CHAPTER II

PROCESS SELECTION
2.1. Process Synthesis
The synthesis of sodium gluconate from biomass requires 2 (two) steps of
process. The first one is the conversion of starch (amylum) into glucose, and the
second one is the conversion of glucose into gluconic acid, which is then
neutralized become sodium gluconate salt. The selected raw material from the
previous section is seaweed. As stated in chapter 1, seaweed contains high
percentage of carbohydrate (amylum) and glucose. It contains 60% of amylum
and 30% of glucose. Besides that, the availability of seaweed is high because it is
cultivated in purpose. In order generate the most effective route to produce
sodium gluconate from seaweed, the basic scheme of this synthesis is:

Seaweed

Conversion of
glucose into gluconic
acid

Conversion of starch
into glucose

Sodium
Gluconate

Figure 2.1Process synthesis

(Source: Personal data)

There are plenty of process alternative from each of the steps in the processing of
seaweed into sodium gluconate. The process selection is then will be analyzed by
using heuristic approach and the scoring method.
2.2.

Proces Selection and Description

2.2.1

Washing
Washing process is the first process for treating biomass. As mentioned in

sub-chapter 1.3.1, the biomass used is seaweed. The ordered seaweed from
supplier will be transported by using truck, and there will be some dirt particles
that is left on seaweed, especially when the seaweeds supply takes directly from
the sea and kept on a warehouse. The purpose of washing process is to remove or
separate unwanted particles from seaweed such as gravel and sand. The input for
this process is raw seaweed and washing water. After washing process, the output
for this process is cleaned seaweed (minus sands and dirt particles) and dirty

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water. The waste water can be recycled to be used in this process again to
minimize the cost. The temperature used in washing process is room temperature
(25-300C) and the pH is neutral (6,5-7). Washing process is conducted using
conveyor washing machine. The washing process is done in a belt conveyor.
2.2.2

Grinding
After that, clean seaweed enters the grinding machine to be grinded.

Grinding is used for particle size reduction from 2 cm to around 1,3 mm. It will
increased surface area, therefore it increases the reaction rate. It will also makes
the further processes easier. The temperature used is room temperature (25-30 0C)
and the pH is neutral (around 6,5-7). The grinding unit used is grinder. The input
for grinding process is raw seaweed with larger particle size, while the ouptut is
seaweed with smaller size.
2.2.3

Homogenization
After grinding, the starch/ amylum from seaweed need to be extracted by

using homogenization process. But before homogenization, the seaweed must be

blend with water to make it become seaweed slurry (the previous condition of
seaweed is dry). The seaweed is mixed with water with water concentration of
70% w/w. The temperature used is room temperature (around 27-30OC) and the
pH is neutral (around 6,5-7).
After blending with water, starch or amylum can be obtained from
seaweed by using high pressure homogenizer. The homogenizer valve will be
open and the starch enter to pressing area. The operating condition used in this
process is about 140 bar at 45oC for 2 hour. Homogenization process produce two
phase of product (solid and liquid). Native starch is solid phase. The solid phase
we call starch slurry, and starch slurry as feed for the next unit process.
2.2.4

Hydrolysis
Hydrolysis is the conversion process to convert starch (amylum) into

glucose. Basically, hydrolysis is the reaction of water addition into starch so that
starch can be broken down into smaller molecules such as dextrose or glucose.
The starch hydrolysis reaction is mentioned below.

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( C 12 H 22 O11 )n +n H 2 O n C6 H 12 O6
Water will attack starch at 1-4 glucosidic linkage become dextrin, syrup and
glucose based on the decomposition degree of polysaccharide chain in starch.
Hydrolysis reaction basically will run very slow, therefore the reaction needs to be
catalyzed in order to increase the reaction rate. There are 2 (two) common
alternatives for the catalysts, such as by using acid or enzyme. Both catalyst have
advantage and disadvantage, therefore both process will be compared and scored
based on several parameters.
Alternative 1: Acid hydrolysis
In acid hydrolysis, the acid acts as a catalyst to increase the reaction rate of
starch decomposition into glucose. Acid condition may caused the glicosidic bond
between amylum is broken down into glucose molecule. The most affecting
factors in acid hydrolysis is the concentration of H + ion, therefore it is preferrable
to choose strong acid than weak acid, because strong acid can be ionized into H +
and its negative ion easily compared to weak acid.
However, the usage of acid as hydrolysis agent must consider the Heuristic
aspects. According to Heuristic 1, we should select raw materials and chemical
reactions to avoid, or reduce the handling and storage of hazardous and toxic
chemicals. Acid such as HCl and H 2SO4 is hazardous if in contact with humans
skin, human eye and in case of ingestion. To reduce the toxicity risk, the operator
needs protective instrument to protect themself. It also must concern the handling
and storage issue. Acid must be stored in a cool and well-ventilated area. The
operator that is handling with acid substance must be well-equipped with
protective equipment such as gloves and glasses due to hazardousness to skin and
eye.
Another consideration is Heuristic 2. According to Heuristic 2, use an
excess of one chemical reactant in a reaction operation to consume completely a
valuable, toxic, or hazardous chemical reactant. To fulfill heuristic 2, the acid
must be neutralized by neutralizing agent to completely consume hazardous
chemical reactant, such as acid catalyst. Therefore, if we use acid as hydrolysis
catalyzing agent, we must use neutralization process to fulfill heuristic 2.

