Enzymic oxidation of fatty acids on a porous bed solid support

A process for enzymic oxidation of a fatty acid to produce an oxidation product and an apparatus for carrying out the process are presented. The process is carried out in the presence of water and oxygen wherein the fatty acid, water and enzyme for the oxidation are substantially uniformly distributed throughout a porous bed of solid suppport material in the substantial absence of a continuous liquid phase. Oxygen is passed through the bed and the oxidation product is recovered from the porous bed. By this process, methyl ketones can be produced from saturated fatty acids. The porous bed is contained within an apparatus which includes a sealable vessel, a gas inlet for supplying the oxygen gas to the reactor and a gas outlet. The gas is forced through the inlet and into passage means defining a plurality of upwardly extending passages that can open along their length into the bed and that are spaced 5 to 20 cm apart.

This invention relates to a process for aerobic enzymic reactions for 
oxidation of fatty substrates that can be carried out in relatively high 
concentrations and can permit the recovery of high yields of desired 
product. 
BACKGROUND OF THE INVENTION 
Enzymic hydrolyses and oxidation processes occur naturally both in solid 
and liquid media, and generally give a range of products. The crude 
mixture may be unpleasant, but individual components may be desirable. 
Therefore, it is known to perform the processes artificially for oxidation 
of naturally occurring materials. 
Commercial aerobic enzymic processes are normally carried out in solution. 
This has to be relatively dilute to allow oxygen to be distributed easily 
throughout the mixture, as otherwise aeration becomes difficult. Thus, the 
biotransformation solution must be a dilute solution and the yields 
resulting from such a dilute solution are relatively low. 
Solid state processes are also known. The solid phase can be the substrate 
that is to be converted (e.g., malting of barley grains) or can be a 
support for the aqueous medium in which the oxidation occurs. In Process 
Biochemistry, July 1966, J. Meyrath describes production of amylolytic 
enzymes by growth of various molds which produce such enzymes on bran and 
on vermiculite to obtain a yield of the amylase enzyme. In addition, Oriol 
et al., in the Journal of Fermentation Technology, volume 66, number 1, 
pages 57 to 62, describe how Aspergillus niger fungus was grown on a solid 
support of sugar cane bagasse. 
The solid state process described by Meyrath is for the production of the 
enzyme itself. However, it is also known to use the enzymes formed to 
obtain the biotransformation products that they produce. Another solid 
state reaction is discussed in Advances in Applied Microbiology, volume 
28, pages 201 to 237, for the production of citric acid on inert 
materials. Sawdust and sugar-free sugar cane bagasse were used as the 
growth support, and this was impregnated with sugar solution. The inert 
materials were inoculated with mold culture, and citric acid was produced. 
Other growth supports mentioned are rice hulls and wheat bran. 
It is known that natural atmospheric oxidation of fats and oils produces a 
crude, unpleasant product (e.g., rancidity), but that some components of 
the product can be useful. 
In Phytochemistry (1984) volume 23, number 12, pages 2847 to 2849, 
Kinderlerer describes natural oxidation of coconut due to the action of 
molds natural to the coconut as giving a series of aliphatic methyl 
ketones and secondary alcohols. In Phytochemistry (1987) volume 26, number 
5, pages 1417 to 1420, she described their production in a liquid medium, 
following growth of Aspergillus ruber and A.repens fungi with coconut oil 
as the sole carbon source. However, low yields were obtained. 
It is also known to use milk and milk compounds as a growth medium for 
biotransformation reactions. For example, ketones have been produced by 
biotransformation processes on milk and milk compounds. In U.S. Pat. No. 
3,100,153, a process is described wherein pasteurized homogenized milk 
containing milk fat is fermented under submerged aerobic conditions using 
Penicillium roqueforti to produce ketones, and in U.S. Pat. No. 3,720,520, 
ketones giving a blue cheese flavor are produced by growing Penicillium 
roqueforti with aeration in an aqueous medium of sodium caseinate and fat. 
Such blue cheese flavors are also described in pages 285-287 of volume 40 
(1975) of the Journal of Food Science, where R. Jolly and F. V. Kosikowski 
report studies of biotransformation of coconut fat and butter fat with 
whey powder, carried out in an aqueous medium, again using Penicillium 
roqueforti. 
U.S. Pat. No. 4,769,243 describes the preparation of green aroma compounds 
by reaction of at least one unsaturated fatty acid (which may be produced 
in situ from fat and lipare) with enzymes from soy beans. An aqueous 
solution of water and ground germinating soy beans is mixed with linseed 
oil, and the mixture is allowed to react while stirring rapidly. Air or 
oxygen is supplied to the mixture throughout the reaction. The resulting 
reaction is due to the enzymes present in the germinating soy bean. This 
type of liquid process must be stirred rapidly to ensure that the mixture 
remains substantially homogeneous. In addition, it is difficult to provide 
oxygen throughout the whole reaction mixture, and so liquid mixtures as 
described above must be relatively dilute and require some form of 
intermixing. If the amount of water is reduced, the fat tends to form a 
very viscous continuous phase between the particles of ground bean, and 
this will prevent adequate oxidation. 
It would be desirable to devise a process of enzymically oxidizing fats 
and/or fatty acids and that can be performed easily and to give high 
yields of the desired products. It would also be desirable to provide 
novel apparatus suitable for such processes. 
SUMMARY OF THE INVENTION 
According to the present invention, there is now provided a process for 
enzymic oxidation of a fatty acid in the presence of water and oxygen to 
produce an oxidation product characterized in that the fatty acid, water, 
and an enzyme for the oxidation are substantially uniformly distributed 
throughout a porous bed of solid support material in the substantial 
absence of a continuous liquid phase; oxygen is passed through the porous 
bed, and the oxidation product is recovered from the porous bed. The 
oxidation product may undergo further reaction.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The starting material is a fatty substrate which can be fatty acid alone, 
or mixed with fat or other ester, or fat alone (which is hydrolysed to 
acid prior to the oxidation process). 
