Rice Hull Composition

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1517-83822012000200005

http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0104-66322010000200013

Raw material preparations

http://www.academia.edu/7300084/MODIFICATION_OF_RICE_HUSK_TO_IMPROVE_THE_INTERFACE
_IN_ISOTACTIC_POLYPROPYLENE_COMPOSITES

The rice husk was soaked in a 0.5N NaOH solution atroom temperature maintaining a ratio of (500mL
alkalisolution /50g rice husk) rice husk was kept immersed inthe alkali solution for 2 h. The fibers were
then washedseveral times with fresh water to remove any NaOH sticking to the fiber surface, neutralized
with dilute hy-drochloric acid and finally washed again with distilledwater. Final pH maintained was 7.
The fibers were thendried at 70oC for 24 h

rice husk was supplied by rice company San Jos, Jojutla, Mor., Morelos varie-ty A70 (20, 30, 40 and 60
mesh)
http://www.journalofanimalscience.org/content/38/1/140.full.pdf

RH used in this study were obtained

from the Arkansas Rice Grower Cooperative

Association, Stuttgart, Arkansas. Ap-

proximately 500-g samples were ground

through a 20-mesh screen, composited and

mixed for subsampling. All in vitro de-

terminations and chemical analyses were then

conducted on subsamples obtained from the

composited sample.

The objectives of this experiment were to

study the effect of drying and/or washing or

neutralization after a 12% NaOH treatment on

the IVDMD of GRH. The design utilized was a

completely randomized design with a 2 x 4

factorial of factor A, control-no drying (al) and

drying (a2) following NaOH treatment; and of

factor B, control-no washing or neutralization

(bl), washing (b2), hydrochloric acid (HC1)

neutralization (b3) and acetic acid (HAc) neu-

tralization (b4), following treatment A (al or

aa2). Sodium hydroxide treatment consisted of

adding 0.75 rnl of 4% NaOH solution to 0.25 g

GRH in a 50-ml centrifuge tube. The added

NaOH represented 12% of the air-dried weight

of GRH in each tube. The mixture (NaOH and

GRH) was incubated at room temperature for

24 hours. Drying (a2) was accomplished in an

oven at 50 C for 24 hours. This material, as well

as the undried material, was then subjected to

treatment B. Treatment b l required no treat-

ment prior to IVDMD determination. Treat-

ment b2 included mixing the NaOH-treated

material with 40 ml distilled water and cen-

trifuging at approximately 2,000 g for 15

minutes. The supernatant was removed by

aspiration and the entire procedure was re-

peated three times. Treatment b3 and b4

consisted of adding either 0.55 ml of 1.0 N

HC1 or 1.0 N HAc per tube. Each treatment

combination (ab) was replicated three times.

http://www.researchgate.net/publication/229415353_Fractionation_and_characterization_of_alk
ali-extracted_hemicelluloses_from_peashrub

Alkali-soluble hemicelluloses were extracted from delignified peashrub (Caragana

korshinskii) by sequential treatments with 0.5, 1.0 and 2.0 mol L1 KOH under a solid
to liquid ratio of 1:25 (g mL1) at 25 C for 10 h. lkali-soluble hemicelluloses were
extracted from delignified peashrub (Caragana korshinskii) by sequential treatments
with 0.5, 1.0 and 2.0 mol L1 KOH under a solid to liquid ratio of 1:25 (g mL1) at 25
C for 10 h.
http://proj3.sinica.edu.tw/~chem/servxx6/files/paper_13402_1278665738.pdf

100 g of rice husk was mixed with a liter of sodium

hydroxide solution (2, 4, 6 and 9n). Then, the mixture was

heated to boiling for 1 or 3 h. The rice husk were separated

by filtration and rinsed with distilled water until neutral pH

was achieved (five washes, on the average). After rinsing,

the RH was dried in an oven for 24 h at 100 C.

Hydrolysis of Cellulose to Glucose

http://www.eng.umd.edu/~nsw/ench485/lab4.htm

A. Equipment

Erlenmeyer flasks
Graduated cylinder
Pipets, 1ml, 10ml
Test tubes
Incubator or thermostated shaker
Temperature bath (or heat source -- Bunsen burner or hot plate)
Thermometer
Balance
Syringe
Filter holder
Filter paper
Spectrophotometer

B. Reagents

Cellulose source (filter paper, wood chips, carboxymethyl cellulose, cotton)

Cellulase, buffered at pH=5.000.01, 10g/l solution
HCl, 5% solution
H2SO4, 5% solution
KOH
Reagents for sugar analysis

