producing microorganisms.
Thus,
the method also has the advantage of allowing the microorganism to be selected
within a wide range, for instance on the basis of lipid production capacity,
yield of
biomass, type of culturing or culturing conditions. The other constituents of
the
microorganism, besides the lipid, can be used energy-efficiently in many
different
ways, thus improving the overall economic performance of the process according
to
the invention. Preferred ways of using the lipid-free microorganism mass are
hydrolysis and recycling into the culture medium of the lipid-producing
microorganism or use as forage or nutritive substance. It is also possible to
separate
various components, such as (special) sugars, colouring agents, (3-glucan,
sterols,
sterol esters or proteins, from the lipid-free microbial mass.
The micro-organism is selected from natural or genetically modified fat-
accumulating microorganisms, preferably from yeasts, moulds, bacteria and
algae,
more preferably from yeasts and moulds, most preferably from yeasts. It is
essential
that the microorganism to be utilized is capable of producing lipid from
hexose or
pentose sugars or from both. The invention therefore encompasses all
microorganisms in which lipid accumulation is based on the ATP:citrate lyase
activity (EC 2.3.3.8) they contain.
Lipid-synthesizing yeast genera that are applicable for the invention comprise
the
following genera: Candida, Yarrowia, Lipomyces, Rhodotorula and Cryptococcus,
which include strains that synthesize the pentose sugar xylose into lipid,
such as
Candida curvata (D) (Evans, C.T. and Ratledge, 1983. A comparison of the
oleaginous yeast, Candida curvata, grown on different carbon sources in
continuous
and batch culture, Lipids 18 623-629), Rhodotorula gracilis (Yoon, S., Rhim,
J.,
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23
Choi, S., Ryu, D. and Rhee, 11982. Effect of Carbon and Nitrogen Sources on
Lipid Production of Rhodotorula gracilis, J. Ferment. Technol. 60, 243-246)
and
Rhodosporidium toruloides, Rhodotorula glutinis, Rhodotorula
graminis,Lipomyces
starkeyi, Lipomyces lipofer, Candida lipolytica, Cryptococcus, Cryptococcus
albidus, Trichosporon cutaneum and Trichosporon pullulans (Fall, R., Phelps,
P.
and Spindler, D. 1984, Bioconversion of Xylan to Triglycerides by Oil-Rich
Yeasts.
Appl. Environ. Mircobiol. 47, 1130-1134) as well as a strain that synthesizes
the
pentose sugar arabinose into lipid, Lipomyces starkeyi (Naganuma, T., Uzuka,
Y.
and Tanaka K. 1985. Physiological Factors Affecting Total Cell Number and
Lipid
Content of the Yeast, Lipomyces starkeyi, J. Gen. Appl. Microbiol. 31, 29-37).
Correspondingly, the fat-accumulating mould genera that are applicable to the
invention comprise, among others:
- Aspergillus
- Chaetomium
- Clodosporidium
- Cunninghamella
- Emericella
- Fusarium
- Mortierella
- Mucor
- Penicillium
- Pythium
- Rhizopus
- Trichoderma
Correspondingly, the fat-accumulating bacterial genera that are applicable to
the
invention comprise, among others:
- Acinetobacter
- Actinobacter
- Anabaena
- Artlirobacter
- Bacillus
- Clostridium
- Flexibacterium
- Micrococcus
- Mycobacterium
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- Nocardia
- Nostoc
- Oscillatoria
- Pseudomonas
- Rhodococcus
- Rhodomicrobium
- Rhodopseudomonas
- Shewanella
- Streptomyces
- Vibrio
Correspondingly, the fat-accumulating micro-algae genera that are applicable
to the
invention comprise, among others:
- Botryococcus
- Brachiomonas
- Chlamydomonas
- Chlorella
- Crypthecodinium
-Dunaliella
- Euglena
- Nannochloris
- Nannochloropsis
- Navicula
- Nitzschia
- Schizochytrium
- Sceletonema
- Scenedesmus
- Tetraselmis
- Thraustochytrium
- Ulkenia
According to a preferred embodiment of the invention, microorganisms that
synthesize fatty acid-containing lipid into their cells in an amount, which
preferably
is 12-65% by weight of the dry weight of the cells, are used for the lipid
synthesis.