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Alternative 2: Enzymatic hydrolysis

There are two types of linkage in starch structures: -1,4 and -1,6,
linkages. Amylose is one kind of starch that is unbranched, single chain polymer
containing 500 to 2000 glucose subunits with only -1,4glycosidic links (Kanlaya
et al., 2004). Amylopectin is another kind of starch that has a branched structure
that is cause by the presence of -1,6glycosidic linkages (Amutha et al, 2001).
The breaking down of the -1,4 and -1,6 linkages to small units of glucose
(monosaccharide) is made possible by the actions of - amylase and glucoamylase
(enzymes) respectively (Wong et al., 2001).-amylase splits -1,4 bonds in
amylose and amylopectin. It is an endo-acting enzyme and its action is often
considered to be random.However, -amylases rapidly decrease the viscosity of
starch solutions. Glucoamylase is an exo-acting enzyme, hydrolyzing -1,4 and 1,6, glycosidic linkages in amylose and amylopectin (Kosaric, 2001).
Enzymatic hydrolysis by using -amylase can be done in 90 0C for 1 hour
of operation for liquefaction process (to decrease the viscosity of the slurry). The
liquefied starch is cooled at 600C, and added by glucomalyase enzyme for
saccharification process. In saccharification process, the pH is 4,5 while the
previous reaction has pH 6-6,5. Therefore the pH needs to be reduced first by
using hydrochloric acid (HCl). If we use HCl, the special handling will be similar
as acid hydrolysis, so we also need self protection equipment to protect ourselves
from contacting with HCl.

Figure 2.2The hydrolysis of starch to glucose catalyzed by -amylase

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(Source: Sigma Aldrich)

Below will described a brief review based on Woiciechowski (2002) and

Carta (1999) for the comparison of two alternatives; acid hydrolysis or enzymatic
hydrolysis, by comparing several parameters such as; reaction condition, product
conversion, and by-product.
Tabel 2.1Comparison of acid and enzymatic hydrolysis

Hydrolysis
Catalyst
Acid

Reaction condition
0

95 C, 105 min
0

Enzymatic (-

1) Alpha-amylase: 90 C, pH

amylase and

6.5, 1 hr

glucoamylase)

2) Glucoamylase: 600C, pH

Product

By-product

conversion
46.00%

Salt

40,79%

neutralization)
-

(due

to

Ref
[1]
[2]

70,11%

4.5, 24 hr
3)

Enzyme

inactivation:

100 C, 10 min
(Source: [1] Woiciechowski et al., 2002 [2] Kanlaaya et al., 2004)

The table above shows the comparison between acid hydrolysis by using
hydrochloric acid and enzymatic hydrolysis by using alpha-amylase enzyme and
glucoamylase. Based on the time consumed, enzymatic hydrolysis is done in 25
hour and 10 minutes, while acid hydrolysis only takes 105 minutes. The product
conversion of acid hydrolysis is slightly higher than enzymatic, which is 46% and
40,79% respectively. The minor difference between the yield can be neglected
because both of them are high, and the the next process need small concentration
of glucose (apporoximately 20%), so both of them need to be added by water to
reach the specified concentration. The temperature used for acid hydrolysis is
950C, while the temperature for enzymatic hydrolysis is 90 0C, 600C, and 1000C, so
the difference is not too large. Another aspect that can be compared is by-product.
Acid hydrolysis will produce salt as by-product, due to neutralization process.
This comparison will be discussed further in scoring and decision part below.