1. Enzymatic Hydrolysis: Repeat the same procedures for shredded wood chips (a
complex and impure mixture of cellulose, lignin, and a variety of others),
carboxymethyl cellulose (a model amorphours-structured cellulose), and cotton
(90 % cellulose, mostly crystalline-structured). If time permits and if there is
extra enzyme solution, try other sources of biomass and waste materials such as
newsprint, grass, straw, and corn stalk. See Note 1.
o Shred a 10 cm2 piece of cellulose filter paper and weigh 0.1 g. (As
opposed to other type of papers with binding materials, a piece of
cellulose filter paper without wetting agents has minimum impurities and
 is almost pure in cellulose. The result of a quantitative analysis using a
filter paper would have been very unreliable had impurities leached out
into the filtrate.
o Submerge the shredded paper in 10 ml of the buffered cellulase solution
in a test tube. Note the starting time.
o Incubate the mixture at 40C. (The enzyme is most active at a
temperature of 40C and a pH of approx. 4.5.)
o This reaction should last for approximately 24 hours. Take 1 ml samples
at some predetermined appropriate intervals. Note that one does not have
much to waste because the starting sample is small. (A volume of 1 ml is
actually considered as a huge sample when working with biochemicals.)
o Stop the hydrolysis reaction in the sample. The first method of stopping
the reaction is to deprive the mixture of substrate. This can be easily
achieved by filtering out the residual solid material from the solution.
The individual samples may be stored frozen for later analysis. The
samples are thawed and brought to room temperature before they are
subjected to measurements. However, this first method is not applicable
to soluble cellulose, e.g., CMC. Alternatively, the enzymatically
catalyzed reactions can be halted either by adding a strong enzyme
inhibitor or by raising the temperature of the mixture to 90C for 5-10
minutes in a heated bath to inactivate the enzyme.
o Measure the glucose concentrations of the samples with the
dinitrosalicylate colorimetric method. (Reference: Gail Lorenz Miller,
Use of dinitrosalicylic acid reagent for determination of reducing
sugar, Analytical Chemistry, 31, 427, 1959.) See Note 2.
2. Acid Hydrolysis (Sulfuric Acid): Use the same cellulose sources as in
enzymatic hydrolysis.
o Add 0.2g cellulose to 10ml of 5% H2SO4 solution in a lightly capped test
tube. See Note 3. One may choose to carry out the reaction at 90C
instead of at room temperature.
o This reaction should last for 2 hours. Take 1 ml samples at some
predetermined appropriate intervals.
o Stop the hydrolysis reaction in the sample by neutralizing the acid and
slightly reversing the pH with the addition of a small volume of a
concentrated potassium hydroxide solution. Make a quick calculation to
see how much KOH is needed for this purpose. Note that one has to keep
a close track of the volume of KOH solution added because this
information will be needed to calculate the glucose concentration in the
original undiluted sample.
o Measure the glucose concentration of the alkaline sample.
 3. Acid Hydrolysis (Hydrochloric Acid): Substitute 5% sulfuric acid with 5%
hydrochloric acid and repeat the same procedures as in sulfuric acid.

http://www.google.com/patents/US4174976

The pretreatment of the cellular materials can be accomplished by selective solvent extraction as follows:
cellulosic materials are first extracted with a dilute acid or alkali to remove hemicellulose; the residue is
then dissolved in Cadoxen made up of 25 to 30% ethylenediamine and 70 to 75% water with about 4.5 tp
7% cadmium added as oxide or hydroxide (for example, about 10 grams of cellulose may be dissolved in
100 grams of Cadoxen); separating lignin, which does not dissolve, from the cellulose-Cadoxen mixture
by filtration or centrifugation; and precipitating cellulose from the cellulose-Cadoxen mixture (as by adding
water or an acid).

In addition, the cellulosic materials may be pretreated by contacting the materials with a chelating metal
caustic swelling solvent. Such an agent may include an aqueous solution of 17% sodium tartrate, 6.6%
ferric chlorideand 7.8% caustic which is stabilized by 6.2% sodium sulfite (all in weight percent). While
particularly, such an agent may be formed by:

______________________________________FeCL 3 6H2 O about 10 gTartaric Acid about . -NaOH

about 22 gNa2 SO3 about 13 g+ water to a total of 200
g.______________________________________

The pretreatment of the cellulosic materials in this case involves contacting the materials with solvent
in a 1 to 4 weight ratio of residue to solvent.

The acid hydrolysis of cellulose can be envisioned as a sequential reaction:

A k.sbsp.1 B k.sbsp.2 C

where A is cellulose, B is glucose, and C is undesirable side products. Reports on the acid hydrolysis
of woods, based on research conducted during World War II by J. F. Saeman of the U.S. Forrest
Products Laboratory, states that the rate of Ak.sbsp.1 B is about the same as B k.sbsp.2 C. In other
words the formation of side products from glucose occurred at about the same rate as formation of
glucose from cellulose. Consequently, the maximum glucose level in the hydrolysate was only 23% of
the sugar potentially available from the cellulose. Over the years some improvements in yields have
been obtained by reducing reaction times, increasing temperature and pressure, and modifying
processing equipment. Yet with all these improvements, the best yields obtained to date, using this
"conventional" technology, are less than 50%.