According to a particularly preferred embodiment of the invention, the lipid-
free
biomass formed in the invention, treated in a way suited for the
microorganism, is
used as nutrients in the culture medium. In addition to these components, the
culture
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medium can be supplemented with components preferable for the microorganism
employed. In order to produce lipid, the microorganism generally requires,
among
others, a carbon source, which it in the present invention acquires from the
source
material, a nitrogen source, such as an inorganic ammonium salt (e.g. ammonium
5 sulphate) or an organic nitrogen source (e.g. amino nitrogen, yeast extract,
or
hydrolyzed cellular mass), and a micronutrient source, such as a phosphate,
sulphate, chloride, vitamin or cation source (e.g. a Mg, K, Na, Ca, Fe or Cu
ion
source), whereby these components can be added to the medium when needed.
When the method of the present invention is applied, the lipid concentration
of the
10 cells is preferably 40% by weight, most preferably 65% by weight.
Manufacture of biofuel
The fatty acid ester contained in the microorganism-derived lipid can be
treated to
be suitable as biodiesel fuel by any known method. One preferred approach is
to
carry out a transesterification with short-chained alcohols, preferably
methanol, to
15 obtain the alcohol ester of the fatty acid.
The impure side flow generated in the manufacture of biodiesel or renewable
diesel
taking place with transesterification, which side flow contains alcoholic
compounds, such as glycerol or non-esterified fatty acid salt, and is
difficult to
exploit in a energy-efficient way, can be re-used for the production of single-
cell
20 lipid, which lipid is usable as such or recyclable into other glycerolipid-
containing
materials of biological origin.
Advantages of the invention
The advantages of the invention include the fact that the equipment needed for
the
method is simple and the associated technology is known as far as manufacture
and
25 operation are concerned. The method according to the invention is not
limited to
any production scale, but can be easily scaled up or down according to the
carbohydrate content and amount of the source material to be treated. Carrying
out
the method to produce lipid does not require energy-consuming heating,
pressurized
unit operations or other chemical catalysts in addition to acid, alkaline or
enzyme
catalysts. The method only requires the use of chemicals that can be
incorporated in
the internal cycle of the method according to the invention, or the processing
of
such biomaterials. Nor does the method require cost-increasing water-removal
from
the usable sugar solutions, since dilute carbohydrate solutions are suitable,
as such,
for use in or as the microorganism culture medium. The overall economic
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26
performance of the method is improved by the fact that the lipid-free biomass
generated in it has, in addition to the internal cycle, many different uses,
such as the
production of individual organic constituents, as forage or as the raw
material of the
forage, or as a supplementary culture medium for the production of
microorganisms. The method is also suited for the production of single-cell
biomass
and ethanol.
Advantages of the stagewise treatment of biomass according to the present
invention are the more comprehensive hydrolysis of organic material and, thus,
the
better usability of the organic material for the production of single-cell
lipid
compared to the present technology. Further, the filtrates or precipitates
obtained
from the acid and alkali treatments neutralize each other and reduce the use
of
chemicals required in a neutralization.
The solutions of the prior art describe methods containing microbiological
steps for
ethanol production, wherein carbohydrate-containing materials are used as raw
materials, as such or converted into monosaccharides. Patent application
publication
US 2002/0185447 and patent US 5637502 also describe carbohydrate treatment
methods, such as treatment with acid or alkali, these treatments being
followed by
alcoholic fermentation. Regarding microbiological treatment, both of the said
methods are limited to ethanol production, wherein hexose sugar formed from
polysaccharides is used. In patent application publication US 2003/0096385,
prenyl
alcohol (geranyl and farnesyl derivatives of alcohol) is produced using
microorganisms. Patent application publication WO 03/038067 describes a
method,
wherein modification of the genome of a fungous microorganism can yield an
organism capable of utilizing pentose sugars. The publication is only aimed at
ethanol production.
The invention provides a new possible solution to the exploitation of the more
significant aqueous side flow of thermo-mechanical wood processing
particularly
taking place within the pulp and paper industry, particularly as raw material
for
traffic fuel. The invention allows for the biological loading of the
carbohydrate-
containing side flow generated in connection with the manufacture of
mechanical
wood pulp, and hence the energy costs of wastewater treatment, to be reduced.
In
concrete form, the invention is a lipid production process providing an
environmentally friendly solution to producing raw material for a traffic
fuel,
biodiesel or renewable diesel, for instance from the dilute, carbohydrate-
containing
aqueous fractions generated in TMP processes or corresponding mechanical
treatment of wood.