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Selection and Decision

Based on the comparison between the use of acid or enzyme on the
hydrolysis process before, there will be a process selection by using scoring
method. Some parameters that is going to be assessed are; product conversion,
reaction time, energy, material cost, hazardous effect, and by-product.
Tabel 2.2Scoring table for acid and enzymatic hydrolysis

No.
1.
2.
3.
4.
5.

Parameter

Criteria weight

Product conversion
Reaction time
Energy (temperature)
Material cost
Material safety (less

point
2
4
3
4
3

4
4
2
5
3

8
16
8
20
9

12
73

hazardous)
6.
By-product
Final Score

Acid hydrolysis

Enzymatic
hydrolysis
4
8
1
4
3
12
1
4
3
9
5

15
52

(Source : Personal data)

Product conversion
5

= Product conversion is above50%

= Product conversion is 40-50%

= Product conversion is 30-40%

= Product conversion is 20-30%

= Product conversion is below 20%

Reaction time
5

= Reaction occurs such in <60 min

= Reaction occurs for 1 2hours

= Reaction occurs for 2 4 hours

= Reaction occurs for 4-8 hours

= Reaction occurs for >8 hours

Energy
5

= Reaction happens in ambient temperature

= The reaction temperature is about 30 60oC

= The reaction temperature is about 60 90oC

= The reaction temperature is about 90 120oC

= The reaction temperature is about >120oC

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Total Material Cost

5

= The total material cost is <$50

= The cost is about $50-$200

= The cost is about $200-$500

= The cost is about $500-$2000

= The cost is about >$2000

Hazardous effect
5

= The material is not hazardous at all

= The material is less hazardous

= The material is hazardous if in case of contact with skin, eye and

ingestion

= The material is strongly hazardous

= The material is strongly hazardous and explosive

By product
5

= 100% main product

= Yields one by product

= Yields about 2 3 by product (doesnt require a specific separation and

non-toxic if it becomes a waste)

= Yields about 2 3 by product (requires a specific separation and toxic if

it becomes a waste)

= Yields more than 3 by product

Based on table 2.2, acid hydrolysis has the better total score than

enzymatic hydrolysis. The highest criteria weight point are from the material cost
and reaction time parameter. These parameters are very important because it is
one of the aspect that will determine whether this plant is deficit or not. The first
parameter that is evaluated is conversion value (in percentage). Both of catalyst
provide a conversion percentage of about 40%, therefore the score is equal. The
next parameter is reaction time. Based on table 2.1, acid hydrolysis takes 105
minutes, while enzymatic hydrolysis takes up to 24 hour. It is due to slow reaction

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of enzyme to break the glucosidic bonds in starch. The short reaction time of acid
hydrolysis makes acid has the better score than enzymatic hydrolysis, because
reaction time is also an important aspect in economic evaluation. The longer the
time required for reaction, the higher the cost that is needed to operate the
machine. Also, the longer it takes to gain the product and it will decrease the
production capacity of sodium gluconate plant if the reaction time takes too long.
The temperature used is also an important aspect to consider. Acid hydrolysis
requires slighlty higher temperature than enzymatic hydrolysis, which is 95 0C and
enzymatic hydrolysis requires 900C and 600C. However, the difference is only
about 50C and it doesnt affect too much the economic evaluation.
The most important aspect of economic evaluation later is the material
cost. According to the research by Woiciechowski (2002), the acid hydrolysis of
150 kg cassava baggase spent $ 34,27, while enzymatic hydrolysis of 150 cassava
baggase spent $2470,99 for the materials described above. Acid hydrolysis is
much less expensive than enzymatic hydrolysis, therefore the score is much
higher. Economic aspect is very important, therefore the weight of scoring for this
parameter is high. Another aspect that is compared is by-product. For the
utilization enzyme as catalyst, there is no by-product formed. While in acid
hydrolysis, the acid needs to be neutralized by base and there is salt formed as byproducts. Based on the consideration above, the selected process is acid
hydrolysis.
Selection of Catalyst
There are several kinds of catalysts that often used in industry for
hydrolyis, however the most common catalysts that is used are Hydrochloric Acid
(HCl) and Sulfuric Acid (H2SO4). There are several advantages and disadvantages
of these catalysts, and below is the comparison between HCl and H2SO4.