The fatty acids used by be saturated or unsaturated hydrocarbon fatty 
acids, or may be saturated or unsaturated hydroxy carboxylic acids. 
Saturated fatty acids are oxidized in the beta position and will 
subsequently undergo decarboxylation. Unsaturated fatty acids undergo 
oxidative cleavage to give products having carbonyl groups at the point of 
cleavage. These aldehydes or ketones may undergo subsequent reduction to 
the corresponding alcohols. Decarboxylation of the oxidation products of 
saturated fatty acids is usually spontaneous due to the thermodynamically 
unstable nature of the oxidized compounds, but may result from a further 
enzymic reaction. Saturated, unsaturated, and hydroxylated fatty acids 
react to produce methyl ketones, aldehydes, and lactones, respectively. 
Secondary alcohols can be produced as the final end product by reduction of 
product methyl ketones or primary alcohols from aldehydes: for example, if 
a reductase enzyme is present. 
Unsaturated aldehydes and alcohols can be produced from doubly or 
polyunsaturated higher fatty acids such as linoleic or linolenic acid. 
Oxidative attack at one of the unsaturated groups breaks the double bond 
to produce, for instance, two fragments, each terminating with a carbonyl 
group. Particularly useful products of this type of reaction are hexenals 
and hexenols. For example, linseed oil contains doubly and triply 
unsaturated C.sub.18 glycerides which result in the formation of 
unsaturated fragments, giving rise to hexenals and hexenols. 
The invention is of particular value for the production of methyl ketones 
from saturated fatty acids. 
A vital feature of the invention is that the oxidation is conducted on a 
support that is porous throughout the reaction, since this permits easy 
aeration and contact of substantially all parts of the material with 
oxygen. To maintain this condition, the support cannot be oil-logged or in 
the form of an aqueous suspension or slurry, and so this is in contrast to 
U.S. Pat. No. 4,769,243, which uses an aqueous slurry. 
Since the liquid phase is carried on the porous bed of solid support, its 
aeration is controlled by the porosity of the bed. The concentration of 
the fatty acid in relation to the aqueous phase can be relatively high, 
and so the yield of product from the process is maximized. It would be 
difficult to obtain satisfactory aeration of a bulk liquid phase of the 
same concentration because of its physical properties. For instance, the 
maximum yield obtainable by a conventional liquid phase process would be 
around 15% or less, but in the invention it is possible easily to obtain 
yields of above 20%, and much higher if optimum conditions are selected. 
The process has an additional advantage in that since it is a bio-process, 
as long as the starting products are of natural origin and not 
artificially produced, the products of the process can be classified as 
natural products. 
The support is a porous matrix which may be fibrous (for instance an open 
web of fibers), but is preferably particulate. The particles may be 
non-porous, but preferably they are a porous particulate substance so that 
the surface area of the support is maximized. The bed can then be regarded 
as both macroporous (due to the interstices between particles) and 
microporous (due to porosity within particles). The particle size is 
generally from 0.001 to 5 millimeters, preferably from 0.01 to 2 
millimeters. Examples of suitable support materials are inert, 
non-carbohydrate materials that may be inorganic or organic (generally 
synthetic polymeric) materials such as vermiculite and fibers or foams 
which may be made of plastic materials. Preferably the support is a 
carbohydrate, preferably a cellulose such as cellulose powder, rice flour, 
maize starch, wheat flour, woodpulp, or other carbohydrate porous matrices 
fibers or other particulate materials. 
In the invention there must be substantially no continuous aqueous or other 
liquid phase. When a particulate material is used, the support preferably 
remains substantially friable throughout most (and preferably all) the 
process. Although some aggregation of particles is acceptable, the 
individual particles or aggregates of them should remain substantially 
separate from one another in order that porosity and aerobic conditions 
prevail substantially throughout the bed. 
The enzymes may be pre-formed, or may be grown in situ. They must be 
suitable to induce the desired oxidative reaction of fatty acids. 
Pre-formed enzymes can be obtained from any suitable source, and should be 
mixed substantially uniformly throughout the carbohydrate or 
non-carbohydrate bed. The pre-formed enzyme may be obtained from a plant 
source, or may be of microbial origin. Examples are ground germinating soy 
beans in aqueous suspension or enzymes isolated from them, or alcohol 
oxidase from Pichia pastoris, or alcohol dehydrogenase from Baker's yeast. 
Preferably, however, the enzymes are generated in situ, for example, by 
growth in the porous support of micro-organisms capable of producing the 
desired enzymes for effecting the desired conversion. This is of 
particular value when the fatty acid is part of a fatty substrate during 
the process which comprises both a fat and a fatty acid derived from the 
fat. The micro-organism should be distributed uniformly throughout the 
bed, as otherwise a proportion of the fatty substrate will not be exposed 
to the aerobic biotransformation. This uniform distribution can be 
achieved by for instance absorbing it into the particles while it is 
carried in, for instance, an aqueous nutrient medium. It is particularly 
preferred to use a filamentous micro-organism since then the growth of the 
micro-organism on the support results in the growth of filaments which 
carry the micro-organism to other parts of the bed. The micro-organism can 
be any of the molds that grow naturally on the fatty substrate or can be 
any other micro-organism capable of effecting the enzymic 
biotransformation of the fatty acid. When the micro-organism is being 
grown in situ, and especially when filamentous, the support is preferably 
unstirred during the process. The porous bed itself may move (e.g., on a 
conveyor), but the relative movement within the bed should be kept to a 
minimum. 
