FIELD OF INVENTION:
The present invention relates to the method for biological conversion of aromatic
acids with propenoic chain to their corresponding aromatic aldehydes and its
system thereof. More particularly, the present invention provides system and
method for biological conversion of aromatic acids with propenoic chain to their
corresponding aromatic aldehydes with high productivity and high yield.
DESCRIPTION OF THE RELATED ART:
Vanillin (3-methoxy-4-hydroxybenzaldehyde), also known as vanillin, citronellal,
relative molecular mass 152.15, white to yellowish needle or powder crystal, is
the main component of vanilla, mainly Used in food, beverage, fragrance,
pharmaceutical, leather, electroplating and pharmaceutical industries.
The production method of vanillin is mainly chemical synthesis, and its main
source is chemical synthesis of phenolic lignin and lignin from petrochemical
products. The vanillin synthesized by this method is not only scented, but also has
environmental pollution during synthesis. Seriously, people are worried about
safety. Another method of producing vanillin is plant extraction. The natural
vanillin extracted from vanilla bean pods is only 40t per year, which is far from
meeting people's needs. 16000t); therefore, the study of microbial vanillin,came
into being.
Reference may be made to the following:
Publication No. CN103060392 relates to a method for converting ferulic acid to
produce vanillin by immobilized amycolatopsis, and belongs to the technical field
of biology engineering. Cellulosic materials such as sawdust, bagasse and the like
are used as carriers, and an amycolatopsis cell is fixed by an adsorption way. An
immobilized cell can be recycled for 10 times, and the mole conversion rate for
converting ferulic acid into vanillin is kept at more than 50%. A resin is coupled
and adsorbed in a conversion system. The output of vanillin reaches 19.65g/L,
and the production strength of vanillin is 0.13 1 g1L.h.
Publication No. CN108 138 125 relates to a strain Amycolatopsis derived from the
strain Amycolatopsis Zyl 926 having at least one additional copy of the genes
-3 -
FCS and ECH encoding feruloyl-CoA-synthetase and enoyl-CoA
hydratase/aldolase integrated at the integration site of the phage Phi C31. The
invention also relates to the method for producing such strains, the integration
cassette of the genes FCS and ECH, the use of said strains in a method for
producing vanillin, and a method for producing vanillin
Publication No. US2018030407 relates to an amycolatopsis sp. strain (zhp06),
and a method of using the whole cell preparation of the strain for vanillin
production. The strain was deposited in China Center for Type Culture Collection
on Jul. 26, 2011 with the number of CCTCC NO: M 2011265. Under high
concentrations of ferulic acid substrate, the vanillin production by this method can
reach more than 10 g/L. The molar conversion rate of ferulic acid is more than
50% and the purity of vanillin is from 80% to 95%. The advantage of this
invention includes: repeated use of biocatalyst cells, mild biotransformation
condition, low environmental pollution, short production cycle, high product
purity and simple purification procedure
Publication No. CN107075465 relates to a culture of Amycolatopsis sp. ATCC
391 16 - mutant F33 or mutant F86 which converts ferulic acid into vanillin
Publication No. US20 163 12250 relates to bioconversion method of making
vanillin including expressing VaoA gene in a mixture, expressing MtSADl gene
in the mixture, feeding eugenol to the mixture, and converting ferulic acid to
vanillin by incubating with a microbial Amycolalopsis sp, strain (Zhp06) andor a
recombinant E. coli strain.
Publication No. EP2772142 relates to vanillin obtained by (a) treating a residue
obtained from the processing of corn with alkali or ester-cleaving enzymes and
hydrolyzing the gamma -0ryzano1 contained in it to ferulic acid and sterols, (b)
isolating ferulic acid from the mixture, (c) fermenting the isolated ferulic acid
with microorganisms of the type Amycolatopsis sp. DSM 9991 andor DSM 9992
in a culture medium, and (d) isolating vanillin, after completion of the reaction.
Independent claims are included for: (1) producing vanillin, comprising the steps
(a)-(d) as above per se; (2) a composition comprising vanillin and vanillyl vanilla;
(3) food containing vanillin obtainable by the above process, or the composition;
-4-
and (4) aromatization of foodstuffs, comprising adding vanillin or the
composition.