Table 2.3 Comparison of H2SO4 and HCl as catalyst

Neutralization agent
By-Product
By-products easiness to

H2SO4
Ca(OH)2
CaSO4 (gypsum)
Easy

HCl
NaOH
NaCl
Hard

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remove
Method of removing by-

Clarification

Nanofiltration or Ion

product

(sedimentation)

exchange chromatography

Source: Reproduced

Based on the consideration above, it is easier to separate CaSO 4 compared than

NaCl. CaSO4 can be separated by using clarification thorugh sedimentation
process because CaSO4 is a solid insoluble salt, while NaCl need to be separated
by using nanofiltration or ion exchange chromatography because it is a soluble
salt. Also, the separation by using nanofiltration or chromatography is very
expensive due to the equipments price. Therefore, the selected acid catalyst is
H2SO4.
2.2.5

Neutralization
As mentioned before, neutralization process is conducted to fulfill

Heuristic 2. Neutralization is a process of neutralizing pH of previous reaction.

Hydrolysis reaction use H2SO4 and will be conducted in pH 3, therefore it needs to
be neutralized until the pH reaches 7. The neutralizing agent for this process is
Ca(OH)2, and the neutralization product is CaSO 4. The neutralization process is
following this reaction:
H 2 SO 4 +Ca(OH )2 Ca SO4. 2 H 2 O
2.2.6

Clarification
Since the previous reaction will produce CaSO4.2H2O (calcium sulfate) as

by-product, it needs to eliminated. Actually CaSO 4.2H2O salt will not affect
further process, but the if the concentration of CaSO4.2H2O is high and
accumulated, it needs to be eliminated. Calcium sulfate will also affect the final
products purity of sodium gluconate if it is not eliminated. Clarification is used to
eliminate CaSO4.2H2O salt from the mixture by using settling principle, therefore
the feed of clarification process is glucose solution and calcium sulfate salt, and
the output from nanofiltration process is glucose solution without calcium sulfate.
This output will be used as a feed for next process. The principle of clarifier is by
settling the particle by gravity to the bottom of the tank and stay there. During
settling, the force acting on a particle are; gravity, overflow up velocity, drag force

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by fluid, and buoyancy. The clarification process is also affected by the hydraulics
of the tank.
2.2.7

Storage
Storage is needed to store the glucose solution that is formed from

previous reaction (hydrolysis). Storage is needed before the feed enters

fermentation tank, because fermentation held in batch mode. Storage tank is also
needed to adjust the glucose concentration in the tank. According to FAO (2013),
yeasts grow well in solutions containing 20% sugar. If the glucose is more than
20%, If the glucose concentration in storage tank is still above 20%, the osmotic
pressure is high and A. niger cell membrane can be burst. So, the solution needs to
be added by water to reach the concentration of 20%.
2.2.8

Aspergillus niger culture tank

In order to use A.niger as the catalyst, it needs to be cultured first. A. niger

is a yeast that is easy to find and to culture. The culture happens in a seed
fermentation tank. This tank is common to use as a fermentation tank. A.niger
able to live in an optimum temperature of 35-37 oC and usually needs to be in
innoculated for 4-5 days. Inside the fermentation tank, there will be nutrient
needed. Based on US patent 1,849,053, the media needed in order to culture
A.niger are:

Glucose 10%

Peptone 0.15%

Potassium biphosphate 0.1%

Magnesium sulphate 0.05%

Calcium Chloride 0.01%

This yeast needs oxygen in order to grow (aerobic). The pH needed in order for
A.niger to grow is aroung 5,5. The aeration needed for the culture is arounf 2
liter/minute. Later, the yeast will be transfered to the fermentation reactor where it
will meet the substrate, glucose.

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2.2.9

Conversion of Glucose into Sodium Gluconate

To produce sodium gluconate, there are several process available. But all

the process actually is an oxidation process. In order to select the process used, we
must look through the heuristic of process synthesis. Heuristic 1 explains that we
must select raw material and chemical reaction thoroughly to avoid difficult
handling and storage. According to heuristic 1, if we have to use a process where
if we use a hazardous, valuable or toxic reactant, the operation reaction must be
able to consume all reactant in order to prevent unwanted activities. For this
reaction, an endothermic reaction is preferable since it decreases the chances of
overheating. Also its preferable to use a process that does not include hazardous,
or toxic reactant, because according to heuristic 2, the reaction that needed those
reactant must be able to eliminate all the hazardous or toxic reactant. The usage of
valuable reactant must be used wisely because we dont want the process to cost
too much. Since the basic reaction to convert glucose to sodium gluconate does
not include hazardous or toxic reactant, the heuristic 2 should not be a problem.
The main reaction of glucose oxidation into the salt form does not produce
impurities within the product, the usage purge stream are unnecessary.
The selection process must also consider the efficiency and capability of
the process to be used in an actual plant. Based on the consideration above, we
have selected three conversion process which are: oxidation with chemical
catalyst,