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The method also provides alternative solutions for the exploitation of other
source
materials. In accordance with the method, recycled fibre, such as printing
paper,
packaging material and comparable cellulose-based materials, can be used as
source
material.
Compared with prior art, the invention is, therefore, in compliance with the
principles of sustainable development, by increasing the availability of lipid
raw
material and reducing the total demand for organic lipid from other sources.
The
invention thus enhances the availability of renewable natural resource-based
biofuel
raw materials and is conducive to bringing their production costs to an end-
user
level acceptable to consumers.
Where the total use of source material is concerned, the invention is not
limited only
to the total use of the contained carbohydrate. The method also includes
stages
allowing the lipid components of the extractive fraction in the wood matter to
be
recovered. When the source material is treated with water, the filtrate
separates a
lipid component which can be recovered from the aqueous solution by methods
familiar to those skilled in the art. As a result of the acid treatment of the
source
material, the lipids in the extractive fraction yield free fatty acids which
are
separated from the filtrate in the form of insoluble alkaline earth metal
salts, such as
Cam salts, and which, after separation of the precipitate, are transesterified
into
alcohol esters. Correspondingly, the alkaline extraction of the source
material,
produces fatty acid salt from the extractive fraction, which salt is water-
soluble and
is thus mixed in the filtrate. In the invention, said salt is converted into
the form of a
water-insoluble salt, such as a Cam salt, and the precipitate is separated
from the
aqueous phase and is used to manufacture fatty acid alcohol esterids.
The following examples are intended to illustrate the invention, and they are
not to
be construed as limiting the scope of this invention in any way. Nor is the
invention
in any way limited to the microbial strains used. The invention can be
implemented
not only by means of the strains used but also by means of other strains of
the same
species or genus, or the strains of other microbial genera or species or by
means of
genetically modified microbe strains. Lipid-producing micro-organisms are
commonly available and they can be found in several strain collections, e.g.
ATCC,
DSM, etc. Lipid-producing microorganisms and lipid production processes using
microorganisms (including algae) are described in the literature, for example
in the
writings: Single Cell Oils, eds. Z. Cohen and C. Ratledge, AOCS Press, 2005
and
Microbial Lipids, eds. C. Ratledge and S.G. Wilkinson, vol. 1 and 2, Academic
Press, 1988.
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Examples
Example 1
100 g each of wood fibre, ground wood, TMP pulp, sawdust and mechanical pulp
were kept in 1 litre of boiling water, 90-100 C, for 2 hours. The solutions
were
filtered away from the fibre material (hereinafter precipitate). The
precipitates were
recovered and treated using various alternatives to increase the
monosaccharide
yield according to one of the alternatives presented in Examples 4 or 5. The
carbohydrate yields of the solutions ranged from 2% to 6% depending on the
source
material, being typically 4% when spruce fibre ground at a high temperature
(over
170 C) was used as source material. Some of the solutions were reused without
preceding evaporation for extracting the following batches of fibre and some
of the
solutions were concentrated by evaporation until a dry-matter content of about
20%
by weight was achieved. The concentrated filtrates were recovered for the
production of single-cell lipid. The dissolved carbohydrates were typical for
the tree
species extracted, dividing into hexoses and pentoses.
Example 2
Wood fibre, ground wood, recycled fibre, TMP pulp, sawdust and mechanical pulp
were each added to one litre of 5% alkaline solution (NaOH) and stirred at 90-
100 C for 3 hours. Filtrations were carried out at normal temperature and the
precipitates were subjected to further treatment for the production of
monosaccharides according to Example 4. The extraction solutions were treated
so
that a part of each solution was reused in treating the next batch of fibre, a
part was
neutralized, and a part was conducted to hydrolysis according to Example 4
since
some lignin as well as some oligo- and polysaccharides had dissolved in the
alkaline extraction solutions. The solutions obtained from different source
materials
by the described alkaline treatment had retained an average of 5% of the
weight of
the source material.
The precipitates obtained in the original alkaline treatment were also
retreated with
alkali by repeating the previous alkaline treatment and grinding for 6 minutes
at a
pressure of 8 bar, at a temperature of 170 C. Using this further grinding,
the
amount of material dissolving in the alkaline solution could be increased to
an
average of 27% of the original amounts of source materials. The solutions
became
black and, following neutralization, hydrolysis and concentration, they were
treated
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with activated charcoal and ion exchanger for decolourization. The mixtures
were
recovered and used to produce single-cell lipid.