We have found that microcrystalline cellulose dissolved in the solvent Cadoxen can be converted,
almost instaneously, to glucose and other soluble products when concentrated sulfuric acid is added.
 Using an acid: cadoxen/cellulose ratio of 10:1, a 69% yield of glucose was obtained within 1 minute
after the two solutions were mixed together. Therefore, under the right conditions, cellulose dissolved
in a solvent such as Cadoxen is very susceptible to acid hydrolysis and gives good yields of glucose.

http://www.google.com/patents/CA1118380A1?cl=en

the cellulose-cadoxen solution is physically stable but this stability apparently requires;a close balance
among cellulose, water, ethylenediamine and cadmium. The exact nature of the balance is not precisely
known, but if, for example, excess water is added, cellulose will re-precip-itate from the solution. cadoxen
i~ of high pH, and upon addition of an acid, cellulose also re-precipitates from the solution. The re-
precipitated cellulose~ like other linear polymers, may spontaneously re-crystallize.
The re-precipitated cellulose can then be-readily hydrol-yzed to give a high yield of glucose by either an
acid or cellulose enzymes. For example, by adding an acid to a cellulose-Cadoxen solution to bring its pH
down to about 5 (whereupon cellulose re-precipitates), followed by adding a Tricoderma viride cellulase
solution, the re-precipitated cellulose is hydrolyzed to glucose.
The ethylenediamine and cadmium salt in the solution apparently do not inactivate the enzyme.

In order to improve the economics of the process~ Cadoxen 10 needs to be recovered and reused. By
different recycle techniquesj the process can be further divided into several alternatives which are
described as follows:
(a) Adding water to precipitate cellulo~e from a cellulose-Cadoxen solution. Separate cellulose by filtr-15
ation or centrifugation. The cellulose is then hydrol-yzed to glucose by acids or enzymes or both. The
liquid can be added with C0 to precipitate cadmium carbonate which in turn~can be converted into
cadmium oxide for reuse. The amine left in the aqueous solution can be 20 recovered by standard
methods such as di~tillation or extraction.

(b); Adding C0 directly into a cellulose-cadoxen solution wheraupon both cellulose and cadmium
carbonate will precipitate. After separation from the liquid 25 which c'ontains ethylenediamine that is to be
recovered as in ~a), the mixture of the two solid precipitates can be treated with cellulases to hydrolyze
and dissolve away cellulose to produce glucose. Cadmium carbonate precipitate left can be converted
into cadmium oxide for reuse.
(c) Adding a water immicible solvent to extract ethylenediamine for recycle, whereupon cellulose will
precipitate. ThUs, we can in one step separate the mixture into 3 fractions: amine in an organic layer~
cadmium in an aqueous layer and cellulose as a precipi-10 tate. The 3 fractions are to be processed
separately.
(d) The re-precipitated cellulose can be hydrolyzed to glucose by either an acid-enzyme two-step process
or an all-enzyme process. An acid or a cellulase enzyme ~most likely an endo-l, 4-beta-glucanase but the
cellul-` 15 ase mechanisms are still a subject of considerable cont-roversy) can "solubilize" cellulose (by
forming cellod-extrins). To complete the hydrolysis to glucose, either free or immobilized cellulases are
then utilized.
(e) cellulose re-precipitated from cadoxen has been 20 observed to spontaneously form beads if left to
stand in solution for several days. Such beads may have potential as a spport for enzyme
immobLlization.

In a further embodiment of the process of our invention, cellulose is dissolved and re-precipitated "in situ"
an~
25 therefore never physically removed from the lignin. The process is now thought to be preferred where
separation 11iL838~

of cellulose is not desired.

In this process, the cellulose is hydrolyzed to glucose and the lignin in solid form which remains is filtered
from the resulting glucose solution. This has been found to be easier than is separating the lignin from the
cellulose and then hydrolyzing the cellulose.
Enzyme can be used to hydrolyze the cellulose, and it is believed that acid hydrolysis can also be
utilized.

For this process, the following steps are utilized:

lQ 1. Treat cellulosic materials to remove hemicellu-lose (this treatment can be the same as described
herein-above with respect to the first discussed embodiment);

2. Dissolve the cellulose materials in solvent (hereagain, this step can be identical to that described 15
hereinabove with respect to the first discussed embodi-ment);

3. Add water, acid, or another liquid such as methanol to re-precipitate cellulosc;

4. Hydrolysis with enzyme or acid to yield glucoseg 20 leaving behind only water insoluble lignin; and 5.
Separation of lignin from glucose syrup by filtration.