fermentation

with

Aspergillus

niger

and

fermentation

with

Aureobasidium pullulans. other that the capability of these processes to fulfill the
heuristic above, these processes also have a literature reasoning behind them. The
production of sodium gluconate from glucose by using chemical catalyst is
Alternative 1: Oxidation by Using Chemical Catalyst Palladium/-Al2O3
This process contain an selective oxidation using oxygen molecules that
are activated using a chemical catalyst called palladium/ -Al 2O3 . This process
happens room temperature. The gluconic acid conversion of this method is
approximately 36% (Andriayani, 2005). The down side of this process is the
usage of palladium/ -Al2O3 which is a little expensive. The catalyst used is 1.5%
of the total glucose weight. The NaOH is used to higher the pH to 10. After the

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gluconic acid is produce, sodium chloride is added in order to produce sodium

gluconate. The reactor usage for this process is two, one for catalyst adding and
the other one for sodium chloride adding. The reaction will take at approximately
4-6 hours.
Alternative 2: Fermentation using Aspergillus niger
This process is going to run by adding Aspergillus niger as the biocatalyst.
Aspergillus niger is used because they are able to produce four enzymes which
will be needed for the hydrolysis which are glucose oxidase, catalase, lactonase
and mutarotase. Glucose oxidase is used for converting glucose to gluconic
acid.As the reaction leads to an acidic product, it is required that it is neutralized
by the addition of neutralizing agents, otherwise the acidity inactivates the glucose
oxidase, resulting in the arrest of gluconic acid production. The process for
sodium gluconate is highly preferable as the glucose concentration of up to 350
g/L can be used without any such problems. pH is controlled by the automatic
addition of NaOH solution. Sodium gluconate is readily soluble in water (39.6 %
at 30 C).
During the process of glucose conversion, glucose oxidase present in A.
nigerundergoes self-reduction by the removal of two hydrogens. The reduced
form of the enzyme is further oxidized by the molecular oxygen, which results in
the formation of hydrogen peroxide, a by-product in the reaction. A.
nigerproduces catalase which acts on hydrogen peroxide releasing water and
oxygen. Hydroly- sis of glucono-d-lactone to gluconic acid is facilitated by
lactonase. The reaction can be carried out spontaneously as the cleavage of
lactone occurs rapidly at pH near neutral, which are brought about by the addition
of sodium hydroxide.
Alternative 2: Fermentation using Aureobasidiumpullulans
Aureobasidiumpullulans (de Bary) Arnaud is a yeast- like mould that has
been found to be very osmotolerant and thus can be processed in economically
profitable concentration ranges. Surprisingly, it has been determined that, under
appropriate fermentation conditions, the yeast proves to be a potential gluconic
acid producer. Produc- tion of gluconic acid can essentially be conducted

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continuously using free-growing cells for a very long period of time. Gluconic
acid formation by A. pullulans isolate 70 occurs during and after growth.Various
process parameters for the continuous and discontinous production of gluconic
acid such as pH, oxygen, temperature and medium composition, air saturation,
etc. were studied. According to Ramachandran et al. (2006), the highest glucose
conversion of 94 % and product yield of 87.1 % was achieved at an optimum pH
of 6.5. At pH=4.5, the product selectivity and yield were very poor, reaching 67.8
and 20.7 %, respectively. Temperature range of 29 to 31 C was found to be
suitable for the production of gluconic acid by the yeast. Increase of temperature
by 1 C, namely to 32 C, dramatically influenced the reduction in steady state
concentration of biomass and product.
Below will described a brief review for the comparison of three
alternatives: oxidation with chemical catalyst, fermentation with Aspergillus niger
and fermentation with Aureobasidiumpullulans. However the enzyme produced
by the microbes is still unclear, the study of using Aureobasidiumpullulans as the
catalyst has not produced a certain outcome. The cell culture process is more
complex compared to other fungi culture process. Using this microbe is still
consider risky.
Tabel 2.3Comparison of hydrolysis catalyst

Oxidation Method
Catalyst

Palladium/-

Reaction

Product

By-product

Ref.

condition
0
24 C, pH 5.5

conversion
36%

Salt

[1]

Lower

95 %

Hydrogen

[2]

Al2O3 (metal catalyst)

Aspergillus niger

than

50C, pH 6-6.
Aureobasidium

30C, pH 6.5

peroxide
87.1 %

unknown

[3]

pullulans
(Source: [1] Andriayani. 2005 ; [2] Yulianto, M.E. et al. 2007 ; [3] Anastassiadis.Set al. 2002)

Based by the comparison above, we can see that all three process have almost
similar reaction condition. But the difference is seen on the product conversion
and the by product. In order to determine which process to be selected, we can use

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scoring with specific parameters as the solution.