Publication No. US2015252397 relates to a novel method for biological reduction
of the carboxylic acids to their corresponding aldehydes andlor alcohols with high
productivity and high yield by using fungus in the category of basidiomycetes.
This reduction is specific and selective for its functional group (-COOH),
without affecting other functional group such as-R groups (-OH, -NH2, -
alkyl, -alkyoxy) and their position, number on aromatic ring. The method of the
invention relates to reduction of aryl acids to aldehyde and/or alcohols by
en~ploying a white rot fungus-Pycnoporus cinnabarinus, an organism of
basidiomycete species, grown in vessel/column.
Publication No. W02014106 189 relates to a method of making vanillin including
expressing 4- hydroxyphenylacetate 3-hyrdroxylase, caffeic acid 3-0-
methyltransferase, methionine synthase, feruloyl-CoA synthetase, and enoyl-CoA
hydrataselaldolase in a mixture, feeding p-coumaric acid to the mixture, and
collecting vanillin. It further includes an enzyme, such as caffeic acid 3-0-
methyltransferase, that facilitates the increased conversion of caffeic acid to
fenilic acid, wherein the enzyrne has been modified at a residue that allows for
increased methylation of ferulic acid, and a method of using the enzyme in the
making of fenilic acid followed by vanillin
Publication No. W02014045298 relates to a method for microbial fermentation
and biotransformation of aromatic acids to aromatic acids with reduced carbon
atoms of wide commercial importance using a culture of actinomycete species.
Amycolatopsis sp or the mutant thereof is employed in the present invention to
convert natural as well as synthetic aromatic acids to reduced carbon aroillatic
acids with wide applications. The said culture in the disclosed invention is
adapted to grow at 37-46OC to achieve the biotransformation of aromatic acid to
reduced carbon aromatic acid is accomplished at 37-46OC to obtain a higher yield
of thc product.
-5 -
Publication No. US201 1065156 relates to unique, innovative, and efficient system
in order to convert ferulic acid into vanillin which involves the immobilization of
actinomycetes in an effective solid support structure which is comprised of one or
more pieces of a material, that is configured so as to allow effective aeration. For
the immobilization of actinomycetes a surface culture is employed in which these
microorganisms develop a mycelial growth pattern and adhere to the solid support
structure. The use of surface culture also allows us to benefit from enhanced
physiological responses such as increased growth rates and production yields of
the actinomycetes when they are cultivated in this surface culture. Since the
microorganisn~s are held captive in the supports, the step of biocatalysis can be
repeated up to 15 times before the biocatalytic capacity is lost. An evaluation of
the whole transformation process results in a higher vanillin yield.
Publication No. US2004203123 relates to a method for the transformation of
Amycolatopsis sp. DSM9991 and DSM9992 and the use of the strains
transformed in this way for the preparation of vanillin, preferably for the
preparation of vanillin from ferulic acid.
Patent No. US6133003 relates to microorganisms of the family
Pseudonocardiaceae, vanillin is produced in high yields from ferulic acid. The
microorganisms are two strains of Amycolatopsis.
The culture stated in the prior art are immobilized. There is selective capture
column attached for selective product formation and the yield is low. Also the
Vanillin obtained by conventional method gives byproduct like vanillic acid
vanilly alcohol, guaiacol and resin was added which restrict reuse of culture. The
adsoption and fermentation pH is same hence yield and conversions are low.
In order to overcome above listed prior art, the present invention provides a
system and method for biological conversion of aromatic acids with propenoic
chain to their corresponding aromatic aldehydes with high productivity and high
yield.
OBJECTS OF THE INVENTION:
-6-
The principal object of the present invention is to provide a system and method
for biological conversion of aromatic acids with propenoic chain to their
corresponding aromatic aldehydes
Another object of the present invention is to provide a method for
biological conversion of aromatic acids with propenoic chain to their
corresponding aromatic aldehydes with high productivity and yield
Yet another object of the present invention is to provide a method for biological
conversion of aromatic acids with propenoic chain to their corresponding
aromatic aldehydes which reduces the production of byproduct and the culture
can be reused
Still another objective of the present invention is to use a hydrophobic adsorbent
in capture column to selectively capture product in presence of substrate without
losing organism's activity and thereby avoiding end product inhibition effect
andlor formation of by product
Yet another object of present invention is to change pH of capture column by
adding alkali continuously to favor selective binding of product and reducing
affinity of substrate.