Example 3
100 g each of wood fibre, ground wood, TMP or MDF pulp, sawdust, mechanical
pulp, recycled fibre and neutralized extract prepared according to Example 2
were
hydrolyzed in one litre of 5% mineral acid, pH 1, at 90-100 C for 3 hours. The
precipitates were separated from the solutions by filtering. The solutions
contained
monosaccharides on average 10% of the amounts of source material. Each
solution
was treated so that a part was neutralized, filtered and concentrated by
evaporation
until a monosaccharide amount of about 20% by weight was achieved, and the
concentrated solutions were recovered for the production of single-cell lipid.
A part
of the solutions was reused as such for hydrolysis of the following batches of
source
material as described in this example. The precipitates were further treated
to
increase the monosaccharide yield according to the following Example 5.
Example 4
125 g each of wood fibre, ground wood, TMP pulp, MDF pulp, sawdust,
mechanical pulp, straw, grain husk and precipitate according to Examples 1-3
were
added to a sulphuric acid solution having a concentration of 10%, and the acid
was
allowed to be absorbed for 2 hours. Excess acid was decanted off and the
mixtures
were conducted into a wing refiner, the temperature was raised by presteaming
for
11 minutes without letting the condensate flow out, after which the
temperature was
raised 160 C and the pressure to 8 bar and the grinding was performed using
the
wing refiner. A mixture grinding time of 11 minutes was selected for the
spruce
fibre. The precipitates were separated from the solutions after the grinding.
Each
precipitate was divided so that a part was used for incineration and a part
was
reground to increase the monosaccharide yield according to the following
Example
5. Correspondingly, the solutions were neutralized, filtered and concentrated
by
evaporation until monosaccharide concentrations of about 20% were achieved.
These solutions were recovered for the production of single-cell lipid.
Example 5
1D..125 g each of wood fibre, ground wood, TMP pulp, sawdust, mechanical pulp,
straw and grain fibre as well as remaining precipitates according to Examples
1-4
were added to a 40% sulphuric acid solution and allowed to absorb at room
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temperature for 2 hours. Excess acid was decanted off and the precipitates
were
ground in a wing defibrator so that the temperature of the precipitates was
raised by
presteaming for 11 minutes without letting the condensate flow out. The
temperature was then raised to 170 C and the pressure to 8 bar. The most
suitable
5 grinding time for straw and grain fibre was 11 minutes. The precipitates
were
separated from the solutions. The proportions of dissolved substances were on
average more than 50% of the source material. The solutions were neutralized,
filtered and concentrated by evaporation, and recovered for the production of
single-cell lipid. The precipitates, i.e. the residual fibres, were partly
conducted to
10 incineration and partly reground according to this example to increase the
monosaccharide yield.
Concentrated sulphuric acid (72%) was also separately absorbed into the above
described source materials, the treatment time being 2 hours, at room
temperature.
Subsequently, the acid was diluted to 5% and boiled at normal pressure at 100
C
15 for 4 hours. Further treatment and use of the generated fractions took
place as
above.
Example 6
Monosaccharide suitable for producing single-cell lipid can be produced from
chaff,
straw and grain husk by direct hydrolysis using 5% acid or be impregnated with
a
20 stronger one and hydrolyzed in a 5% solution or first be treated with
alkali and the
hemicellulose and cellulose can be hydrolyzed separately as presented in the
above
examples. These treatments can be combined with impregnation treatments with
acid or alkali and subsequent repeated grindings in a wing or disc refiner to
produce
thermo-mechanical pulp. In this example, however, chaff, straw and grain husk
25 (125g each) were pre-steamed for 11 minutes at a pressure of 8 bar and
treated into
thereto-mechanical pulp in a wing refiner at a temperature of 170 C. The
generated
thereto-mechanical pulps were hydrolyzed in 5% acid (sulphuric acid) in a
volume
of one litre at 90-100 C at normal pressure for 4 hours. The solution
fractions were
separated by filtration and neutralized. Subsequently, the solutions were
30 concentrated and recovered for the production of single-cell lipid. The
monosaccharide yield was on average 50% of the source material. To increase
the
carbohydrate yield, the precipitates remaining after the filtration were
thermo-
mechanically re-treated according to Example 5 and a part was conducted to
incineration.