An important feature is that when the enzyme is produced in situ, a limited 
source of carbohydrate for growth of the enzyme-producing micro-organism 
should be present throughout the bed. This source may be a soluble 
carbohydrate such as dextrins, glucose, or various monosaccharides. It may 
be added to a non-carbohydrate support, but preferably it is released from 
an otherwise insoluble carbohydrate support that may, for instance, be 
contaminated with for instance 0.1 to 5%, often about 1%, soluble 
carbohydrate before use. The preferred carbohydrates listed above 
generally provide a suitable source as well as being suitable supports. 
It is generally desirable that the support material releases or otherwise 
provides part of the source of carbohydrate available to the 
micro-organism during at least most of the process, especially when the 
support is static and the micro-organism filamentous. Generally, the fatty 
substrate also serves as a source of carbon. However, in some instances it 
can be desirable to include a small amount of soluble saccharide, such as 
sucrose or glucose, in the aqueous medium to promote initiation of the 
process. If soluble saccharide is included after the process has been 
initiated, the product yield is liable to drop, possibly because the 
micro-organism is metabolizing the soluble saccharide in preference to the 
fatty product and the insoluble carbohydrate. 
A mold that will provide the desired enzyme is preferably mixed into the 
support as spores which germinate on the support to form mycelia. 
Alternatively, mycelia may be added to the support; but this is generally 
not preferred, as mycelia are much less robust than mold spores and are 
liable to be damaged during distribution throughout the support. Growth 
conditions should be carefully regulated during the germination stage to 
obtain optimum results as the micro-organism is very sensitive to adverse 
conditions during this stage. 
The germination stage produces mycelia which generally takes from around 1 
to 6 days, and the optimum temperatures for this stage are ambient: for 
example, 15.degree. to 30.degree. C., preferably 18.degree. to 25.degree. 
C. In addition, the germination stage is sensitive to acidity, and 
although it has been found that free fatty acids can be added to the 
support bed for enzymic oxidation, the smaller chain fatty acids have been 
found to tend to prevent or impair the rate of germination. Accordingly, 
germination should preferably be conducted in the substantial absence of 
fatty acids of less than 8 carbon atoms. During this initiation phase, 
little or no oxidation of fatty acid takes place. 
When germination is conducted in the presence of fatty acids, germination 
is followed by a lipolysis stage in which fat is hydrolysed to the fatty 
acid components. 
The micro-organism then induces oxidation of fatty acid and this can cause 
a rise in temperature typically from ambient to between 35.degree. to 
50.degree. C. 
In order to limit excessive temperature rise, a cooling means may be used, 
and this is preferably a cooling coil and/or jacket. The temperature in 
the bed is generally below 50.degree. C., preferably below 40.degree. C., 
and most preferably below 35.degree. C., throughout the reaction. 
The fatty acid may all be pre-formed by any natural process (and the fatty 
substrate will then consist solely of fatty acid), especially when 
pre-formed enzyme is used. When the fatty acid is pre-formed, preferably 
it is added to the support bed gradually, as conversion occurs, because if 
the acid content in the bed is too high, the enzyme action may be 
affected. This is a particular problem with short chain fatty acids, and 
the problem can be minimized by using only fatty acids having a carbon 
chain length of ten or above. Preferably, some or all of the fatty acid is 
generated by hydrolysis in situ of a corresponding ester (for example an 
ethyl or other alkyl ester, but preferably a glyceride) either by a 
hydrolyzing enzyme produced by micro-organism grown on the support, or 
with the addition of some pre-formed lipase to effect the initial 
hydrolysis of fat or oil to fatty acid. In situ production of fatty acid 
is advantageous in that it gives a means of controlled release of fatty 
acid to the support bed and acts as a rate-limiting step i the process. 
The fatty substrate during the process will then contain both fatty acid 
and fat, but at the start of the process (i.e., before the lipolysis) may 
consist solely of fat. 
Preferably the process comprises introducing the fatty acid as a fat (which 
may be a solid fat or an oil) and growing a micro-organism that provides 
both a lipase for hydrolysis of the fat and an oxidase for oxidation of 
the resultant fatty acid to ketone or aldehyde. 
It is often preferred for some of the fatty acid to be produced from a fat 
or oil in situ and some of the fatty acid to be added to the reaction 
mixture, since this can increase yield. The fatty substrate can then 
comprise different types of fatty acids, and generally also fat. When the 
fatty acid is added to the support bed after the germination stage, it is 
distributed into the support bed in order to produce a substantially even 
distribution throughout the bed while aiming for the minimum disturbance 
of the bed. For instance, the fatty acid can be blended with separate 
porous support material, and this can then be blended with the bed 
containing the germinating microorganisms since this reduces the amount of 
stirring needed to obtain uniform distribution. 
A separate, first lipolysis stage may be provided in a separate reactor in 
which fat and lipase enzymes such as Candida lipase are reacted to produce 
fatty acid which is subsequently added to the bed. Preferably the 
subsequent addition of fatty acid to the bed is regulated. 
Preferably, the fat used is coconut oil, but other suitable fats or oils 
include castor oil, linseed oil, arachis oil, sunflower oil, maize oil, 
palm leaf oil, palm kernel oil, or any edible oil. The amount of fatty 
substrate added as fatty acid and the amount added as fat can vary 
considerably. The source of fatty acid may comprise 100% fat or it may 
comprise 100% pre-formed fatty acid. However, in the preferred process 
where the hydrolyzing enzyme is formed in situ, the ratio of fat to fatty 
acid is generally between 1:10 and 50:1, and preferably from 1:5 to 10:1 
by weight. 