Exampls of our process are as ~ollows:

33~

EX~P~E 1 Pretreatment and HydrolysLs of - Cellulose (a) Preparation of 1:he Solvent (Cadoxen):
Ethylene diamine, 25 to 30~/c in water, was saturated by adding an excess of cadmium oxide which was
previously oven dried. Cadmium oxide which did not dissolve formed a hydroxide which appeared as a
white precipitate. The precipitate was filtered or centri-fuged off. The procedure of adding cadmium oxide
and separating out the hydroxide was performed twice to lO ensure that a solution saturated with
cadmium oxide resulted. In this way an aqueous solution of 25 to 30~/O
ethylenediamine and 4.5 to P/~ cadmium resulted. Due to its cadmium and ethylene diamine content, this
solvent is commonly known as Cadoxen.
(b) Dissolution of ~-Cellulose ~
B (1) Crystalline ~-cellulose (Avicell~ O l g~ was added to lO ml of the solvent, prepared as in (a), and
mixed. Dissolution of the cellulose was complete within lO minute~s at room conditions.
20~ ~t2) Dissolution tests were repeated for O.l g~
~0.2~g, 0.3 g, 0.4 g, and 0.5 g portions of crystalline cellulose (Avicell), each in lO ml solvent~ by a
procedure where the cellulose~was mixed with solvent at room conditions and then cooled by placing on
ice.
25 Times required for complete dissolution to occur are summarized in Table l.

~, _ ` ~ ' 38~

TAsLE 1 % Avicell Dissolution in Solvent Time 1 2 minutes 2 2 minutes 3 7 minutes 4 9 minutes 15

minutes (3) Dissolution tests, performed as described in (2) above, were repeated. In this case, however~
amorphous cellulose,~instead of crystalline cellulose, was dissolved. Using the same quantities of
cellulose, 10 and the same procedures as in (3j, the dissolution times summarized in Table 2 were
observed.

~ TABLE 2 % cellulose Dissolution in Solvent Time 1 2 minutes 2 ~ 2 minutes 3 7 minutes 4 9 minutes 15

minutes (4) Solutions containing greater amounts of cellulose were made by modifying the compo~ition of
the solvent and by altering the dissolution procedure.

20 Sodium hydroxide was added to the solvent in (a) to give a solution which is 0.5 M in sodium
hydroxide. To 1 3~

milliliter of the modifiecl solvent, 300 milligrams ~vicell were added, followed by an excess of CdO.
Mixing of this solution was carried out resulting in an almost clear, very viscous solution having a
cellulose concentration of approximately 30D/
(c) Preparation of Enzyme.
(1) Cellulase enzyme from the Enzyme Development Corporation, New York, was processed as follows to
give enzyme preparation "CS". Enzyme~ 10 g, was added to 10 25 ml water. Using an ultrafiltration
membrane the enzyme solution was concentrated and diluted with excess water and then concentrated to
a volume of 25 milliliters again. This was repeated several times until the salt and carbohydrate level o
the enzyme preparation was 15 negligibleO
(2) Enzyme preparation "CW" was made as follows.
Enzyme, 10 gram, was dissolved in 100 milliliters water.
Mext 57 gram ammonium sulfate was added. Upon mixing, the ammonium sulfate dissolved and a white
precipitate 20 formed. This precipitate was separated by centrifugation and re-dissolved in 30 milliliters of
w~ er. The solution B was then desalted using Sephadex G-25 ~Pharmacia Corpor-ation) and made up to
a final volume of about 100 ml.
(d) Hydrolysis of Crystalline ~-Cellulose.
~5 (1) To 1 volume of ~/c crystalline ~-cellulose dissolved in the solvent described in (a~, 2 volumes of
3~/0 HCl was added causing the dissolved cell~lose to 38~:) re-precipitate. This was followed by addition
of 5 volumes of sodium acetate buffer and 1 volume ~nzyme preparation "CS'. Upon mixing and
incubation at 50C~
up to 500/O conversion of cellulose to glucose was obtained in 30 minutes. Apparently, the presence of
cadmium and ethylene diamine did not inactivate the enzyme.
(2) One volume of 3% Avicell in solvent (aj was mixed with 2.5 volumes water causing the cellulose to 10
re~precipitate. The re-precipitated cellulose was washed with water after which the cellulose concentrat-
ion was 0.5%. After adjusting the solution to pH 5 wit~
buffer, sufficient en~yme preparation "CW" was added td give an enzyme volume- cellulose weight ratio
of 18 : 1.
15 Incubation of this solution at 50C gave 80/c conversion to glucose in 5 hours and 90k in 50 hours. In
comparison, a control experiment using cellulose which had not been dlssolved in solvent and re-
precipitated, i.e., untreated cellulose, gave conversions of only 15% after 5 hours 20 and 47% after 50
hours.
EX~MPLE 2 Pretreatment~and Hydrolysis of Agricultural Residues Preparation of the solvent and
enzyme solutions was done exactly as described previously in Example 1.
(a) Dissolution of cellulose in corn cob. Corn 25 cobs~ ground to 40 mesh size particles, were combined
with solvent in a weight ration of 1:55 corncob: cadoxen.