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Example 7
Chaff, straw and grain husk (16 kg each) were each treated in 100 litres of
alkaline
solution (1.2 M NaOH) at 90-100 C for 4 hours. The mixture was divided into a
precipitate and a solution. On average 49-57% of the dry matter of the fed
source
materials ended up in the solutions. The alkaline solutions were rendered 5%
in
terms of the acid, using sulphuric acid, and they were hydrolyzed as in
Example 6.
Mixtures of xylose and arabinose, which also contained small amounts of
glucose
and galactose, were mainly formed. The monosaccharide content of the solutions
became 23-33% of the fed source material. The colour of the solutions was
reduced
by treatment with activated charcoal before they were recovered for the
production
of single-cell lipid.
The precipitates obtained in the filtration were impregnated in 5% acid
(sulphuric
acid) and ground in a wing refiner for 11 minutes at a pressure of 6 bar and a
temperature of 150 C. The precipitates degraded into soluble carbohydrates,
mainly
glucose and, galactose, which after neutralization and filtration were
recovered for
the production of single-cell lipid. Parts of the precipitates remaining after
the
filtrations, carried out after the acid hydrolysis, were still re-ground and
then
hydrolyzed in 10% acid according to Example 4. Solutions were obtained after
these treatments, which contained monosaccharides in a total of 55-65% of the
source material. The solutions were recovered for the production of single-
cell lipid.
Example 8
Steam-dried beet pulp was poured into a tub and enough boiling water was
poured
on top of it so that the water covered the beet pulp. The solution was allowed
to
cool before separating the water from the solids by decanting. 3.6 mg/ml of
dry
matter had dissolved in the water. The aqueous fraction was partly used to
treat a
new batch and partly, after concentrating, recovered for the production of
single-cell
lipid. The obtained solid fraction, the precipitate, containing 16.7% of dry
matter,
was weighed 748g, corresponding, as dry matter, to 125g of dried beet pulp,
was
impregnated with 0.4M phosphoric acid (H3PO4, 500 ml) at room temperature for
18 hours. Excess acid solution was decanted off and pre-steamed for 11 minutes
before initiating the thermo-mechanical grinding. The mixture was ground for
11 minutes using a wing refiner. During grinding, the pressure was 8 bar and
the
temperature was initially 172 C and, when 10 minutes of grinding time had
lapsed,
162 C. The mixture was removed from the refiner and filtered. The solution
fraction, 2.26 litres, contained 2.75% dry matter. 50% of the dry matter fed
to the
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grinding was present in the solution after the filtration. The solution
contained
mainly monosaccharides, glucose, galactose, arabinose and xylose. In addition,
minor amounts of oligosaccharides in the molecular weight range of 500 to 3000
and higher molecular weight compounds were observed. The solution fraction was
neutralized, concentrated and recovered for the production of single-cell
lipid.
The solid fibre pulp, the precipitate (125g dry matter), obtained from the
above
treatment of the beet pulp was impregnated in 15% hydrochloric acid at room
temperature for 4 hours. Excess acid solution was decanted off and the pulp
was
reground under the same conditions. After the filtration stage, it was
observed that
an additional 28% of monosaccharides had entered the solution phase. The
precipitate was discarded.
Example 9
The source material was thermo-mechanically treated spruce fibre, of which
46.8g
(about 1 litre in volume) was weighed, and 0.2N NaOH was added on top of it,
to
which a further 5.6g of Na2CO3 and 1.3g of MgSO4 6H20 (500m1) was added. The
mixture was heated to 50 C,--yielding minor amounts of glucose, xylose,
galactose,
arabinose, mannose and additionally oligomers in the solution. The pH of the
solution was about 11, it was filtered and washed with water. The washing and
the
filtrate were combined and the solution was evaporated to 320m1. The amount of
dissolved substance was 4.5% (dry matter) of the source material. To increase
the
amount of monosaccharides, acid was added to the solution, resulting in a
precipitate. The precipitate was separated from the solution, the latter being
recovered and used to produce single-cell lipid. The precipitate was
hydrolyzed into
monosaccharides in 5% acid as described in Example 3. Using the hydrolysis, 1%
monosaccharides was generated from the mass of the precipitate, which
monosaccharides entered the solution and were, after decolourization,
recovered for
the production of single-cell lipid.