Tabel 2.4Reaction scoring

No

Parameter

1
Product conversion
2
Reaction time
3
Temperature (energy)
4
Availability (cost)
Final score

Criteria
weight
point
4
3
4
2

Metal
catalyst
1
5
5
1

4
15
20
2
39

Aspergillus
niger
5
2
4
4

20
6
16
8
50

Aureobasidiump
ullulans
4
2
4
2

(Source: Personal data, 2014)

Product conversion.
5

= Product conversion at range 90 99%

= Product conversion at range 80 89%

= Product conversion at range 70 79%

= Product conversion at range 60 69%

= Product conversion < 60%

Reaction time
5

= Reaction occurs such in a short time (6 hours)

= Reaction occurs for 6 12 hours

= Reaction occurs for 12 24 hours

= Reaction occurs for 24 48 hours

= Reaction occurs such in a very long time (> 48 hours)

Temperature (energy)
5

= Reaction happens in ambient temperature

= The reaction temperature is about 30 50oC

= The reaction temperature is about 50 70oC

= The reaction temperature is about 70 100oC

= The reaction temperature is about >100oC

By product

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16
6
16
4
42

= Easy to find, easy production and easy handling

= Rarely found, easy production and easy handling

= Hard to find, needs to be process, easy handling

= Hard to find, needs to be process, hard handling

= Hard to find, needs complex process to produce, hard handling

Based on table 2.4.we can see that after the scoring process of those three
alternatives, fermentation using Aspergillus niger has been selected as the most
effective reaction to be applied. Generally, this reaction doesnt require high
temperature and with an almost neutral pH, this reaction can occur with a high
conversion.
Glucose fermentation using Aspergillus niger
Due to utilization of A. niger, the innoculum needs to be cultured in a seed
tank before it enters the fermentation tank. The seed tank needs to be aerated and
agitated. The glucose from glucose tank is moved into seed tank and fermentation
tank. In fermentation tank, the A. niger is enriched with nutrition in order to
improve the growth. The fermenatation tank is also supplied by humidified air
(contains oxygen), because the reaction is aerobic. The reaction in fermentation
tank is:
C6 H 12 O6 +O2 + H 2 O Glucono deltalactone + H 2 O2
1
Glucono delta lactone C 6 H 12 O7+ H 2 O+ O 2
2
Since for many technical purposes, the desirable to produce gluconic acid
in the form of soluble salts, there is where sodium gluconate cameini.
Fermentation of sodium gluconate from the glucose is accomplished by the
addition of sodium bases such as sodium hydroxide to the fermenting medium
during the fermentation. Their function therefore was twofold, they served as
buffering agents to control the hydrogen ion concentration and also served as an
agency for the convenient recovery of gluconic acid in the form of its salts. As
the gluconic acid is formed in these prior process, it is immediately neutralized on
coming in contact with sodium ions. Gluconate salts are then formed which have a

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relatively low solubility in the aqueous medium and are recoverable from the
process by relatively simple methods of crystallization due to their reduction. In
prior processes these sodium bases had an important technical function of
controlling the pH of the media within optimum limits for the production of
gluconic acid by fermentation. The sodium ion may be aadded in the form sodium
bases such as sodium hydroxide or other sodium salts. For this we decided to use
sodium hydroxide since it will later produce the by product of hydrogen and
oxygen. But the difference of adding sodium ions into this process is that it is not
capable of performing pH control like the calcium and magnesium bases. By
adding sodium bases in the medium, the pH of fermentation will increase
drastically. According to Kennetet al (1950) in order to be able to perform well,
the addition of the sodium bases must be at a specific range periodically.
Aspergillus niger are capable of concerting nutrient glucose into gluconic
acid with sodium bases within the limits of pH 5.0 7.5.the fermentation are
conducted with the help of aeration and agitation under submerged conditions.
The gas used for aeration is preferable sterilized atmospheric air or oxygen.
Aspergillus niger is used for its capability to produce the needed enzyme for the
converting process. Here describes the oxidation process with the help of
Aspergillus niger.

