Chapter II

CHAPTER IIPROCESS SELECTION2.1. Process SynthesisThe 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:Conversion of starch into glucoseConversion of glucose into gluconic acidSodium GluconateSeaweed

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 Description2.2.1 WashingWashing 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 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. 2.2.2GrindingAfter 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 (25-300C) 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.2.2.3HomogenizationAfter 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. 2.2.4HydrolysisHydrolysis 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.

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 hydrolysisIn 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 H2SO4 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.

Alternative 2: Enzymatic hydrolysisThere 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 900C 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

(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 hydrolysisHydrolysis CatalystReaction conditionProduct conversionBy-productRef

Acid950C, 105 min46.00%Salt (due to neutralization)[1]

Enzymatic (-amylase and glucoamylase)1) Alpha-amylase: 900C, pH 6.5, 1 hr2) Glucoamylase: 600C, pH 4.5, 24 hr3) Enzyme inactivation: 1000C, 10 min40,79%

70,11%-[2]

(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 900C, 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.

Selection and DecisionBased 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 hydrolysisNo.ParameterCriteria weight pointAcid hydrolysisEnzymatic hydrolysis

1.Product conversion24848

2.Reaction time441614

3.Energy (temperature)328312

4.Material cost452014

5.Material safety (less hazardous)33939

6.By-product3412515

Final Score7352

(Source : Personal data)Product conversion5= Product conversion is above50%4= Product conversion is 40-50%3= Product conversion is 30-40%2= Product conversion is 20-30%1= Product conversion is below 20%Reaction time5=Reaction occurs such in 8 hoursEnergy5=Reaction happens in ambient temperature4=The reaction temperature is about 30 60oC3=The reaction temperature is about 60 90oC2=The reaction temperature is about 90 120oC1=The reaction temperature is about >120oC

Total Material Cost5=The total material cost is $2000Hazardous effect5= The material is not hazardous at all4= The material is less hazardous3= The material is hazardous if in case of contact with skin, eye and ingestion2= The material is strongly hazardous1= The material is strongly hazardous and explosive

By product5=100% main product 4=Yields one by product 3=Yields about 2 3 by product (doesnt require a specific separation and non-toxic if it becomes a waste)2=Yields about 2 3 by product (requires a specific separation and toxic if it becomes a waste)1=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 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 950C 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 by-products. Based on the consideration above, the selected process is acid hydrolysis. Selection of CatalystThere 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 catalystH2SO4HCl

Neutralization agentCa(OH)2NaOH

By-ProductCaSO4 (gypsum)NaCl

By-products easiness to removeEasyHard

Method of removing by-productClarification (sedimentation)Nanofiltration or Ion exchange chromatography

Source: ReproducedBased on the consideration above, it is easier to separate CaSO4 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. 2.2.5NeutralizationAs 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 CaSO4. The neutralization process is following this reaction:

2.2.6ClarificationSince the previous reaction will produce CaSO4.2H2O (calcium sulfate) as by-product, it needs to eliminated. Actually CaSO4.2H2O 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 by fluid, and buoyancy. The clarification process is also affected by the hydraulics of the tank. 2.2.7StorageStorage 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 tankIn 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 35-37oC 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. 2.2.9Conversion of Glucose into Sodium GluconateTo 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/-Al2O3This process contain an selective oxidation using oxygen molecules that are activated using a chemical catalyst called palladium/ -Al2O3 . 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 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 nigerThis 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 AureobasidiumpullulansAureobasidiumpullulans (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 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 catalystOxidation MethodReaction conditionProduct conversionBy-productRef.

Catalyst Palladium/-Al2O3 (metal catalyst)240C, pH 5.536%Salt[1]

Aspergillus nigerLower than 50C, pH 6-6.95 %Hydrogen peroxide[2]

Aureobasidium pullulans30C, pH 6.587.1 %unknown[3]

(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 scoring with specific parameters as the solution.

Tabel 2.4Reaction scoringNoParameterCriteria weight pointMetal catalystAspergillus nigerAureobasidiumpullulans

1Product conversion414520416

2Reaction time35152626

3Temperature (energy)4520416416

4Availability (cost)2124824

Final score395042

(Source: Personal data, 2014)Product conversion.5=Product conversion at range 90 99%4=Product conversion at range 80 89%3=Product conversion at range 70 79%2=Product conversion at range 60 69%1=Product conversion < 60%

Reaction time5=Reaction occurs such in a short time (6 hours)4=Reaction occurs for 6 12 hours3=Reaction occurs for 12 24 hours2=Reaction occurs for 24 48 hours1=Reaction occurs such in a very long time (> 48 hours)

Temperature (energy)5=Reaction happens in ambient temperature4=The reaction temperature is about 30 50oC3=The reaction temperature is about 50 70oC2=The reaction temperature is about 70 100oC1=The reaction temperature is about >100oC

By product5=Easy to find, easy production and easy handling4=Rarely found, easy production and easy handling3=Hard to find, needs to be process, easy handling 2=Hard to find, needs to be process, hard handling1=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 nigerDue 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:

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 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 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 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 hoursOne 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 MicrofiltrationThe 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, 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 CrystallizationCrystallization 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 60-80oC (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 CentrifugationCentrifugation 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. 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 ConclusionTabel 2.5 Process ConclusionNo.ProcessUnitOperating Condition

1Raw material washingConveyor washer Temperature: Room temperature (30oC)

pH: Neutral (around 6.5-7.0)

2Raw material grindingGrinder Temperature: Room temperature (30oC)

pH: Neutral (around 6.5-7.0)

3Raw material soaking in waterVessel Temperature: Room temperature (30oC)

pH: Neutral (around 6.5-7.0)

4Starch extraction from seaweedHomogenizer Pressure: 140 bar

Temperature: 40-50oC

pH: Neutral (around 6.5-7.0)

5Hydrolysis by HClContinuous stir tank reactor pH 3.0

Temperature: 95OC

Time: 105 minutes

6Neutralization by NaOHContinuous stir tank reactor pH 3.0

Temperature: 95OC

7Glucose mixing with waterVessel Temperature: 35oC

pH: 6.5-7.0

(Source: Personal data, 2014)

Tabel 2.6Process Conclusion (continue)No.ProcessUnitOperating Condition

6Aspergillus niger cultureSeed reactor pH 6.5

Tempeature: room temperature

Aeration: 0.25 vvm

Agitation speed 420 rpm

7Glucose fermentation to Sodium gluconateContinuous stir tank reactor pH 6.5

Tempeature: room temperature, approximately 30oC.

Aeration: 0.25 vvm

Agitation speed 420 rpm

Duration: 20 hours

8Aspergillus niger filtrationMicrofiltration Temperature: Room temperature (30oC)

pH: Neutral (around 7.0)

10Sodium gluconate crystallizationCrystallization Temperature: 70oC

(Source: Personal data, 2014)21