Continous Immbolizied Cell Reactor

MANOJ SELVARAJ

CONTENTS

CHAPTER 1
1.1

INTRODUCTION

Introduction. 1

CHAPTER 2 LITERATURE REVIEW

2.1 Fermentation of bioethanol 2.1.1 2.1.2 2.1.3 2.2 2.3 2.4 2.5 Microbes for fermentation .. 5 Fermentation aspects .. 6 Fermentation Pathway . 7

Immobilization . 8 Batch Reactors .. 11 Packed Bed Reactors . 13 Fluidized Bed Reactor 15

CHAPTER 3 METHODOLOGY
3.1 Immobilization procedure 20 3.2 Procedure for glucose assay by Dinitrosalicylic Colorimetric Method (DNS) 3.2.1 Solutions preparation 21 3.2.2 Procedure for standard plot ... 21 3.2.3 Procedure to determine unknown glucose concentration .. 22 3.3 Procedure for ethanol assay 3.3.1 Solutions preparation . 22 3.3.2 Titration procedure . 23 3.3.3 Calculation .. 24 3.4 Fermentation medium preparation . 25 3.5 Batch reactor ...26
I

3.6 Procedure to measure dry weight of yeast cells for growth curve .. 27 3.7 Immobilized Cell Reactor (ICR) setup 28 CHAPTER 4 RESULTS 4.1 Introduction .. 30 4.2 Bakers yeast growth curve .. 31 4.3 Standard plot for glucose assay 34 4.4 Batch reactor ..................................................................................................................... 36 4.5 Immobilization .. .. 40 4.6 Immobilized Cell Reactor 43 4.7 Sample calculation for ethanol concentration .. 45 CHAPTER 5 CONCLUSION 5.1 Discussion . 47 5.2 Future work ... 47 REFERENCES

II

CHAPTER 1 INTRODUCTION
1.1 Introduction: The term bio-fuels can refer to fuels for direct combustion for energy production, but is generally used for liquid fuels in transportation sector. The use of bio-fuels can contribute to the mitigation of greenhouse gas emissions, provide a clean and therefore sustainable energy source, and increase the agricultural income for rural poor in developing countries. Today, bio-fuels are predominantly produced from renewable biomass resources. Biomass appears to be an attractive feedstock for three main reasons [18],

(1) It is a renewable resource (2) It appears to have formidably positive environmental properties resulting in no net release of carbon dioxide and negligible sulfur content which reduces acid rains. (3) It appears to have significant economic potential provided that fossil fuel prices increase in the future.

Bio-fuels are liquid or gaseous fuels made from plant materials and residues, such as agricultural crops, municipal wastes and agricultural and forestry by-products. Liquid bio-fuels can be used as an alternative fuel for transport, as can other alternatives such as liquid natural gas (LNG), compressed natural gas (CNG) and liquefied petroleum gas (LPG). Bio-fuels could significantly reduce the emissions from the road-transport sector if they were widely adopted. They have been shown to reduce carbon emissions, and may help to increase energy security. There are many

different types of bio-fuels, which are produced from various crops and via different processes. Bio-fuels can be classified broadly as bio-diesel and bio-ethanol, and then subdivided into conventional or advanced fuels.

With increasing gap between the energy requirement of the industrialized world and inability to replenish such needs from the limited sources of energy like fossil fuels, an ever increasing level of greenhouse pollution from the combustion of fossil fuels in turn aggravate the perils of global warming and energy crisis. [18]

The principle fuel used as a petrol substitute for road transport vehicles is bioethanol. Bioethanol fuel is mainly produced by the sugar (especially from glucose) fermentation process, although it can also be manufactured by the chemical process of reacting ethylene with steam. The main sources of sugar required to produce ethanol come from energy crops. These crops are grown specifically for energy use and include corn, maize and wheat crops, waste straw, willow and popular trees, sawdust, reed canary grass, cord grasses, Jerusalem artichoke, myscanthus and sorghum plants. There is also ongoing research and development into the use of municipal solid wastes to produce bioethanol fuel.