Still another object of present invention is to provide a method for recycling the
cells in the continuous manner and multiple cycles of the biotransfomation
resulting in cost effective process.
Yet another object of present invention is to provide a method for aseptic and
competitive growth of sensitive culture of Actinomycetes species and using the
grown culture for biotransfonnation of aromatic acids containing propenoic acid
side chain to their corresponding two carbon reduced aldehydes
Still another objective of the invention is to provide a method for gradual feeding
of sterile media into the fennenter containing seed culture in its exponential
phase, whereby the feed flow rate is adjusted according to the lower than the
-7-
doubling time of the respective culture/ flow rate is increased exponentially or
with constant feeding according to doubling time of culture or growth curve
phases of culture
Yet another object of the present invention is to provide a mild reaction condition,
easy downstream processing and eco friendly and greener product
Still another objective of the present invention column was eluted with organic
solvent to get concentrated product solution. The product in organic solvent was
distilled and re-crystallized to get purelcrude product.
At the outset of the description that follows, it is to be understood that the
ensuing description only illustrates a particular form of this invention.
However, such a particular form is only an exemplary embodiment and is
not intended to be taken restrictively to imply any limitation on the scope of
the present invention.
SUMMARY:
The present invention discloses a continuous method for biotransformation of
natural and/or synthetic aromatic acids with propenoic chain to respective
aldehydes using the growing or resting cells of aseptically grown cells of
Actinomycetes species with high yield and high purity. This conversion is specific
and selective for its functional group propenoic carboxylic (-CH=CH-COOH),
without affecting other functional groups such as-R groups (-OH, -NH2, -alkyl, -
alkoxy,-Br, -Cl,-H ) and their position, number on aromatic ring.
In other embodiment of the present invention, the method employs a system to
produce only aldehyde with high purity and high yield. The design includes use of
in-situ filter arrangement in the fermenter for continuous circulation of cell free
broth containing product and substrate, through the capture column, wherein, the
filterlmembrane having porosity 0.01 micron to 20 micron, thus making
dowilstreiirri processing of the product easier.
-8-
The other embodiment of the present invention the growth of cell culture by the
microorganism may be carried out on a carbon and nitrogen source, wherein
carbon source is selected from the group such as without limitation glucose,
molasses, starch, sugar hydrolyzate, corn steep liquor, glycerol, propanediol most
preferably glycerol.
Another embodiment of the present invention the nitrogen source used may be
organic and/or inorganic source, wherein organic source is selected from the
group consisting of group such as without limitation yeast extract, malt extract,
peptone, beef extract, soy protein extract, maize gluten protein extract andlor
inorganic source is selected from the group consisting of ammonium sulphate,
urea, and sodium nitrate.
In most preferred embodiment of the present invention there is provided a process
for biotransformation of aromatic acid to aromatic aldehyde, wherein recovery
and recycling of cell culture may be carried out for subsequent biotransformation
malting the process continuous with multiple bio-transformations using the same
cell culture.
BREIF DESCRIPTION OF THE INVENTION:
It is to be noted, however, that the appended drawings illustrate only typical
embodiments of this invention and are therefore not to be considered for
limiting of its scope, for the invention may admit to other equally effective
embodiments.
Figure 1 shows system drawing and compound structure
Figure 2 shows system/ process steps for conversion of aromatic acids with
propenoic chain to their corresponding aromatic aldehydes
DETAILED DESCRIPTION OF THE INVENTION:
The present invention discloses method for biological conversion of aromatic
acids with propenoic chain to their corresponding aromatic aldehydes with high
productivity and high yield using bacteria of the category of actinomycetes. This
reduction is specific and selective for its functional group (-CH=CH-COOH),
-9-
without affecting other functional groups such as-R groups (-OH, -NH2, -alkyl, -
alkyoxy) and their position, number on aromatic ring (figure 1).
The present invention also discloses a process for selective conversion of natural
andfor synthetic aromatic acids to aldehydes by employing sensitive microbes of
Actinomycetes species namely Amycolatopsis thermojlava NRRL 24140 or
mutant thereof, grown competitively at a larger scale. There is provided a method
for growing (use different wording - slow growing culture of Actinomycetes
species, namely Arnycolatopsis thermojlava with a competitive advantage by
reverse media addition strategy which gives the advantage of contamination free
growth, pH regulation and foam suppression.