Particularly preferred fatty acids are those which occur naturally (as 
glycerides), and the preferred fatty acids are C.sub.6-18 aliphatic 
carboxylic acids which may be saturated or unsaturated and may have one or 
more hydroxy groups. 
In the case when the micro-organism is also the source of lipase and 
oxidase, and the fat used is, for example, coconut fat, the fatty acid 
preferably has a carbon chain length less than C.sub.16, and most 
preferably less than C.sub.14, because these lower carbon chain lengths 
appear to be preferentially oxidized by the micro-organisms. However, 
using other fat sources and/or other micro-organisms, preferred fatty 
acids may differ. We hypothesize that the reason for this is that initial 
fat hydrolysis is an extracellular reaction resulting from the lipase 
enzymes which are secreted by the micro-organism and act outside the cells 
to produce fatty acids, and the resultant fatty acids must be capable of 
migration into the cells of the microorganism through the cell wall, for 
the subsequent oxidation and decarboxylation, which is an intracellular 
reaction. 
Suitable micro-organisms for producing enzyme or enzymes for hydrolyzing 
and oxidizing oils, fats, and fatty acids such as those discussed above, 
are molds from the Phycomycetes class, or the Ascomycetes or Fungi 
imperfecti classes. Preferably, those used are food grade molds such as 
Penicillium roqueforti, Penicillium cameberti, Aspergillus niger, 
Aspergillus oryzae, Rhizopus japonicus, or Rhizopus oryzae. Non-food grade 
molds are also suitable for the biotransformation, and examples are 
Penicillium cyclopium and Eurotium herbariorum. They conveniently can give 
methyl ketones. 
Enzymes that are particularly suitable for converting linseed oil and 
similar polyunsaturated materials can be provided by any green leaf plant. 
Grass or germinating soya beans are examples of preferred materials 
because they have a relatively high content of the enzyme. 
Octenols and hexenols can be obtained from germinating soya beans with 
unsaturated fatty oils such as linseed oil. Castor oil and carotenoids may 
also be used as substrates in conjunction with grass, since this 
vegetation provides suitable enzymes. 
For preparation of the support bed, the fatty substrate (which may be a 
solid or an oil at ambient temperature) may be warmed to assist loading by 
ensuring that the viscosity is low so that it will mix easily onto the 
support. Preferably, it is mixed with the particulate material before 
addition to the vessel in which fermentation is to occur. The mixing can 
be achieved using a mixing machine: for example, a Nauta or Winkworth 
mixer. Water or aqueous nutrient is also loaded onto the support in this 
way and may be added either prior to or following the addition of the 
fatty substrate. 
Alternatively, the fatty substrate and water or aqueous nutrient may be 
mixed to form an initial emulsion which is then loaded onto the support. 
It may be necessary to add a suitable emulsifier to maintain the emulsion 
until it is loaded onto the particulate material to ensure that both 
components are distributed substantially evenly throughout the support. 
Suitable emulsifiers are, for example, lecithin, casein, and quillaja. 
Artificially produced emulsifiers may also be effective, but are less 
preferable for production of food grade products. 
When the enzyme is pre-formed, either fat or fatty acid can be loaded onto 
the support. However, when the enzyme is produced in situ by the 
micro-organism, it is important to select the optimum conditions for 
enzyme induction in order to maximize growth and bioconversion. As 
explained above, due to the sensitivity of the germination stage, it is 
generally essential that the acid present should be at a relatively low 
concentration, and in particular there should be substantially no free 
fatty acid present having a short carbon chain length. It has been found 
that while the short chain fatty acids, for example C.sub.8, are damaging 
or retarding to the germination stage, a C.sub.10 fatty acid may be 
present and the germination can still take place. 
When the germination stage is substantially complete, pre-formed fatty 
acids can be added directly to the support. These are generally C.sub.6 
-C.sub.18, and preferably C.sub.8 and/or C.sub.10 fatty acids. Preferably 
the fatty acids are added as a mixture of different carbon chain length 
fatty acids, such as occurs naturally. The acids can be added as, for 
instance, alkali metal salts. 
The weight ratio of fatty substrate to water on the support should be 
carefully controlled for optimum yields, and is generally from 1:10 to 
7:10, preferably from 1:3 to 4:7; and most preferably around 2:3. If there 
is too much fatty substrate, a fatty layer tends to separate out into the 
bottom of the reaction vessel and the process loses efficiency. If the 
amount of water is too high, the yield is reduced and there is a tendency 
for the bed to become saturated with water if the amount of fluid is 
increased to compensate for the low yield. 
When the enzyme is formed in situ and a fatty acid is added to the support 
bed prior to germination, the substrate is preferably added at a 
concentration of from 2% to 30%, and most preferably the concentration is 
from 5% to 10%, based on the total weight of loaded support bed. 
Where the fatty acid is added to the support bed after germination of an 
enzyme-producing mold, it is again generally added at a concentration of 
from 2% to 30%, and preferably 5% to 15%. 
Concentrations of fatty acid below 2% can be used, but tend to be 
impractical, while the upper limit of fatty substrate that can be used is 
generally imposed by the possible loading of fatty substrate and water 
onto the support material and the moisture requirement of the 
micro-organism for effective growth. 
In choosing suitable support materials, it should be noted that the 
micro-organism can affect the capacity of the support for the aqueous 
phase. For example, it has been found that for a particular fatty 
substrate-to- water ratio, the tendency for separation of the two phases 
is increased following inoculation of micro-organism into the bed of 
support material and its consequent rise in temperature. 
In addition, the ratio of water to the support material and fatty substrate 
to the support should be controlled. Although it is desirable to have a 
high content of liquid phase, the support bed should not be fully 
saturated to the point where it will tend to form a continuous phase and 
reduce the passage of oxygen so that some parts of the support bed will, 
at least in part, no longer be aerobic. 