Ethanol or ethyl alcohol (C2H5OH) is a clear colorless liquid; it is biodegradable, low in toxicity and causes little environmental pollution if spilt. Ethanol burns to produce carbon dioxide and water. Ethanol is a high octane fuel and has replaced lead as an octane enhancer in petrol. By blending ethanol with gasoline the fuel mixture gets oxygenated so it burns more completely and

reduces polluting emissions. Ethanol blended fuels are widely sold in the United States. The most common blend is 10% ethanol and 90% petrol (E10). Vehicle engines require no modifications to run on E10 and vehicle warranties are unaffected also. Only flexible fuel vehicles can run on up to 85% ethanol and 15% petrol blends (E85). The Brazilian car manufacturing industry developed flexible-fuel vehicles that can run on hydrous ethanol (E100).

In the past decades, microbial ethanol production has been focused and considered as an alternative fuel for future since fossil fuel is depleted. Several microorganisms, including Clostridium bacterium, the well-known yeast ethanol producers, Saccharomyces cerevisiae and Zymomonas mobilis are suitable candidates to produce ethanol [3].However the yeast, Saccharomyces cerevisiae, is the predominant industrial microorganism responsible for alcoholic fermentations. The organism is also known as bakers yeast or brewers yeast, is a unicellular micro fungus that plays important roles in industry, the environment and medical science. It is the main cell factory in modern bioethanol production processes. [18]

This project concentrates on increasing the concentration of the bioethanol produced. Conventionally by batch production around 8% (v/v) is produced. There are various other reactors which could produce higher ethanol concentration and in lower time. The purpose of this research was to compare the ethanol production with various different types of reactors under the same operating condition. First and foremost, ethanol was produced using a 500 ml batch reactor with Saccharomyces cerevisiae yeast. Ethanol concentration of about 7.7% (v/v) was produced. Next the Saccharomyces cerevisiae yeast was immobilized using an entrapment technique utilizing alginate as a porous wall to retain the yeast cells. Later an immobilized cell reactor (ICR) was used to produce ethanol concentration up to 12.2% (v/v).

CHAPTER 2 LITERATURE REVIEW

2.1 Fermentation of Bioethanol 2.1.1 Microbes for fermentation Graeme M Walker (2010) claims that the yeast, Saccharomyces cerevisiae, is the predominant industrial microorganism responsible for alcoholic fermentations. This organism, also known as bakers or brewers yeast, is a unicellular micro fungus that plays important role in industry, the environment and medical science. It has been exploited for millennia in food and beverage fermentations and is the main cell factory in modern bioethanol production processes. He also explains that in order to grow and ferment, yeast cells require a range of essential nutrients [6]. These can be categorized as: Macronutrients (sources of carbon, nitrogen, oxygen, sulphur, phosphorus, potassium, and magnesium) required at the millimolar level in growth media; Micronutrients (sources of trace elements such as Ca, Cu, Fe, Mn, Zn) required at the micromolar level. Most yeast grows quite well in simple nutritional media, which supplies carbon and nitrogenbackbone compounds together with inorganic ions and a few growth factors. The latter are organic compounds required in very low concentrations for specific catalytic or structural roles in yeast, but are not used as energy sources. Growth factors for yeast include vitamins, which serve vital functions as components of coenzymes; purines and pyrimidines; nucleosides and nucleotides; amino acids; fatty acids; sterols; and other miscellaneous compounds (e.g., polyamines).[6]