The cells were grown in fermenter by reverse addition of media to seed culture
(figure 2). The vessellcolumn has the provision for pH monitoring and pH
control, impellors for efficient mixing of air and nutrients and energy source with
stirrer. The sterile aeration is provided using a sparger at the bottom. The vessel or
column is provided with dissolved oxygen monitoring system and dissolved
oxygen control unit. The dissolved oxygen concentration is maintained by
aeration andlor sparging andlor agitation to provide minimum formation of
byproducts.
An in-situ filter system is provided in vessel1 column/ fermenter for broth
filtration before going to capture column. The filter separates the
cells/spores/mycelia/pellets from broth allowing only clear liquid to pass to the
column for product adsorption. Membrane filterlperforated plateslperforated
filterlpoly ethylene filter bags of pore size of about 0.Olmicron to 20 micron is
used as a filter system.
The capture column is used for extraction of the product is packed with
polystyrene divinyl based hydrophobic resin and connected to the reaction vessel.
The type and volume of adsorbent is based on selectivity of the resin towards the
product and its binding capacity. The adsorbent use includes a hydrophobic
polystyrene divinyl benzene based or any polystyrene methacrylate based
polymeric adsorbent for product capture
-10-
The present invention includes a system wherein the aromatic acid is selectively
converted to aromatic aldehyde of high yield and high purity by resting or
growing cells of Actinomycetes species, namely Amycolatopsis thermoj7ava
NRRL 24140, wherein said process comprises: preparing the 24-72 hour grown
seed culture of Amycolatopsis thermoflava NRRL 24140; preparing the fermentor
for aseptic growth with a volume of water, 10-25 % (minimum agitable volume)
along with a insitu filter; adding the seed suspension of Amycolatopsis
thermoflava NRRL 24140 in the fermenter in a volume of 5-25 % of the total
final volume; sterilizing the growth media of the remaining volume separately;
adding the said growth media in a drop wise fashion into the fennenter over the
time interval of 12-20 hours; and then continuing the incubation for further 18-30
hours, to obtain the grown cullre in pelleted form for biotransformation;
Filtering the cell broth through the in situ filter while retaining the cell pellets
inside; re-suspending the cell pellets in sterile distilled water; adding aromatic
acid substrate to the fermenter of the above step to obtain the final concentration
of 1-20 g/L and maintaining the fermenter pH in the range of 7.5-9.5; wherein the
aromatic acid is consumed within 24-40 hours; connecting the capture column
containing hydrophobic adsorbent to the fermenter and circulating the cell free
broth to continuously and selectively extract the aromatic aldehyde and recirculating
the aromatic acid substrate back into the fermenter for hrther
biotransformation with residance time of aromatic aldehyde in fermentor 16-24
hrs; eluting the capture column with an organic solvent to obtain the crude
aromatic aldehyde in high concentration; and crystallizing the crude product to
. obtain pure aromatic aldehyde.
In another aspect, there is provided a process for biotransformation of natural
and/or synthetic aromatic acids to its aromatic aldehyde, wherein said natural
andlor synthetic aromatic acid is selected from the group consisting of cinnamic
acid, 3-methoxy-4-hydroxy cinnamic acid, 4-hydroxy cinnamic acid, 3-hydroxy
cinnamic acid, caffeic acid, sinapinic acid, hydrolyzate of lignin, hydrolyzate of
phenolate acid, most preferably 3-methoxy-4-hydroxy cinnamic acid or caffeic
acid or p-coumaric acid.
-1 1-
In another aspect, there is provided a process for biotransformation of natural
andlor synthetic aromatic acids to its aromatic aldehyde, wherein said aromatic
aldehyde is selected from the group consisting of cinnamaldehyde, 4-hydroxy-3-
methoxy cinnamaldehyde, 4-Hydroxy cinnamaldehyde, 3 -hydroxy
cinnamaldehyde, 3,4-dihydroxy cinnamaldehyde, 3,5-dimethoxy-4-hydroxy
cinnamaldehyde, most preferably 4-hydroxy-3-methoxy benzoic acid or
protocatechuic acid or p-hydroxy benzoic acid.