Figure 2.3 Oxidation of glucose by Aspergillus niger

(Source: Ramachandran. 2006. GluconicAcd: A Review)

Aspergillus niger is used because it is able to produce all of the enzyme needed
for the conversion to happen which are: glucose oydase, catalase, lactonase and
mutarotase. While the glucose conversion is happening, autoreduction will happen
to

glucoseoxydase by the separation of two hydrogen ions. Then it will be

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oxidized by oxygen which will the produce hydrogen peroxide as the by product
of this process. In order to fasten the reaction, the appearance of mutarotase is
needed. The catalase enzyme will the help the hydrogen peroxide to be converted
into hydrogen and oxygen. The hydrolysis of D-glukono--lactone to gluconic
acid will be facilitated by the help of the lactonase enzyme. In order to the
lactonase enzyme to work, the pH must be near to normal, so the addition of
sodium hydroxide is needed. The outcome of this reaction is sodium gluconate.
Removal of lactone from the medium is recommended as its accumulation in the
media has a negative effect on the rate of glucose oxidation and the production of
gluconic acid and its salt. There are reports stating that the enzyme
gluconolactonase is also present in A. niger, which increases the rate of
conversion of glucono-d-lactone to gluconic acid. Ramachandran et al. (2006)
said that the general optimal condition for the production is by having glucose at
concentrations between 110-250 g/L, ideal temparture is approximately at 40C,
Nitrogen and Phosphorus sources at a very low concentration (20,M), pH value of
medium around 4.5 to 6.5 and a very high aeration rate by the application of
elevated air pressure of 4 bar.
There are two key parameter for this process which are the concentration
of dissolved oxygen and the mediums pH. The oxygen is needed in a big amount
since its the key substrate in the glucose oxidation. In this process, the oxygen
concentration could be monitored by looking at the oxygen concentration gradient
and the oxygen volumetric transfer coefficient. The concentration of the oxygen
depends on the oxygen transfer rate inside the medium from the gas phase to the
aqueous phase. Also during a fungi growing period, the oxygen distribution is not
even, it is shown by the low oxygen absorption while the myselium concentration
increases. The aeration rate and the agitator speed are the factors that effected the
oxygen transfer rate. Based on Sakurai et al, by using a pure oxygen with the
pressure of 6 bar and the dissolved oxygen roughly around 150 ppm, the
immobilized Aspergillus niger mycelium will produce more gluconic acid
compared to the usage of air in the atmospheric pressure. Kapalet al. also said that
the most optimal agitation speed is at 420 rpm with the aeration of 0.25 vvm of
dissolved oxygen. pH is also an important factor since it will effect whether the

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reaction will happen or not. Because Aspergillus niger will produce a weak
organic acid, the pH of the medium will decrease time after time. If the pH is not
increased to the normal range, there will be a unwanted cycle called the TCA
cycle where the cycle will facilitate the production of citric acid that is hazardous.
The pH range for this process is around 4.5-7.0, but 6.5 is considered to be the
most optimal pH in order for the sodium gluconate to be produced.
Based on the information above, we can conclude that the operation
condition during the fermentation process is:

pH 6.5
Temperature: room temperature, approximately 30oC.
Aeration: 0.25 vvm
Agitation speed 420 rpm
Duration: 20 hours
One of the problem by using this method is that the oxygen concentration

is low when there is mycelia growth. It is because the oxygen supply will be
quickly depleted by the microorganism. An alternative method written by
Fedureket al (2001) to increase the dissolved oxygen concentration in culture
media by the addition of hydrogen peroxide that later will be decomposed by
catalase to oxygen and water. But since the hydrogen peroxide itself is hazardous
and the oxidation process beforehand also

produce hydrogen peroxide, the

method wont be used. Other suggested method is by using a terpene-treated

Aspergillus niger spores. Other method suggested by Fedureket al is by
immobilizing the Aspergillus niger.
2.2.10 Microfiltration
The product received from the previous process is sodium gluconate solute
in water which also contains Aspergillus niger and several enzyme used in the
process. In order to be crystallized, the fungi and enzyme must be separated first.
There is where microfiltration comes in. Microfiltration has the range of 0.1 3
micrometer and it is able to filter fungi and enzymes. Microfiltration is a low
pressure cros flow membrane process for separating colloidal and suspended
particles. When recovering enzyme from a mixture, the permeability and the flux
can be influenced by interaction between the membrane surface and the product,