Most yeast thrives in warm, dilute, sugary, acidic, and aerobic environments. Industrial S. cerevisiae strains grow best at a temperature range of 20-30C and between pH 4.5 and 5.5. Concerning oxygen requirements, S. cerevisiae is not, strictly speaking, a facultative anaerobe and is generally unable to grow well under completely anaerobic conditions. This is because oxygen is needed as a growth factor for membrane biosynthesis, specifically for fatty acid (e.g. oleic acid) and sterol (e.g.ergosterol) biosynthesis. [6] 2.1.2 Fermentation Aspects According to Greame M Walker (2010) the traditional way of producing Bioethanol would be to mix sugar, water and yeast bacteria, which are then allowed to ferment in warm environment. Gradually the mixture becomes a liquid that has an approximate of fifteen percent alcohol. As and how the alcohol percentage increases, the yeast consumes itself in the process and dies out eventually which stops the process altogether. Then the liquid mash that is created is distilled and purified to get approximately ninety-nine point five percent Bioethanol. Thus this process of fermentation is a series of chemical reactions wherein the simple sugars are converted into ethanol. Yeast or bacteria, which feed on the sugars, cause the reaction and thus fermentation occurs. Ethanol and carbon dioxide are produced as and how the yeast consumes the sugar. [6][13] The above reaction can be represented as,

C6H12O6

(glucose)

>

CH3CH2OH

(ethanol)+

CO2

(carbon

dioxide)

In this kind of a process of production, bioethanol is derived from a variety of sugar and starchrich crops, which includes grain, corn, sugar cane, and sugar beet. The process of traditional

production of this kind of substitute fuel is well-known and easy process that only consists of the fermentation of the sugar, similar to the process used to prepare beverages like whisky or vodka. 2.1.3 Fermentation Pathway CH3CHO + CO2 Acetaldehyde + CO2 CH3CH2OH Ethanol

C6H12O6 Glucose

CH3COCOOH Pyruvate

Michael L Schuler (2009) explains the fermentations pathway of glucose getting converted to ethanol. The oxidation of glucose is known as glycolysis. Glucose is oxidized to either lactate or pyruvate. Under aerobic conditions, the dominant product in most tissues is pyruvate and the pathway is known as aerobic glycolysis. When oxygen is depleted, as for instance during prolonged vigorous exercise, the dominant glycolytic product in many tissues is lactate and the process is known as anaerobic glycolysis. The overall reaction of glycolysis is: [13] Glucose + 2 ADP + 2 NAD+ + 2 Pi > 2 Pyruvate + 2 ATP + 2 NADH + 2 H+

Where, ATP = adenosine tri phosphate (biological energy) NAD =Nicotinamide adenine dinucleotide NAD+ = oxidized form of NAD NADH = reduced form of NAD Pi =inorganic phosphate

2.2 Immobilization Pilkington P H (1997) explains that immobilization may be used as a tool to confine intact cells to an inert carrier within a bioreactor. Pilkington P H (1997) lists out that this "tool" will further increase the efficiency of a continuous fermentation system by providing: [16] High cell densities per unit bioreactor volume which result in very high fermentation rates. The reuse of the same biocatalysts (yeast cells) for extended periods of time due to constant cell regeneration. a continuous process which may be operated beyond the nominal washout rate A discrete phase in which cells may be manipulated. Easy separation of biocatalyst from the liquid phase where the desired products are present thus minimizing separation costs. Higher cell densities combined with operation at high dilution rates, decreasing the risk of reactor shutdown due to contamination. Improved tolerance or protection of cells from product inhibition. Smaller bioreactor volumes which may lower capital costs. Pilkington P H suggests that an immobilized cell system should have the following properties for large scale industrial application: [16] The carrier material must be nontoxic, readily available and affordable; The system should be efficient, easy to operate and give good yields; The carrier material should allow for high cell loading and physical strength; The cells should have a prolonged viability in the support.