The growth of cell culture by the microorganism is carried out on a carbon and
nitrogen source, wherein carbon source is selected from the group consisting of
glucose, molasses, starch, sugar hydrolyzate, corn steep liquor, glycerol,
propanediol most preferably glycerol. The carbon source used is in the
concentration range from 1-30 g/L, more preferably in the range of 2-15 g/L.
The nitrogen source used may be organic and/or inorganic source, wherein
organic source is selected from the group consisting of yeast extract, malt extract,
peptone, beef extract, soy protein extract, maize gluten protein extract andor
inorganic source is selected from the group consisting of ammonium sulphate,
urea, and sodium nitrate. The nitrogen source used may be in the range of
concentration of 1-30 g/L, most preferably in the range of 2-20 g/L.
The growth media is added drop wise to seed suspension in fermenter, flow rate
of media addition being increased exponentially according to the lower than
doubling time of the culture. The growth media addition rate is adjusted in the
range of 0.5 mllmin to 20 ml/min. The incubation time after complete media
addition is continued till 16-40 hours. The substrate concentration used for
biotransformation is in the range of 1-20 g/L, more preferably 5-12 g/L.
The alkaline pH of the substrate solution is adjusted in the range of 6-9, more
preferably 8-9.5. The pH of the growth media is formulated in the range of 6-9,
most preferably 7-8.5.The pH of the biotransformation media was adjusted to 7-
8.5 and pH of capture column was adjusted to 8-9.5 where only aldehyde bind to
column and acid recycle in fermentor
Aromatic carboxylic acid substrate is added in its salt form to achieve required
final concentration. The biotransformation of aromatic acid to their respective
aromatic aldehydes is carried out in fed batch andlor continuous manner.
Biotransformation in a fed batch is carried out by addition of subsequent doses of
substrate with concentration in the range of 2-5 g/L after complete consumption
of the substrate that is added in first batch with same parameters of
biotransformation.
In the fed batch operation of the invention, the biotransfonnation of aromatic acid
to aromatic aldehyde, is perfomled by directly feeding the substrate solution i.e.
the aromatic acid to the biotransfonnation medium and the aldehyde product is
continuously captured on column in continuous operation, while the substrate
concentration is kept constant in biotransformation medium by stepwise or
dropwise addition of the aryl substrate. The filtration is carried out using
membrane filterlperforated filter1 perforated plates, having pore size at least 0.22
micron before adsorption on capture column. The filtration is done to obtain
retentate comprising culture cells/mycelia/pellets, and permeate comprising liquid
containing aromatic aldehyde which is passed through the column for adsorption.
The flow rate of the circulation of the fermentation broth through the column is in
the range of 6 hr to 24 hrs residance time product formed is completely adsorbed
onto the colun~n and fiu-ther biotransformation of the product is paused. The
capture column is packed with hydrophobic adsorbent.
The hydrophobic adsorbent used in capture colun~nis hydrophobic macroporous
polystyrene divinyl benzene based or any polystyrene methacrylate based
polymeric adsorptive resin. The first elution of capture column was done with
NaHC03 to remove the traces of the bound un-reacted aromatic acid substrate and
then with organic polarlnon polar solvent depending on the solubility or
miscibility of the product. The elution of capture column is carried out using
organic polar solvent selected from the group consisting of methanol, ethanol,
isopropanol, butanol, water, acetone, ethyl acetate, most preferably ethanol.
The crude product is obtained by concentrating the elution solvent by vacuum
distillation at temperature in the range of 55" C to 65' C and allows the
concentrated product to cool down gradually for 6-12 hrs and/or at temperature in
the range of 10-30" C. The crystallization of crude product is done in distilled
water or potable water. The yield and purity of aromatic aldehyde and is in
between 90-97% and 96-99.5% respectively.
There is provided a process for biotransformation of aromatic acid to aromatic
aldehyde, wherein recovery and recycling of cell culture is carried out for
subsequent biotransformation making the process continuous with multiple
biotransformations using the same cell culh~re.
Another embodiment of the present invention, wherein cell cultures are
~~sedlrecyclefodr 10- 15 cycles of biotransformation without losing the organism's
activity for biotransformation.
Advantages of the technology
1. The present invention provides selective and continuous production of
aromatic aldehyde from aromatic acid in high yield and high productivity.