The ratio of water to the support material is generally from 1:10 to 6:3, 
preferably from 1:3 to 5:3, and most preferably around 4:3. 
It is naturally desirable that the load of fatty substrate and aqueous 
nutrient solution, or water, be as high as practicable without saturating 
the support and destroying its porosity. Generally, the total amount of 
fatty substrate and the aqueous nutrient solution or water that is 
absorbed into the support is from 15% to 99%, often 50% to 90%, by weight 
of the amount required to cause complete saturation as indicated by the 
formation of a substantially continuous phase. For instance, when as is 
often preferred, the support medium can absorb around 2 to 3 parts 
solution and fatty substrate per part support material, it is often 
convenient to absorb into the particles 0.7 to 1.3 (often around 1) part 
fatty substrate and 0.7 to 1.5 (often around 1.3) aqueous nutrient 
solution or water per part by weight support. 
All the above ratios are optimum for a support bed of cellulose powder. 
However, they are also suitable for most supports, although some may 
required slightly different amounts for optimum results. 
Where the enzyme is produced in situ, an appropriate nutrient source must 
be included: for instance, to ensure that all of the standard essential 
trace elements of biotransformation are available. Some essential elements 
will already be available as inevitable trace elements in, for example, 
the support material. Examples of essential elements are iron, copper, 
manganese, molybdenum, and selenium. The nutrient source is preferably 
included in the water that is loaded onto the support, in order to promote 
growth of the enzyme-producing micro-organism. Thus, the nutrient source 
and water are preferably an aqueous solution that provides adequate 
inorganic nutrients for the biotransformation, but typically can be Czapek 
type medium (often at 1.5 to 2.5, preferably about 2, times the normal 
concentration). Thus, the aqueous solution typically contains nutrients 
such as sodium, magnesium, potassium, zinc, copper, and iron; and 
generally contains 1.5 to 6 (preferably 3 to 5) g/l sodium nitrate, 0.3 to 
2 (preferably about 0.8 to 1.2) g/l magnesium sulphate heptahydrate, 0.5 
to 2 (preferably about 0.8 to 1.2) g/l potassium chloride, and trace 
quantities of ferrous sulphate, zinc sulphate, and copper sulphate. 
It is naturally important that there should be no extraneous poisoning 
contamination present in the porous bed. It is a recognized problem in 
many microbiological processes that some contaminants may interfere with 
or stop desired microbiological processes at certain concentrations, and 
so the materials and conditions used in the invention should not be such 
as to produce contamination by toxic concentrations of potentially toxic 
materials. For instance, if a toxic impurity might be leached from 
apparatus used in the process by contact with an acidic medium, contact 
between fatty acid and that apparatus should be avoided. 
The aqueous medium may be buffered to a pH at which the process proceeds 
most efficiently. Although the process can be performed at pH values in 
the rage of, for instance, 4 to 8, best results are generally achieved 
when the pH is in the range of 6 to 7.5, and most preferably when it is 
buffered with phosphate to a pH of about 6.8. However, as discussed above, 
fatty acids such as C.sub.10 may be added to the support bed after the 
initial germination, and in these cases the pH will be lower. 
The desired product formed during the process may escape from the porous 
support bed as it is produced and thus can be collected from gas escaping 
from the bed during the process. However, this may be unnecessary since 
the amount of product evolved as vapor may be very low, and the bulk of 
the product may be trapped in or on the support and the bed that it forms; 
and the desired products can be recovered from the bed at the end of the 
process. The trapping can be either by absorption into any porous 
particles in the support, or by dissolution or adsorption with the organic 
materials (especially mycelium) therein, or both. It is particularly 
surprising and convenient that the process can be substantially run to 
completion before recovering the products from the support bed. 
Recovery of the product of the bioconversion can be achieved by 
distillation, preferably by mixing the contents of the bed in water, 
distilling the mixture, and recovery of the distillate. 
The collected product is a mixture containing natural components, and this 
mixture itself can be very useful. For example, the product from coconut 
oil is a mixture containing natural ketones having excellent blue cheese 
flavor properties. However, the individual components of the product 
mixture are particularly useful; and these can be obtained from the 
mixture by a normal procedure of fractional distillation, preferably under 
vacuum. 
The process may be carried out in a biotransformation vessel which contains 
the support bed and that includes means for aerating the bed so as to 
ensure sufficient oxygen is available for the required oxidative process 
at substantially all positions in the bed. 
The process generally may be carried out in a semicontinuous or continuous 
process (for example on a conveyor), but is preferably conducted 
batch-wise. 
It is generally desirable for the bed to be static and unstirred, since 
stirring the support bed may tend to help form a continuous liquid phase 
which will reduce porosity and may prevent air permeation through the bed, 
in addition to causing damage to mycelia. 
Generally, the bed is less than 2 meters (preferably less than 1 meter) 
deep since increasing the depth of the bed increases the pressure in the 
bed and the risk that the friable mixture will compress and tend towards a 
continuous phase. Generally the bed is greater than 0.1, and preferably 
greater than 0.5, meters deep. 
Air or oxygen should be passed through the support bed at a rate sufficient 
to stimulate rapid biotransformation, but insufficient to fluidize the 
bed. A satisfactory rate is 0.25 to 5 liters of air per minute, and more 
preferably 1 to 3 liters of air per minute, per 1000 kilograms of the 
loaded support bed. 
It is often preferred that the concentration of oxygen in the gas supply 
should be below the oxygen content of air since better results are 
obtained using a high volume of a dilute gas than with a low volume of a 
concentrated gas, because it is easier to control the actual amount of 
oxygen delivered to each part of the bed. Preferably the gas contains 1% 
to 10% oxygen, often 2% to 5%, with the balance being inert gas such as 
nitrogen. 