o After stlrring for 2.5 hours, the solid and liquid phases were separated~ The solid phase was washed
with water, dried and weighed. Weight loss, based on initial and final weights of dry solids, was as high as
77%.
This indicated all cellulosic material had been dissolved~
(b) Dissolution of cellulose in corn residue.
Corn residue, ground to 40 mesh, was combined with solvent in a weight ratio of 1:10 corn residue:
Cadoxen.
10 The stirring, washing and recovering of solids was performed as described in Example 2(a). weight
loss was estimated to be between 44% and 94% (dry basis).
(c) In Situ dissolution and hydrolysis of corn residue. Corn residue containing 3~/0~ -cellulose was 15
mixed with Cadoxen in a 1:4.2 weight ratio - corn residue:
Cadoxen. After sitting 12 hours, buffer, water~ and enzyme preparation "CW" wexe added to give a 2.5%
solution of residue at pH 5. Hydrolysis of the mixture at 45C gave 72% conversion of the ~-cellulose to
glucose 20 in 19 hours. Since the solvent pretreatment and subse-guent cellulose re-precipitation was
done without first separating the solvent and dis601ved cellulose from the solid residue, this technique
was referred to as "in situ"
dissolution ~and re-precipitation).
~d) In Situ dissolution and hydrolysis of bagasse (sugar cane residue). Bagasse residue containing 33%
~-ce}lulose was mixed with cadoxen in a 1:4.2 weight ' .~' ~ ' ratio - bagasse: Cadoxen. using the same
conditions as in Example 2(c), 7~/0 conversion was obtained in 19 hours.

Hydrolysis with Immobilized Enzyme (a) Preparation of immobilized enzyme. A filter disk of chemically
activated porous PVC membrane material (supplied by Amerace Corporation~ Butter ~ew Jersey) was
submersed in a solution of l~/o gluteralde-hyde buffered with phosphate to pH 7 for 2 hours at room
conditions. After washing with water and sodium acetate 10 buffer (pH 5), the disk was submersed in
enzyme prepara-tion "CW" for 12 hours at 4C. The disk was then washed with buf~er, placed in a column
apparatus and used as described below.
(b) Hydrolysis of cellodextrins using immobilized 15 enzyme was completely solubilized with 7~/c being
converted to glucose and the balance to cellodextrins. This clear solution was passed through the
immobilized enzyme disk 5 times resulting in an increase in glucose conversion from 7~/0 to 81%.
Glucose was formed due to hydrolysis ~0 of solu~le cellooligosaccharides to glucose~ A control study
using only soluble enzyme was conducted, consecu-tively. In this run~ glucose conversion stayed
constant at 7~/0 during the same time period.

Formation of Cellulose Beads 25 A solution of 2% Avicell in Cadoxen was combined with 3~

50O/c Hcl in a 1:1.5 volume ratio of solution: acid. This caused the cellulose to precipitate. The mixture
was heated to 50C for 30 minutes and then cooled to room temperature. Upon standing for twelve hours
the flocculent cellulose re-precipitate had spontaneously formed small cellulose beads.

As can be appreciated from the foregoing, this invention provides a process for treating cellulosic
materials and yielding glucose therefrom, with one disclosed process 10 dissolving and re-precipitating
cellulose "in situ" while another disclosed proces~ recovers cellulose from cellulosic materials which can
then be utilized to provide a high yield of glucose.

Fermentation of Kojic acid

http://www.hindawi.com/journals/btri/2014/642385/

A 1.5-liter B. Braun stirred tank (Biostat. A) fermentor (from B. Braun Biotech.