2. Selective aromatic aldehyde production by continuous product capture
technology confers the benefits of easy downstream processing in fewer steps
and hence economically feasible.
3. The aromatic aldehyde product, being toxic to cells, is continuously removed
from the cells' contact, thus avoiding cell toxicity as well as end product
inhibition or avoid other metabolic impurity formation which result high
yield.
4. The present invention provides recycling of the once grown cell pellets for
biotransformation, thus malcing it a continuous process resulting in a cost
effective process with higher productivity as it saves the media cost as well as
the time and energy spent in the growth of organism.
5. The present invention provides a method for growing a very sensitive
organism of Actinomycetes species, namely Anzycolatopsis tlzerrnoflava in
contamination free state by reverse media addition strategy thereby also
providing an advantage of pH regulation as well as foam suppression.
The invention is described in detail with reference to the examples given
below. The examples are provided just to illustrate the invention and
therefore, should not be construed to limit the scope of the invention.
EXAMPLES
Example 1
Slant culture of Amycolatopsis thermoflava NRRL 24140: A culture medium
containing 0.5% glycerol, 0.2% yeast extract and 0.1% dipotassium hydrogen
phosphate and 2.2% agar is formulated, pH 7.0. All of the contents of constituents
are percentage of weight to volume i.e g1100 ml (similarly hereinafter). The
culture medium is autoclaved at 121" C. for 20 minutes and made into slants afler
cooling. The strain Ainycolatopsis thermoflava is inoculated on the slants and
incubated at 39" C. for 5 days.
Example 2
Preparation of the preliminary culture: 1000 ml medium containing 0.5%
glycerol, 0.2% yeast extract and 0.1% dipotassium hydrogen phosphate is
formulated, pH 7.0. and 250 ml of the above media was dispensed in four 1000
ml conical flasks and autoclaved at 121" C. for 20 minutes. The strain
Amycolatopsis thermoflava is inoculated into the culture medium using nichrome
loop. The shake flask fermentation is performed at 39" C, 200 rpm for 48 hours.
The resultant culture obtained in the form of pellets is used as seed cultures for
biotransformation.
Example 3
System preparation, Column attachment, pH adjustment
2000 ml of the growth medium containing 0.5% glycerol, 0.2% yeast extract and
0.1% dipotassium hydrogen phosphate is formulated, pH 7.0. and autoclaved in a
separate reservoir that can be later attached to the fermenter. The fermenter or any
other suitable vessel is autoclaved with 1000 ml distilled water. After cooling it
was inoculated with 500 ml of the seed culture medium as per The example 2.
The growth media was then added drop wise into the fermenter with the flow rate
of 2.2 mllmin. Culture conditions were 39" C, 150 rpm and 0.75 L of air per
minute. The complete media addition takes around 16 hours. Growth is continued
for further 24 hours. After which pelleted growth is obtained with clear
surroundinglsupernatant media. This strategy of reverse media addition gives
contamination free growth of Amycolatopsis species with the added advantage of
foam control and pH regulation.
Example 4
Reduction of Aromatic Acid to Aromatic aldehyde selectively
The pelleted cells obtained in Example 3 are used for selective reduction of
aromatic Acid to aromatic aldehyde. The broth containing pellets is filtered
through insitu filter/ membrane of 10 micron porosity while retaining the cells
inside the fernlenter. The alkaline solution of aromatic acid substrate, ferulic acid
in the concentration of 5 g/L is added and reaction continued at the same
conditions. The ferulic acid solution is prepared by dissolving it in an alkaline
solution and pH adjusted to 8.5 using 6M HCI before addition in the fermentor.
After 5 hrs of substrate addition, circulation of broth through capture column was
started by adjusting pH of the solution 8.5 and the flow rate of column was
maintained to 2.2 mllmin. and the process was continued till the total substrate
ferulic acid gets consunled. After complete consumption of substrate column is
detached from reaction vessel and subjected to washing with 100 ml of 0.1M
NaHC03 to elute traces of bound unreacted substrate. Then product vanillin is
eluted selectively with 300 ml methanol. The eluted product is subjected to
vacuum distillation at 60' C. and the residue is allowed to cool down gradually.