One way of achieving this dilute oxygen is by recycling the oxygenating gas 
within a loop consisting of a bio-reactor or including one or more 
reactors, and adding sufficient oxygen or air to the recycle to maintain 
the desired concentration. The recycle will have a high content of carbon 
dioxide; and we have surprisingly found that this does not interfere with 
the process, and that the carbon dioxide is an inert gas in this process. 
Accordingly, a preferred gas is 1% to 10% (often 1% to 5%) oxygen, above 
50% CO.sub.2, and 0% to 49% nitrogen. The amount of CO.sub.2 is often 70% 
to 90%. 
When recycling, it is necessary to prevent escape of too much water from 
its desired location and, in particular, it is necessary to ensure water 
does not condense in the recycle and cause localized waterlogging of a 
bed. 
The biotransformation vessel is preferably supplied with a gas inlet and 
with an outlet. Both connections may be fitted with filter units, 
scrubbers, or valves in order to prevent microbial contamination. 
One way to ensure substantially even distribution throughout the 
biotransformation vessel is to force gas up through the bed from a 
distribution manifold across the base of the bed. 
If the vessel is a substantially airtight cylindrical or other vessel 
having a removable lid, then it can be convenient to have a flexible gas 
supply duct leading down through the lid and support bed to below the 
manifold. A gas outlet is generally provided towards the top of the vessel 
so that waste or product gases can be removed. Simple apparatus of this 
type can be a substantially cylindrical vessel with a capacity of 
approximately 50 to 450 liters, and most preferably from 180 to 340 
liters. 
Instead of passing the oxygenating gas upwards, it is often preferred to 
pass it laterally through the bed from a series of spaced-apart gas supply 
passages that each supplies gas into the bed over substantially its entire 
length. The extent of permeation will depend on the material of which the 
bed is formed. It can be determined by simple experiment, but is generally 
not more than about 10 cm and is often 5 to 8 cm. Thus, the separation 
between two passages should be not more than about 20 cm, often not more 
than 10 to 16 cm. This technique has the advantage of providing more 
uniform oxygen concentration than by upflow from a manifold. 
Preferred apparatus for use in the invention is novel and can be used for a 
variety of fermentations conducted using a porous bed (such as citric acid 
production) or for the fermentations of the invention. It comprises a 
sealable bio-reactor that can contain the porous bed; a gas inlet for 
supplying oxygenating gas to the reactor; a gas outlet; passage means 
defining a plurality of passages that can open along their length into the 
bed and that communicate with the inlet and that are preferably spaced 5 
to 20 cm (preferably 5 to 15 cm) apart; and means for forcing oxygenating 
gas through the inlet and along and out from the passages and into the 
bed. 
The invention includes both the empty vessel and the vessel filled with the 
bed. The passage means can be horizontal and spaced apart both 
horizontally and vertically, but preferably they are substantially 
vertical. 
The spacing of the passage means relative to one another is dependent upon 
the porosity of the bed, and this is largely dependent upon the particle 
size of the support material forming the bed. The passage means are spaced 
so that gases can permeate to substantially all areas of the bed. 
One suitable passage means comprises a plurality of rod shaped passages 
that provide outer annular gas passages. For instance, each passage can be 
defined by a permeable tube, allowing gas to escape along its length, 
preferably at a substantially uniform rate along the length. However, the 
annular passages can also be formed lo by forcing tubes through the bed so 
as to create an annular layer around each tube that is of lower density 
than the rest of the bed. For instance, each tube can be inserted while 
covered by a sheath, and the sheath can then be removed. To prevent 
blockage of the tube during insertion, the tube can have a removable core 
which can be removed after insertion so that air can then flow down the 
center of the tube, out at the lower end, and up through the low density 
annulus around the rod. Additionally, low density annulus may be formed by 
shrinkage of the bed away from the tube during use. 
Another suitable passage means comprises a substantially self-supporting 
open network that preferably extends up through the bed and that 
communicates at its base with the inlet. Gas is forced from the inlet into 
the base of the network and flows up the network and out into the bed from 
most or all of the surface area of the network. Preferably the networks 
are substantially parallel to one another and not more than about 10 to 15 
or 20 cm apart. Generally, two networks define the sides of an annular 
bed. 
In order to maintain the desired humidity in the bed, there is preferably a 
trough in the base of the bed in which water can collect when in use, and 
a wicking layer of fabric extends up from this trough over part or all of 
some or all of the networks, so as to promote evaporation of water into 
the gas surrounding the networks and into the bed. The evaporation also 
helps maintain a uniform cool temperature throughout the bed. 
The process of the invention can also be conducted using flat trays 
carrying a shallow layer of support or a drum method as described in 
Advances in Applied Microbiology, volume 28 (1982), "Solid Substrate 
Fermentations," by K. E. Aidoo, R. Hendry and B. J. Wood, page 209. 
The apparatus in FIG. 1 comprises an air-tight vessel (1) having a sealable 
closure (2) and an inner body (3) defined by cylindrical walls (32) 
extending upwards through the vessel, but which does not extend fully to 
the top of the vessel. This defines an annular space (4) inside the 
vessel. A fan (5) is positioned at the top of the inner body (3) for 
circulating the gases in the vessel down through air ways (6) cut in the 
walls (32) of the inner body around its base and leading into the annular 
chamber (4). A bed of porous support material on which the fermentation 
takes place is positioned in the annular space (4). Gas inlet (12) is used 
to provide oxygen and any other required gases to the apparatus, and 
outlet (13) is used for removal of exhaust gases. 
A gas permeable open network (7) is provided around the inner and outer 
walls of the annular space. The network is provided by an open mesh of 
substantially rigid, self-supporting material which extends from the 
bottom of the vessel upwards towards the top of the vessel. 