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nutrients or other species, ionic strength and pH and also formation of a gel
polarization layer. The first issue can be addressed by membrane choice and
possible changes to antifoam choice and the second by controlling ionic strength.
Gel polarization layer is influenced by the nature of the feed and conditions at the
membrane surface. In order to be able to filter the enzyme, the pore size should be
0.1 micrometer. Since the fungal size is larger than enzyme, the pores should suit
the enzyme needs. There are no specific operating condition for this process. The
output from this process is by having a sodium gluconate solute in water with no
other contaminant.
2.2.11 Crystallization
Crystallization is used in order to separate solid particles. Crystallization is
used in order to eliminate water from sodium gluconate. Crystallization is
basically a method used in order to purify substance with others that has a big
difference in their boiling temperature. Crystallization in three different modes
which are solution crystallization, precipitation and melt crystallization.
According to heuristic 14, in order to separate organic chemicals by the melt
crystallization with cooling, using suspension crystallization, followed by removal
of crystals by the settling, filtration or centrifugation. The crystallization happens
when the temperature is low enough for the wanted product to form crystal and
precipitate. The temperature for crystallizing sodium glucate is 60-80 oC (based on
Patent CN103667375 A) and then it wll be dropped to a lower temperature at
around 25oC. After the crystallization, the product will be in the form of slurry and
mother liquor. The last product will be a crystallized sodium gluconate.
2.2.12 Centrifugation
Centrifugation is used in order to separate slurry and mother liquor. There are
several important factors in the selection of equipment including moisture content
of the cake, solids content of the mother liquor, fragility of the crystals, crystals
particle size, and filtration rate. Based on heuristic 18, since the cakes of low
moisture content are required, use solid-bowl centrifugation if solids are permitted
in the mother liquor. After centrifugation, the wet cakes formed are sent to dryers
in order to remove the remaining moisture.

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2.2.13 Drying
The wet cakes produced from centrifugation needs to be dried in order to
produce a dry crystal. Based by heuristic 19 For granular material, free flowing
or not, of particle sizes from 3 to 15 mm, use continuous tray and belt dryers with
direct heat. For free flowing granular solids that are not heat sensitive, use an
inclined rotary cylindrical dryer, where the heat may be supplied directly from a
hot gas or indirectly from tubes, carrying steam, that run the length of the fryer
and are located in one or two rings concentric to and located just inside the dryer
rotating shell the dryer used is belt dryer.
2.2.14 Process Description Conclusion
Tabel 2.5 Process Conclusion

No.
1

5
6
7

Process

Unit

Operating Condition

Temperature:
Room
o
temperature (30 C)
Conveyor
Raw material washing
washer

pH: Neutral (around 6.57.0)

Temperature:
Room
o
temperature (30 C)
Raw material grinding Grinder

pH: Neutral (around 6.57.0)

Temperature:
Room
o
temperature (30 C)
Raw material soaking
Vessel
in water

pH: Neutral (around 6.57.0)

Pressure: 140 bar

Starch extraction from

Temperature: 40-50oC
Homogenizer
seaweed

pH: Neutral (around 6.57.0)

pH 3.0
Continuous stir
Hydrolysis by HCl

Temperature: 95OC
tank reactor

Time: 105 minutes

pH 3.0
Neutralization
by Continuous stir
NaOH
tank reactor

Temperature: 95OC

Temperature: 35oC
Glucose mixing with
Vessel
water

pH: 6.5-7.0

(Source: Personal data, 2014)

Tabel 2.6Process Conclusion (continue)

No.
6

Process
Aspergillus

Unit
niger Seed reactor

20

Operating Condition

pH 6.5

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Tempeature:
room
temperature

Aeration: 0.25 vvm

Agitation speed 420 rpm

culture

Glucose
Continuous
fermentation to
tank reactor
Sodium gluconate

Aspergillus niger
Microfiltration
filtration

10

Sodium gluconate
Crystallization
crystallization

stir

pH 6.5

Tempeature:
room
temperature,
approximately
30oC.

Aeration: 0.25 vvm

Agitation speed 420 rpm

Duration: 20 hours

Temperature:
temperature (30oC)

Room

pH: Neutral (around 7.0)

Temperature: 70oC

(Source: Personal data, 2014)

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