International, Sortorius group, GmbH, Schwarzenberger, Germany) with one liter
working volume was used in this study. The fermentor was equipped with pH,
temperature, agitation, dissolved oxygen tension (DOT), and foam controllers. Seed
cultures were carried out in 250mL flask containing 50mL of medium, held on a rotary
shaker at 150rpm, at 28C for 48h. Seed culture flask (50mL) from fungal isolates
(Aspergillus flavus number 7), which proved to be the higher kojic acid producer, was
used to inoculate the fermentor at 30C. Fermentation lasted around 14 days. The culture
medium was modified synthetic medium consisting of (g/L): glucose, 100; yeast extract,
5.0; KH2PO4, 1.5; and MgSO47H2O, 0.5. The pH was adjusted to 3.0, temperature at
30C, and agitation at 400rpm, while the DOT in the culture broth was controlled via a
sequential cascade control as air flow rate. The maximum and minimum set points of
permitted airflow rates were 1.2L/min and 0.1L/min, respectively. The DOT during
fermentation was controlled at medium (~50%) of saturation.

http://link.springer.com/article/10.1023%2FA%3A1018593815266

An unstructured model based on logistic and Luedeking-Piret equations was proposed to describe
growth, substrate consumption and kojic acid production by Aspergillus flavus Link strain 44-1 in batch
fermentation and also in a resuspended cell system. The model showed that kojic acid production was
non-growth associated. The maximum kojic acid and cell concentrations obtained in batch fermentations
using the fermenter with optimized dissolved oxygen control (32.5 g/l and 11.8 g/l, respectively) and using
a shake-flask (36.5 and 12.3 g/l, respectively) were not significantly different. However, the maximum
specific growth rate and a non-growth-associated rate constant for kojic acid formation (n) for batch
fermentation using the fermenter (0.085/h and 0.0125 g kojic acid/g cell.h, respectively) were
approximately three and two times higher than the values obtained for fermentation using a shake-flask,
respectively. Efficient conversion of glucose to kojic acid was achieved in a resuspended pellet or
mycelial system, in a solution containing only glucose with citrate buffer at pH 3.5 and at a temperature of
30 C. The resuspended cell material in the glucose solution was still active in synthesizing kojic acid
after prolonged incubation (up to about 600 h). The rate constant of kojic acid production (n) in a
resuspended cell system using 100 g glucose/l was almost constant at an average value of 0.011 g kojic
acid/g cell.h up to a cell concentration of 19.2 g/l, above which it decreased. A drastic reduction of n was
observed at a cell concentration of 26.1 g/l. However, the yield based on glucose consumed (0.45 g/g)
was similar for all cell concentrations investigated.

http://www.academicjournals.org/article/article1380106808_Mohamad%20et%20al.pdf

Purification of Kojic Acid

http://books.google.com.ph/books?id=4ViVUQi7Z60C&pg=PA454&lpg=PA454&dq=kojic+acid+purificati
on&source=bl&ots=7I7w7td8GU&sig=aYEWW328PBfezUlJiFYqpg_88MM&hl=en&sa=X&ei=gvO8U7SoNc
WJogSXzICwBw&ved=0CCcQ6AEwAQ#v=onepage&

Crystallise the acid from MeOH(charcoal) by adding Et2O. It sublimes at 150-200 degree/0.1 torr

http://www.google.com/patents/US3165535

. nique. are removed from the harvested broth, usually by filtra- United' States Patent 3,165,535
RECOVERY F KQEKC ACID James V. Kehoe, Glendale, and Frank Inzerilio, Elmont, N.Y., assignors to
Chas. Pfizer & (30., Inc., New York, N511, a corporation of Delaware No Drawing. Filed Feb. 8, W63, Ser.
No. 257,105 7 Claims. 7 (Cl. ass-345.9

This invention is concerned with kojic acid. More particularly it is concerned with a new and useful
process for the recovery of kojic acid from fermentation media.

Kojic acid (2 hydroxymethyl 5 hydroxy gammapyrone) possesses the following structural formula:

iii-CH OH and may be produced readily by microbiological means. Several molds of the genus Aspergillus
have the ability -to produce kojic acid from suitable carbon-containing nutrient solutions. These include,
for example, A. oryzae, A. flavus Win, A. gymnosara'ae, A. awamori, A. candidus, A. clavzttus, A.
fumigatus, A. gigantus, A. albus, A. efiusus, A. nidulans and others. From among the bacteria, several
species of Acetobacter may also form kojic acid under favorable conditions.

Suitable carbon-containing substances fermented to produce kojic acid include starches; dextrins,
disaccharides, such as sucrose and maltose; hexoses, such as glucose, fructose, mannose and
galacto'se; pentoses such as xylose and arabinose; and sorbitol, dulcitol, inositol, glycerol, glycero-beta-
phosphate dihydroxyacetone, gluconic acid, tartaric acid, and other substances. Particularly good yields
have been obtained from glucose and xylose. V

In the production of kojic acid by'the action of a suitable organism on an essentially carbohydrate
substrate, a temperature range of about 29 to 35 C. is optimum for the fermentation. The fermentation
generally requires from about 9 to 20 days for completion, the period depending on such factors as type
of substrate, species of mold, temperature and pH. The optimum pH for the production of kojic acid under
a given set of conditions can readily be determined experimentally by one skilled in the art. A pH range of
about 2 to 5 has been found to be satisfactory. c

A common practice of obtaining kojic acid from the fermentation medium is the so-called boil down tech-
After the fermentation is complete, the mycelia tion. The broth is thereafter concentrated and subjected 'to
decolorization-and sequestration to improve appearance and purity. The kojic acid is recovered from the
concentrated broth by crystallization.