As heat is produced during biofermentation, water evaporates off the bed 
and is liable to condense on the sides of the vessel and run down into the 
base of the vessel. Therefore, in order to prevent water-logging of the 
bottom portion of the support bed, an annular raised platform (9) is 
provided in the annular space and above air ways (6) so that a trough or 
chamber (10) is provided beneath the bed for collection of condensed water 
and circulation of gases. In addition, in order to provide redistribution 
of this condensed water to the support bed a second layer (11) is provided 
next to the mesh layer (7) inside the vessel around the annular walls and 
comprises a wicking material. The wicking layer (11) is positioned so that 
its lower edge reaches substantially to the bottom of the vessel into 
chamber (10) so that it is in contact with any collected condensed water. 
The wicking action of the layer then draws the condensed water back up the 
side of the vessel where it can be reabsorbed by the porous support bed 
either by evaporation or by conduction. 
It has been found that, using the bed of porous support material described 
in the examples, adequate oxygen distribution is achieved for up to 
approximately 8 cm from the edge of the bed. Therefore, in order to 
achieve effective oxygen distribution throughout the porous bed, the 
distance between the gas permeable layers (7) should be no greater than 16 
cm. This distance may vary with porosity of the support bed. 
The apparatus may be arranged in other ways: for example, a series of 
concentric annuli may be provided for containing the support bed, each 
annulus separated from the next by a gas permeable layer (optionally with 
a wicking layer). 
FIG. 2 illustrates a similar apparatus except that the wicking layer is 
omitted and there are cooling coils (14). Cooling water enters the cooling 
coils via inlet (15) and leaves the cooling coils via exit (16). 
Evaporated water from the support bed condenses on these cooling coils 
and, because of their position above the annular space (4) for containing 
the porous bed of support material, condensed water falls back into the 
support bed and is redistributed. FIG. 2 also shows fluid drive (17) for 
the hydraulic powering of the fan (5). 
The reactor may be any shape, but for convenience is preferably 
cylindrical. The inner body may be integral with the vessel or may be a 
separate body positioned in the bottom of the vessel. The apparatus can be 
made of any suitable material which will not interfere disadvantageously 
with the reaction inside the vessel, but preferably is prepared from a 
material which can be autoclaved for sterilization: for example, plastics 
such as polycarbonate plastic, polypropylene, or other suitable materials. 
The fan (5) may be powered by any suitable means, but is preferably 
powered by pneumatic or hydraulic means as this minimizes safety hazards. 
The gas permeable network may be formed from any material which will not 
inhibit the reaction in the reactor, but is preferably made from 
autoclavable material. Generally, the gas permeable network is formed of, 
for example, plastics materials in which the diameter of the network 
strands is preferably sufficient to make the mesh self-supporting, but 
small enough to leave substantial spacing within the layer. For example, 
the strands may be from 0.5 to 5 mm thick, preferably from 1 to 2 mm 
thick, and preferably at least 50% of both the transverse and longitudinal 
cross-section areas of the layer comprises open pores in the network. 
The wicking layer may be formed from any material which will provide a 
wicking action to redistribute the water: for example, an open wave cloth 
such as linen. 
FIG. 3 is a diagrammatic representation of an alternative reactor system. 
The reactor vessel (20) is a closed, substantially air-tight container 
having at its base a chamber (29) connected to the main body of the 
reactor by an air distribution plate (21). The reactor is also provided 
with cooling coils (22), an exhaust gas outlet at the top of the reactor, 
and a gas inlet (30) at the base of the reactor. The apparatus illustrated 
is a closed-loop circulation apparatus. Pump (23) draws gas out of gas 
outlet (24) and through condenser (25). The escaping gas can carry heat 
out of the bed. As a result, gas is drawn through gas inlet (30) and 
through air distribution plate (21). Instead of relying on vacuum to draw 
gas into the reactor, less preferably gas can be pumped into the vessel at 
a higher pressure. 
The supply of available oxygen can be controlled by its addition to the 
circuit. This addition may be made either by sucking in oxygen into the 
negative pressure side, or by pumping oxygen in on the positive pressure 
side of the pump. 
Gas can be recirculated through the apparatus so that the gases drawn into 
the apparatus are drawn from the pump via the condenser (26) and reservoir 
(27) which will collect any condensed water. Monitors may be introduced 
into the circuit to detect the exact oxygen concentration. 
The initial gas supply may enter the system via flow meter (28). 
FIG. 4 represents a partially cut away perspective view of the portion of 
the apparatus that contains a cylindrical bed of support material having 
an arrangement of rod passage means for distributing gases throughout the 
bed. Gases are supplied from an upper manifold (not shown) to the top of 
each of hollow tube (33) and down the length of the tube. Gases then exit 
hollow tubes (33) through exit holes (34) at the bottom of each tube and 
pass up the length of the tube in an annular zone around each tube and 
that is substantially free of particulate material. Additional holes (34) 
can be distributed along the length of each tube, if required, to provide 
a more uniform gas supply along the length of each tube. The gas migrates 
laterally from around the tubes and into the porous bed. The spacing apart 
of hollow tubes (33) is such that gases can permeate substantially all 
areas of the bed. This is dependent upon the porosity of the bed and 
particle size of the support material. Exhaust gas leaves the support bed 
and exits from the apparatus via a gas outlet. 
FIG. 5 represents an enlarged cross-sectional view of the lower portion of 
a tube with removable core and removable sheath in position, in 
preparation for insertion into a bed. Tube (33) is provided with exit 
holes (34) at its end and along its length. A removable close-fitting 
sheath (35) is provided around hollow tube (33) and a removable 
close-fitting core (36) is provided inside hollow tube (33). This 
arrangement is inserted into a bed of porous support material and, once in 
place, the core (36) and sheath (35) are removed, leaving hollow tube (33) 
free of support bed material and with an empty annulus around the tube. 