Kourkoutas Y (2003) claims that numerous biotechnological processes are advantaged by immobilization techniques and therefore several such techniques and support materials have been proposed. Kourkoutas Y (2003) lists these techniques into four major categories based on the physical mechanism employ, [9] i. ii. iii. Attachment or adsorption on solid carrier surfaces, Entrapment within a porous matrix, Self aggregation by flocculation (natural) or with cross linking agents (artificially induced), iv. cell containment behind barriers

Yekta G Ksungur (2000) says that the most effective and efficient way of immobilizing Saccharomyces cerevisiae is with Ca-Alginate. He further explains that prior to the immobilization step, S. cerevisiae cells should be grown at 30oC for 36 hours in a temperature controlled shaker. The composition of the growth medium was Glucose 15 g/l (NH4)2SO4 - 18 g/l (NH4)2HPO4 10g/l KH2PO4 -5g/l MgSO4 5 g/l yeast extract 1g/l

Fifty milliliters of this growth medium was mixed with an equal volume (1:1, v/v) of 4% (w/v) Na-alginate solution. A 100 ml aliquot of alginate-cell suspension containing 2% Na-alginate was added drop wise to 1000 ml of 2% CaCl2 with a peristaltic pump. Alginate drops solidified upon contact with CaCl2, forming beads and thus entrapping bacterial cells. The beads were allowed to harden for 30 min and then were washed with sterile saline solution (0.85% NaCl) to remove excess calcium ions and cells. Yekta G Ksungur (2000) researched that with the immobilized beads in a packed bed reactor has 4.2 times better productivity than a batch reactor and in the packed bed reactor reduced inhibition from both product and toxic materials in the substrate. [22] The potential use of immobilized cells in fermentation processes for fuel production has been described previously. If intact microbial cells are directly immobilized, the removal of microorganisms from downstream product can be omitted and the loss of intracellular enzyme activity can be kept to a minimum. [5]

2.3 Batch Reactors: According to Greame M Walker (2010) the majority of bioreactions are batch-wise. Furthermore the first phase of batch bioreactions is commonly sterilization, after which the sterile culture medium is inoculated with microorganisms that have been cultivated to achieve a specific result. During this dynamic reaction period, cells, substrates (including the nutrient salts and vitamins) and concentrations of the products vary with time. Proper mixing keeps the differences in composition and temperature at acceptable levels. To promote aerobic cultivation, the medium is aerated to provide a continuous flow of oxygen. Gaseous byproducts formed, such as CO2, are removed, and aeration and gas-removal processes take place continuously. Next the pH is controlled to the required optimum value for maximum efficiency. To keep foaming to acceptable levels, antifoaming agents may be added when indicated by a foam sensor. [6] According to Pramanik K (2003) when a one liter batch reactor is used with an initial sugar concentration of 100 g/l, and with a fermentation time of 72hrs, ethanol concentration up to 45.5 g/l with bakers yeast. If Saccharomyces cerevisiae is separated out from other substances like toddy, under the same conditions ethanol concentrations can go up to 48 g/l. Pramanik K (2003) also found out the effect of various operating conditions like temperature, inoculum time, pH etc on ethanol concentration. The ideal operating conditions for maximum ethanol concentration are, [17] pH = 4.25 Temperature = 30 o C Initial Sugar Concentration = 100 g/l Inoculum time = 24 hrs

Pramnik K (2003) mentions that, the effect of inoculum time is not considerable. However the other factors have considerable effect on ethanol concentration. In particular high initial sugar concentration has a huge effect on ethanol concentration; Pramnik K (2003) explains this as the inhibitory effect of high sugar concentrations for alcoholic fermentation may be due to plasmolysis of yeast cells. [17] Adding to MaziarSafaeiAsli (2009), that media composition also has an effect on ethanol concentration. The maximum ethanol concentration was achieved when K2HPO4 is used as phosphorous source, (NH4)2SO4 is used as a nitrogen source and beef extract is used instead of yeast extract as a nutrient. [12]

2.4 Packed Bed Reactor: Several studies have described continuous ethanol production using Ca-Alginate immobilized yeasts with different bioreactor configurations. In these studies, the most commonly used bioreactors are the continuous flow stirred tank bioreactor, fluidized-bed bioreactor and packedbed bioreactor. Packed-bed bioreactors have become very popular in recent years due to their low manufacturing and operating costs and also due to the ease of process automation in these reactors. [22]