.This processof recovering kojic acid has several disadvantages. If the carbon-containing substrate is a
pure sugar, such as, for example, refined sugar, recovery yields as high as 80% are possible. The use of
a purified sugar substrate, however, is not justifiable from an economic point of view. On the other hand,
molasses substratcs of varied purity are customarily used in the fermentation, since theseare a cheaper
source of carbohydrate substrate. The kojic acid-containing fermentation broth often contains such
impurities as sugars,

organicysalts, inorganic salts, byprod'uct acids, etc. As

a consequence, the ability to recover kojic acid by the boil down technique is seriously impaired. The kojic
acid thus recovered will not meet reasonable requirements of Patented i... 12, 1965 purity. The recovery
yield is, of course, much lower. Some sources of relatively high purity molasses may allow recovery yields
up to about 70%. To obtain reasonably pure kojic acid from the fermentation of molasses sustrates,
recourse must generally be made to yield-consuming and time-consuming recrystallizations.

It is an object of this invention to provide a new and novel method of recovering kojic acid from
fermentation media.

It is 'a further object to provide a process 'for the recovery of kojic acid in relatively high yields.

The objects of this invention are accomplishedby the substantially stoichiometric or slight excess addition
of metal salts to crude fermentation broths, followed by the metathesis of the resulting salts with acids
such as sulfuric or oxalic.

Kojic acid readily forms stable, water-insoluble metal chelates. The most probable structure for the kojic
acid chelate with the metal appears to be:'

wherein M designates the metal.

Such water-insoluble salts of kojic acid containing'barium, calcium, copper, zinc, tin, manganese and
others have been prepared.

It has now been found that kojic acid may be recovered in substantially high yields from fermentation
broths by contacting the fermentation broth with a suitable metallic salt, such as Zinc sulfate, for example,
precipitating the insoluble salt of kojic acid, separating the insoluble salt of kojic acid by filtration;
thereafter contacting the metallic kojate with substantially stoichiometric or slight excess amounts of an
acid such as oxalic acid, for example, and removing the metallic oxalate by filtration. The kojic acid is
obtained from the filtrate by cooling, concentration or other means known in the art. If desired, soluble
salts of kojic acid, such as sodium kojate, may be prepared at this point without isolating the free 'acid.
Yields of kojic acid obtained by this process will vary somewhat depending upon the type subtrate used
and the choice of metallic salt. With a suitable zinc salt, such as zinc sulfate, for example, and crude
molasses as the carbohydrate substrate, yields of about to are obtained.

In the process described hereinbefore, it is noteworthy that after the fermentation reaction for the
production of kojicacid has been completed, the fermentation broth may or may not be filtered prior to the
addition of the metallic salt. It is preferred, however, that the broth be filtered. Also, it is preferred that the
broth be adjusted to a pH of about 10 to 11 prior to the addition of the metallic salt. However, this
adjustment is not critical. After 'the metallic salt has been contacted with-the fermentation broth, the
optimal pH of the broth should be from about 5.0 to 9.0 and preferably from about 6.8 to 7.2. The metallic
kojate which precipitates from solution may be separated from the mother liquor by means well ,known in
the art, such as by filtration 0r centrifugation. I Metathesis may be accomplished by the addition to the
metathesis is accomplished by acidification with an acid which forms a soluble salt with the metal ion of
the metallic kojate, the kojic acid is crystallized out of the solution and the soluble metal salt is rejected in
the mother liquor.

Salts which have been found to be satisfactory for precipitating kojic acid from the fermentation broth
include, for example,the chloride or sulfate in particular, of such metals as magnesium, calcium, strontium
and barium of Group IIA of the periodic table; divalent salts of metals such as zinc, copper, manganese,
iron and lead; and trivalent salts of metals such as aluminum and bismuth. Particularly effective are the
divalent salts selected from the group consisting of zinc, copper, manganese, iron and lead. The metallic
salts need not be limited to the chloride or sulfate, but other salts are also effective, such as, for example,
the nitrate, acetate, phosphate and the like.