Gases can then travel freely down the inside of the tube end and leave via 
exit holes to enter the empty annulus around the tube for travel along the 
outside of the tube and permeation into the bed. 
EXAMPLE 1 
45 kilograms of coconut oil were dispersed onto 45 kilograms of cellulose 
powder and mixed in a Nauta mixer. 60 kilograms of Czapek type medium were 
mixed into the cellulose powder and coconut oil mixture until an even 
texture was obtained. The Czapek type medium comprised sodium nitrate 4 
g/l, magnesium sulphate 1 g/l, potassium chloride 1 g/l, ferrous sulphate 
0.02 g/l, trace elements: zinc sulphate 0.01 g/l and copper sulphate 0.005 
g/l, with 0.075 moles/1 potassium phosphate buffer at pH 6.8. 
The final mixture was discharged into a biotransformation vessel comprising 
an open-top drum having an air-tight lid and a flexible air inlet running 
through the mixture and to a manifold in the base of the drum and a gas 
outlet in the lid of the drum. 
Aspergillus niger had been grown on malt Czapek agar at 25.degree. C. The 
spores were harvested after approximately 10 to 14 days. Between 10.sup.7 
and 10.sup.9 spores per kilogram of loaded support bed were used for 
inoculation. Air was pumped to the vessel at a rate of 3 liters of air per 
minute. 
Germination of the spores occurred over the first 2 to 6 days, and the 
temperature rose from ambient temperatures to a maximum of 50.degree. C. 
Elevated temperature were maintained over the course of the fermentation, 
and as the fermentation came to an end after between 20 and 35 days, the 
temperature in the vessel began to fall gradually. 
The methyl ketone products were separated from the fermentation mixture by 
water distillation. The density of the oily product was 0.82 grams/ml and 
the product separated out from the water and could easily be removed. 
A portion of this product was fractionally distilled under vacuum using a 
column of twelve theoretical plates in order to obtain the individual 
methyl ketones. The total yield of methyl ketones was approximately 16%, 
based on the weight of coconut oil. 
EXAMPLES 2 to 4 
Example 1 was repeated, but with the addition of capric and/or caprylic 
acid into the reactor after germination of the mold. The quantities of 
Czapek type medium, coconut oil, and oxygen were varied. The total yields 
of mixed ketones produced are shown in Table 1. The poor yields obtained 
for examples 2 and 4 were thought to be due to the effects of toxic trace 
elements. 
TABLE 1 
______________________________________ 
Example: 2 3 4 
______________________________________ 
Amount of 22.5 Kg 3.00 Kg 3.00 Kg 
coconut oil 
Amount of 50.0 Kg 6.65 Kg 4.55 Kg 
Czapek medium 
Amount of 37.5 Kg 6.65 Kg 4.55 Kg 
Cellulose powder 
Amount of 15.0 Kg 2.70 Kg -- 
capric acid 
Amount of -- -- 0.90 Kg 
caprylic acid 
Total weight 125.0 Kg 19.00 Kg 13.00 Kg 
Rate of flow 3 1min.sup.-1 
1 1min.sup.-1 
1 1min.sup.-1 
of air 
Yield of 9% 25% 12% 
mixed ketones 
(based on weight of 
fatty substrates) 
Yield of nonan-2-one/ 
17% 46% 25% 
heptan-2-one 
(based on weight of 
capric/caprylic acid) 
______________________________________ 
EXAMPLE 5 
3 kilograms of coconut oil were dispersed into 3.5 kilograms of cellulose 
powder using a Winkworth mixer. 3.5 kilograms of Czapek type medium were 
mixed into the cellulose powder and coconut oil mixture until an even 
texture was obtained (Czapek type medium as before). The final mixture was 
discharged into a biotransformation vessel as shown in FIG. 1. 
Aspergillus niger produced as for Example 1 was inoculated into the 
biotransformation vessel. Air was pumped to the vessel at a rate of 1 
liter of air per minute. Germination of the spores occurred over the first 
4 days, at which time a separately prepared mixture was added. The 
separately prepared mixture comprised 1.5 kilograms of capric acid 
dispersed into 1.75 kilograms of cellulose powder, followed by 1.75 
kilograms of Czapek type medium. This separately prepared mixture was 
added directly to the fermenting mixture in the biotransformation vessel 
and mixed together by stirring. The biotransformation vessel was resealed 
and the air flow continued at 1 liter of air per minute. Elevated 
temperatures were maintained over the course of the biotransformation, and 
the mixture was sampled after a total of 18 days fermentation. 
The volatile products in the sample were isolated by water distillation, 
and the composition of the oily distillate was determined by gas 
chromatography. 
After a total of 29 days fermentation, the process was stopped and a 
further sample taken for distillation and analysis of the oily distillate. 
The coconut fat was found by analysis to contain 6.1% of capric acid, which 
gives 274.5 g of capric acid (as glycerides) in the coconut fat used for 
the experiment. In addition, 1500 g of capric acid was added to give a 
total of 1774.5 g of available capric acid in a total mass of 15 
kilograms. This amount of capric acid is theoretically capable of 
conversion into 1466 g of nonanone. After 18 days fermentation, the total 
content of nonanone in the 15 kilograms of fermenting matrix was found to 
be 907 g, which was a 62% conversion; and after 29 days fermentation the 
content of nonanone was 1209 g, which was an 82% conversion. This example 
illustrates the improved yield obtained by carrying out the process in the 
novel apparatus of FIG. 1 and is due to improved control of temperature 
and aeration throughout the bed.