The crude crystals of vanillin with 90% of purity are obtained which are separated
by filtration from the mother liquor. The crude crystals are re-crystallized in 50 ml
of distilled water. The formed crystals are filtered through vacuum filter and
washed with chilled DW to obtain white, shiny crystals of vanillin with 99.5%
purity and yield of 94%.
Example 5
Reduction of ferulic acid to vanillir~i n fed batch manner
A. thermoflava cells were grown in fermentor as per Example 3. The product
formed is continuously extracted using the capture column as in example 4. Again
a second dose of substrate ferulic acid at a concentration of 5 g/L is added to the
cells in the fe~mentora nd reaction continued at the same conditions. The product
is adsorbed on the adsorbent capture column at pH 8.7and again fresh substrate is
added in the fermentor. Reaction is continued till complete depletion of the
-16-
substrate. After complete substrate depletion, column detached from vessel and
subjected to elution with 100 ml of 0.1M NaHC03 to elute traces of bound
unreacted substrate. Then product vanillin is eluted selectively with 300 ml
methanol. The eluted product is subjected to vacuum distillation at 60' C. and the
residue is allowed to cool. The crude crystals of vanillin with 89% of purity are
obtained which are separated by filtration from the mother liquor. The crude
crystals are recrystallized in 20 ml of distilled water. The formed crystals are
filtered through vacuum filter and washed with chilled DW to obtain white, shiny
crystals of vanillin with 99% purity and yield of 90%
Example 6
Continuous process for reduction of fenllic acid to vanillin
A. thermoflava cells were grown in fermentor as per Example 3. Ferulic acid at a
concentration of 2.5 g/L, was continuously added (flow rate 2 mllmin) using
pump through membrane filter to the fermentor and reaction continued as per
example 4. Four adsorbent columns were attached in parallel. The product is
adsorbed on the capture column. After saturationlcompletion of capacity of first
column, adsorption process is switched over to second column. The first column
detached from vessel and subjected to elution with 100 ml of 0.1M NaHC03 to
elute traces of bound unreacted substrate. Then product vanillin is eluted
selectively with 300 ml natural ethanol. The eluted product is subjected to
vacuum distillation at 60' C. and the residue is allowed to cool. The crude crystals
of 4-hydroxy-3-methoxybenzaldehyde with 89% of purity are obtained which are
separated by filtration from the mother liquor. The crude crystals are
recrystallized in 50 ml of distilled water. The formed crystals are filtered through
vacuum filter and washed with chilled DW to obtain white, shiny crystals of 4-
Hydroxy-3-methoxy benzaldehyde with 99% purity. Overa1143.8 gms of vanillin
was collected after 12 days.
Example 7
Conversion of Mixture of Aromatic Acids to respective Aromatic aldehydes and
purification
The pelleted cells obtained in Example 3 is used for selective reduction of
aromatic Acid to aromatic aldehyde. The broth containing pellets is filtered
through in-situ filter1 me~nbrane of 0.2 micron porosity while retaining the cells
inside the fermenter. The alkaline solution of Phenolic hydrolysate containing
mixtures of aromatic acid substrate, ferulic acid and p-coumaric acid in the
concentration of 5 g/L and lg/L respectively is added and reaction continued at
the same conditions. The substrate solution is prepared by dissolving it in an
alkaline solution and pH adjusted to 8.5 using 6M HCl before addition in the
fermentor.
After 5hrs of substrate addition, circulation of broth through capture column was
started with the flow rate of 2.2 ml/min and the process was continued till the
total substrate mixture gets completely consumed. After complete consumption of
substrate column is detached from reaction vessel and subjected to washing with
100 ml of 0.1M NaHC03 to elute traces of bound unreacted substrate. Then
products vanillin and 4-hydroxy benzaldehyde is eluted selectively with 300 ml
methanol. The eluted product is subjected to vacuum distillation at 60' C. and the
residue is allowed to cool down gradually. The two aldehydes are separated at the
crystallization step. The crude crystals of vanillin with 90% of purity are obtained
which are separated by filtration from the mother liquor. The crude crystals are recrystallized
in 45 ml of distilled water. The formed crystals are filtered through
vacuum filter and washed with chilled DW to obtain white, shiny crystals of
vanillin with 94.5% purity and yield of 90%.