Example 10
A 45g batch (about 1 litre) of thermo-mechanically treated spruce fibre was
weighed, on top of which was added 0.2 N NaOH, to which was added a further
5.6g of Na2CO3 and 1.3g of MgSO4 6H20 (500 ml). The mixture was heated to
50 C, filtered and the precipitate washed with water. The washing and
filtrate
solutions were combined. To increase the amount of monosaccharides, acid was
added to the combined filtrate and hydrolysis was performed according to
Example
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3. After neutralization, the generated hydrolysis product was recovered for
the
production of single-cell lipid: The washed precipitate was compressed to 300
ml
and 1 litre of citrate buffer, pH 4.8, was added, and cellulase enzyme was
added.
Oligomers and glucose were generated in the mixture. The enzyme-treated
mixture
was recovered both as such and as a separated filtrate, a solution, and used
to
produce single-cell lipid.
Example 11
A 125g batch of oat chaff was provided and it was thermo-mechanically ground
at
170 C and a pressure of 8 bar for 2 min. After the treatment, the precipitate
was
separated from the solution by filtration. The solution was recovered for use
in
producing single-cell lipid. A 7 g batch of precipitate (fibre) was used and
rendered
19% in strength in terms of sulphuric acid and refluxed for 4 hours. Analysis
showed that the treatment resulted in the monosaccharides entering the
solution in
56% of the source material, mostly as xylose, mannose, glucose galactose and
arabinose. The solution was recovered for use in producing single-cell lipid.
Example 12
400g of oat chaff was weighed and 3 litres of water and 200g of NaOH were
added.
The mixture was kept at a temperature of 90-96 C under stirring for 2 hours.
The
mixture was filtered through a cloth and the fibres were separated. The
filtrate
fraction (50 ml) was neutralized and its pH adjusted to 4.8 with a citrate
buffer
(40 ml), multifect xylanase (10 ml) is added and the mixture was thermostated
to
50 C. Sugars were released into the solution, of which 25.2% were xylose and
11.8% were arabinose. There were also oligomers in the solution after a
reaction
time of 50 hours. The entire mixture was recovered for use in producing single-
cell
lipid.
Example 13
Beet pulp, 125g, was impregnated with 0.4 M sulphuric acid and excess acid was
decanted off after 12 hours. The pulp was transferred into a wing refiner, in
which it
was pre-steamed for 4 min and ground at 150 C at a pressure of 6 bar for
11 minutes. The mixture was neutralised and citric acid was added until the pH
dropped to 4. 10 ml of pectinase 4450 U, i.e. 178 mg/ml of protein (Sigma),
were
added and the reaction was allowed to proceed for 24 hours at 25 C. After the
reaction, a part of the mixture was recovered for the production of single-
cell lipid
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34
and a part was filtered. The precipitate obtained from the filtration was
returned to
the grinding stage and the solution fraction resulting from this treatment was
treated
with activated charcoal, 2 g/litre, and then conducted to an anion exchange
column.
The monosaccharide solution obtainable from the column was evaporated to a
concentration of 20% in terms of dry matter, was recovered for the production
of
single-cell lipid.
Example 14
Oat chaff was treated according to Example 7, yielding a mixture containing
glucose at 23.8 g/L, xylose at 93.3 g/L, arabinose at 37.1 g/L and galactose
at
9.0 g/L. This mixture was added as a culture medium for the lipid-synthesizing
yeasts Yarrowia lipolytica ATCC 20373 and Rhodotorula glutinis TKK 3031 as
such, as a 1:1 dilution and as a corresponding dilution supplemented with
glucose at
11 g/L. The culture period was 68 hours, the temperature 28 C, shaking at 250
rpm
and culture volume 50 ml. As is observed in Figure 3, the yeasts were able to
grow
and synthesize lipid without requiring the addition of other nutrients.
Example 15
Three yeasts, Rhodotorula glutinis, Yarrowia lipolytica and Kluyveromyces
marxianus (Anam. Candida kefyr) ATCC 42265, were cultured solely with xylose
as the carbon source. The culture medium contained xylose at 20 g/L, yeast
extract
at 10 g/L and peptone at 20 g/L. The culturing was performed in a volume of 50
ml
and at 25 C under shaking at 200 rpm. Figure 4 shows that all three strains
are able
to utilize pentose sugar as a carbon source.