In the examples set forth hereinafter, the fermentation broths were obtained by cultivating Aspergillus
flavzrs (NRRL 484), in a fermentation medium containing crude molasses as the carbon-containing
substrate; The kojic acid content of the various fermentation broths was determined by measuring the
adsorption of the ferric complex of kojic acid. This may be accomplished as follows: One gram of sample
is placed in a 1 liter volumetric flask and diluted to volume with water. A 25 ml. aliquot is taken and diluted
to 100 ml. with water. A 10 ml. aliquot of this is taken and to it are added 40 ml. of water and 5 ml. of
freshly prepared 0.5% ferric chloride solution (aqueous). After waiting 5 minutes for color to develop, the
solution is placed in a Beckman Du Spectrophotometer and read at 498 m against a blank.

per cent kojic acid= optical density X 2000 Slope of Standard Curve Wt. of Sample Example I Eight
thousand gallons of fermentation broth, containing 0.5 lb. of kojic acid per gallon are adjusted to a pH of
10.0 with caustic while maintaining the temperature below 50 C. A zinc sulfate solution (2520 lbs. zinc
sulfate monohydrate in 500 gallons of Water) is added with stirring. The pH is adjusted to 6.8, and the
mixture stirred for an additional 2 hours. The zinc kojate which precipitates out is filtered and placed in a
tank with 200 gallons of water. A solution of 2120 lbs. of oxalic acid dihydrate in 1800 gallons of water is
added to the tank containing the zinc kojate, and the Whole is heated at 65 C. for 1 hour. The mixture is
filtered and the precipitate, which is zinc oxalate, is Washed with water at 65 C. to a total volume of 2350
gallons in the filtrate,

The filtrate, containing approximately 3500 lbs. of kojic acid in solution, is adjusted to a pH of 9.5 by the
addition of 1 N sodium hydroxide solution while maintaining the temperature at 3840 C. The slurry which
forms is then cooled to room temperature and allowed to crystallize further for 2 hours. The mixture is
centrifuged, and the precipitate is washed with cold water. The mother liquor obtained from the
centrifugation is adjusted to a pH of 9.5 with caustic and concentrated in a vacuum pan at 42 C. to about
1100 gallons. After concentration, the mother liquor is cooled to room temperature, crystallized and
centrifuged to remove the precipitate. The precipi- 4 tates are combined to yield the sodium salt of kojic
acid in 75% yield.
Example II Following the procedure of Example I, the filtrate, containing approximately 3500 lbs. of kojic
acid, is concentrated to near dryness. The precipitate which crystallizes out is filtered and dried to yield
kojic acid in about 78% yield.

Example Ill The procedure of Example I is followed except that instead of using a zinc sulfate solution, a
ferrous sulfate solution (3920 lbs. ferrous sulfate heptahydrate in 1600 gallons of water) is used; Kojic
acid is recovered as the sodium salt in about 80% yield.

Example IV Example V Four thousand gallons of fermentation broth, containing 0.4 lb. of kojic acid per
gallon, are adjusted to a pH of 10.5 with caustic while maintaining the temperature below about 50 C. A
zinc sulfate solution (1125 lbs.

zinc sulfate monohydrate in 300 gallons of water) is added with stirring. The pH is adjusted to 7.2 and the
mixture stirred for an additional 2 hours. The zinc kojate which precipitates out is filtered and placed in a
tank with 100 gallons of Water. A solution of 840 lbs. of oxalic acid dihydrate in 715 gallons of water is
added to the tank containing the zinc kojate, and the whole is heated at 65 C. for 1 hour while maintaining
the pH at about 2. The mixture is filtered, and the precipitate, zinc oxalate, is Washed with water at 65 C.
to a total volume of about 800 gallons in the filtrate.

The filtrate, containing approximately 1500 lbs. of kojic acid in solution, is concentrated to near dryness.
The precipitate which crystallizes out is filtered and dried to yield kojic acid in about yield.

Example VI The procedure of Example I is repeated substituting for zinc sulfate monohydrate,
stoichiometrically-equivalent amounts of the following salts:

CuSOl.5HzO CaCl A101 PbSO4 In each case, kojic acid is obtained as the sodium salt.

Example VII The procedure of Example I is repeated using a stoi- 'chiometrically-equivalent amount of
phosphoric acid instead of oxalic acid. The zinc is removed as the insoluble zinc phosphate. Kojic acid is
recovered as the sodium salt.

Example VIII Example IX Following the procedure of Example I, the zinc kojate which precipitates out is
filtered and placed in a tank with enough water to make a slurry. The salt is decomposed by adding nitric
acid until a pH of 1 is reached.

Kojic acid precipitates out and is filtered. Zinc is removed in the mother liquors as the soluble nitrate

PERRYS HAND BOOK 8th ED.

Density NaOH 1.04g/ml

Solubility of MgSO4 .7H2O 40.8g/100g water

PARTICLE SIZE OF KOJIC ACID

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