Numerous modifications and adaptations of the system of the present
invention will be apparent to those skilled in the art, and thus it is intended
by the appended claims to cover all such modifications and adaptations
which fall within the true spirit and scope of this invention.

We Claim:
1. A method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes with high purity and yield comprising:
a) preparing the 24-72 hour grown seed culture of Amycolatopsis
thermoflava NRRL 24140;
b) preparing the fennentor for aseptic growth with a volume of water, 10-25
% (minimum agitable volume) along with a in situ filter;
c) adding the seed suspension of Amycolatopsis thermoflava (NRRL 24140)
in the fermenter in a volume of 5-25 % of the total final volume;
d) sterilizing the growth media of the remaining volume separately;
e) adding the said growth media in a drop wise fashion into the fermenter
over the time interval of 12-20 hours; and then continuing the incubation
for further 18-30 hours, to obtain the grown culture in pelleted form for
biotransformation;
f) Filtering the cell broth through the insitu filter while retaining the cell
pellets inside;
g) re-suspending the cell pellets in sterile distilled water;
h) adding aromatic acid substrate to the fermenter of the above step to obtain
the final concentration of 1-20 g/L and maintaining the fermenter pH in
the range of 7.5-9.5 wherein the aromatic acid is consumed within 24-40
hours;
i) connecting the capture column containing hydrophobic adsorbent to the
ferrnenter and circulating the cell free broth to continuously and
selectively extract the aromatic aldehyde and re-circulating the aromatic
acid substrate back into the fernlenter for further biotransformation with
residance time of aromatic aldehyde in fermentor16- 24 hrs;
j) eluting the capture column with an organic solvent to obtain the crude
aromatic aldehyde in high concentration;
k) crystallizing the crude product to obtain pure aromatic aldehyde
2. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein the
cells were grown in fermented vessel/column by reverse addition of media to
seed culture which has the provision for pH monitoring and pH control,
-19-
impellers for efficient mixing of air and nutrients and energy source with
stirrer, the sterile aeration is provided using a sparger at the bottom, dissolved
oxygen monitoring system and dissolved oxygen control unit and the
dissolved oxygen concentration is maintained by aeration and/or sparging
andlor agitation such that there is minimum formation of byproducts
3. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein the
fermenter comprises in-situ filter for broth filtration before going to caph~re
column to separate the cells/spores/mycelia/pellets from broth allowing only
clear liquid to pass to the column for product adsorption compris'ing the
membrane filterlperforated plateslperforated filter of pore size of about
0.Olmicron to 5 micron is used as a filter system.
4. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein the
capture colunln is used for extraction of the product which is packed with
polystyrene divinyl based hydrophobic resin and connected to the reaction
vessel and the type and volume of adsorbent is based on selectivity of the
resin towards the product and its binding capacity.
5. The method biological conversion-of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein the
adsorbent used may include a hydrophobic polystyrene divinyl benzene based
or any polystyrene methacrylate based polymeric adsorbent for product
capture
6. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein said
nah~raal ndlor synthetic aromatic acid is selected from the group consisting of
cinnamic acid, 3-methoxy-4-hydroxy cinnamic acid, 4-hydroxy cinnamic acid,
3-hydroxy cinnamic acid, caffeic acid, sinapinic acid, hydrolyzate of lignin,
hydrolyzate of phenolate acid, most preferably 3-methoxy-4-hydroxy
cinnamic acid or caffeic acid or p-coumaric acid.
7. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein carbon
source is selected from the group consisting of glucose, molasses, starch,
sugar hydrolyzate, corn steep liquor, glycerol, propanediol most preferably
glycerol.
8. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein
wherein organic source is selected from the group consisting of yeast extract,
malt extract, peptone, beef extract, soy protein extract, maize gluten protein
extract and/or inorganic source is selected from the group consisting of
ammonium sulphate, urea, and sodium nitrate.
9. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 7, wherein the pH
of the biotransformation media was adjusted to 7-8.5 and pH of capture
column was adjusted to 8-9.5 where, only aldehyde bind to column and acid
recycle in fermentor.
10. The method biological conversion of aromatic acids with propenoic chain to
their corresponding aromatic aldehydes as claimed in claim 1, wherein the
yield and purity of aromatic aldehyde and may be in between 90-97% and 96-
99.5% respectively.