GhasemNajafpour (2003) used the term immobilized cell reactor (ICR) for a packed bed bioreactor. The ICR was a plug flow tubular column, constructed with a nominal diameter of 5 cm, ID of 4.6 cm, Plexiglas of 3 mm wall thickness and 85 cm length. 70% of the column volume was packed with Ca-Alginate beads immobilized with yeast. With an initial sugar concentration of 50 g/l and retention time of 6hrs, 16% (v/v) ethanol was produced. Also for high initial sugar concentration like 150 g/l a higher retention time is required. The microbial overgrowth was controlled with carbon dioxide, which is passed through the bed. There was a maximum 30% increase in the beads diameter at the lower part of column because of the high glucose concentration in that region which means the immobilized yeast in that region is the most active. [5]

Yekta G. Ksungur (2000) suggests that in order to study the operational stability of the immobilized packed-bed bioreactor, the system was run continuously for 25 days at a constant dilution rate of 0.22 h-1. Beet molasses medium (pH=3.9) containing 10.90% initial sugar was used as the production medium and the temperature was maintained at 30oC. CaCl2 solution

(2%) was passed through the column every 7 days to prevent disruption and maintain the mechanical structure of Ca-Alginate beads, and air was passed through the column every 2-3 days to remove accumulated CO2. The ethanol concentration was 4.2-4.6% during the 25 days of operation of the packed-bed bioreactor. At the end of 25 days, 4.43% ethanol concentration and 79.5% theoretical yield were obtained. During the continuous fermentation, the structure of the Ca-Alginate beads was remaining same. [22]

2.4 Fluidized Bed Reactor Fluidized bed reactor (FBR) is a type of reactor device that can be used to carry out a variety of multiphase chemical reactions. In this type of reactor, a fluid (gas or liquid) is passed through a granular solid material (usually a catalyst possibly shaped as tiny spheres) at high enough velocities to suspend the solid and cause it to behave as though it were a fluid. This process, known as fluidization, imparts many important advantages to the FBR. As a result, the fluidized bed reactor is now used in many industrial applications. The solid substrate (the catalytic material upon which chemical species react) material in the fluidized bed reactor is typically supported by a porous plate, known as a distributor. The fluid is then forced through the distributor up through the solid material. At lower fluid velocities, the solids remain in place as the fluid passes through the voids in the material. This is known as a packed bed reactor. As the fluid velocity is increased, the reactor will reach a stage where the force of the fluid on the solids is enough to balance the weight of the solid material. This stage is known as incipient fluidization and occurs at this minimum fluidization velocity. Once this minimum velocity is surpassed, the contents of the reactor bed begin to expand and swirl around much like an agitated tank or boiling pot of water. The reactor is now a fluidized bed. Depending on the operating conditions and properties of solid phase various flow regimes can be observed in this reactor. [6]

SinsuphaChuichulcherm (2003) lists out the design considerations of a fluidized bed reactor as follows: [20] a) Support material: Material used should have a high surface area to volume ratio, a rough surface or high porosity with a slightly high density compared to the fermentation broth. Porous support such as clay and activated carbon is suitable for biofilm attachment since

it has a high adsorptive power but its strength is not good enough when to be used in a fluidized bed reactor. Sand is always reported to be used as it has a rough surface and the biomass can attach onto the surface but not inside the sand particle. Other physical characteristics which have to be considered are size, shape, and hardness. It is also have to consider the cost of the material and its poisons to the microbes. The support material used in by Sinsupha Chuichulcherm (2003) is PVC pellets rubbed with sand paper to increase roughness so that yeast can attach to it. More common material to be used is yeast immobilized in Ca-Alginate. [20][9]

b) Support particle diameter and the fluidized bed column diameter: The column diameter should be 30 times larger than average diameter of a support particle to avoid hinder settling velocity effect. Also it is recommended the height of the column should be around ten times of the column diameter. However, the surface velocity in the fluidized-bed system needs to achieve 20% to 40% expansion to get a better mix and higher heat and mass transfer rate. [3]

c) Alkalinity and recirculation: due to an increase of a fermentation product, CO2, pH of the fermentation broth seem to shift to slightly alkaline region. Also, a need of high surface velocity in the fluidized column, sometimes higher than the dilution rate, makes a recirculation and a recycle port necessary. The recycle port is a small box where the feed inlet port, the effluent port, pH probe, are situate and is attached to the fluidized bed column. A recirculation pump can be used to circulate fermentation broth form the recycle port to and from the column with a flow rate higher than the dilution rate, 2000 used a recirculation flow rate 50 times higher than the dilution rate in an anaerobic system to achieve a better yield.[20]

d) Velocity in fluidization operation: Two velocities which have to be considered are V

OM

,a

minimum fluidization velocity and Ut, a terminal fluidization velocity. Minimum fluidization velocity V
OM

can be written as shown in equation (1)

The terminal velocity is proportional to V

OM

and void fraction as can be seen in equation (2).

The two equations can be used when the particles Reynolds number is higher than 1000 (a turbulent regime). If the velocity used in the operation is lower than the V
OM

, the bed would

not expand. On the other hand, when the operating velocity is higher than the Ut, all the support particles would be carried out of the column with the fermentation broth. Then, the volume flow rate of the fermentation broth can be determined by multiply the velocities calculated with a cross-sectional area of the column. Once the volume flow rate known, a suitable recirculation pump and rotameter can be selected. [20]

SinsuphaChuichulcherm (2003) used a two phase fluidized bed reactor where the solid particles which contains yeast is fluidized using the fermentation medium. This is a simple way of using a fluidized bed reactor. With this setup a high ethanol concentration of 13.2g/l was produced [20].Haiyam Mohammed A. Al-Raheem (2009) used the concept of a more complicated three phase fluidized bed reactor. Nitrogen gas was used to fluidize the flocculated yeast. [7] Haiyam Mohammed A. Al-Raheem (2009) constructed the reactor from plexiglass cylinder of 20 cm diameter and 108 cm height. The reactor is fitted with four sampling points, which are placed at equal intervals between the bottom and the top part of the reactor. Feed streams into the reactor are air and glucose solution. The air stream is uniformly bubbled in the liquid phase via perforated ceramic tube. On the other hand, the feed glucose solution is distributed through a coarse bed of glass beads placed in the lower section of the reactor. Phase separation, i.e., solid, liquid, and gas is achieved by fitting the top part of the reactor with a cylindrical tube of large diameter. As a result, solids settle back into the reactor, while gas bubbles coalesce and escape through the reactor top opening. The rising liquid phase exits the reactor through a side part. The feed sugar solution is kept in a storage tank which is fitted with an immersion heater to maintain constant temperature throughout the experiment. Also, another immersion heater is positioned in the reactor top opening, to ensure temperature uniformity. During experiments both heaters are set to the same temperature reading. Fluidization of the reactor content is achieved by the use of a variable speed pump. [7]

With this setup under normal operating conditions and initial sugar concentration of 100g/L, 16 g/L ethanol concentration was achieved which is much higher than the ethanol concentration in the two phase fluidized bed reactor. [7]

CHAPTER 3 METHODOLOGY
3.1 Immobilization Procedure: 1. 100ml growth medium was prepared. Growth medium composition was 15g/l Glucose; 18g/l (NH4)2SO4; 10g/l (NH4)2HPO4; 5gm/l KH2PO4; 5gm/l MgSO4; 1 g/l yeast extract. The pH of the solution was adjusted to 4.25 with sulphuric acid and then sterilized in an autoclave for 15 min at 15 psi pressure. 2. 20 ml of concentrated (0.1g/ml) yeast solution was added. 3. The Saccharomyces cerevisiae cells were allowed to grow for 20 hours to reach their starting of the exponential phase. 4. Na-Alginate Solution was prepared by dispersing 4g of Na-Alginate in 100ml distilled water (4% w/v). Na-Alginate is added slowly with continuous stirring until a clear viscous fluid is formed. 5. The prepared 100ml of the grown medium is added to the 4% w/v Na-alginate solution slowly with constant stirring. 6. 1000 ml of CaCl2 was prepared by dissolving 20g of CaCl2 in distilled water and make up to 1000ml. 7. The 200ml aliquot of alginate-cell suspension containing 2% (w/v) Na-Alginate was added drop wise using a syringe into 2% (w/v) CaCl2 solution. The drops forms as soft beads. 8. The beads were allowed to harden for 30 min and then were washed with sterile saline solution (0.85% (w/v) NaCl) to remove excess calcium ions and cells. 9. The beads were stored at 4oC in 0.2% (w/v) yeast extract solution until use.

3.2 Procedure for Glucose assay by Dinitrosalicylic Colorimetric Method (DNS) 3.2.1 Solutions Preparation (DNS):

1. 1% (w/v) Dinitrosalicylic Acid Reagent Solution (DNS Reagent) is prepared by dissolving10g of Dinitrosalicylic acid, 2g Phenol, 0.5g Sodium Sulphite and 10g Sodium Hydroxide in 1l of distilled water. 2. 40 %( w/v) Potassium Sodium Tartrate solution is prepared by dissolving 4g of Potassium Sodium Tartrate in 10ml of distilled water.

3.2.2Procedure for Standard Plot:

1. Prepare test tubes containing 3ml glucose solution where the concentration varies from 0.2mg/ml to 1 mg/ml. 2. 3 ml distilled water is taken in an oven dried test tube and kept aside. 3. Add 3ml of DNS reagent into each test tube which contains 3 ml of distilled water. 4. Heat the mixture at 90oC water bath for about 15 minutes to develop red brown colour. 5. Add 1ml of 40% (w/v) Potassium Sodium Tartrate solution to stabilize colour. 6. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer at 575 nm. 7. Plot concentration vs. absorbance.

 3.2.3 Procedure To Determine Unknown Glucose Concentration:

1. Dilute the sample glucose solution ten times. 2. Prepare test tubes containing 3ml sample diluted glucose solution. 3. Add 3ml of DNS reagent into test tube. 4. Heat the mixture at 90oC water bath for about 15 minutes to develop red brown colour. 5. Add 1ml of 40% (w/v) Potassium Sodium Tartrate solution to stabilize colour. 6. After cooling to room temperature in a cold water bath, record the absorbance with a spectrophotometer at 575 nm. 7. From the standard plot find the concentration of glucose sample corresponding to the observed absorbance.

3.3 Procedure for Ethanol assay: 3.3.1 Solutions Preparation: 1. Acid dichromate solution: (0.01 molL-1in 5.0 molL-1sulfuric acid) 125 ml of water

acid was added with constant swirling. Flasks were cooled under cold water tap and 0.75 g of potassium dichromate was added. It was then diluted to 250 ml with distilled water.

was added to a 500 ml conical flask. Carefully 70 ml of concentrated sulphuric

2. Starch indicator solution (1.0% (w/v) solution) - 1.0 g of soluble starch was 3. Sodium thiosulfate solution (0.03molL-1) - 7.44 g of Na2S2O3.5H2O was added to 4. Potassium iodide solution: (1.2molL-1) - 5 g of KI was dissolved in 25 ml of water. a 1L volumetric flask, dissolved in distilled water and diluted up to the mark. dissolved in 100 ml of recently boiled water. The mixture was stirred until dissolved.

3.3.2 Titration Procedure:

1. Sample is diluted at 1:20 with distilled water. with rubber stopper. titration.

2. 10 ml of the acid dichromate solution was transferred to a 250 ml conical flask 3. 1 ml of the diluted sample was pipetted into the sample holder. Three samples of 4. The sample holder was suspended over the dichromate solution and held in 5. The flask was stored overnight at 2530C place with the rubber stopper. the beverage were prepared as the entire contents of the flask are used in the

6. Next morning the flask was allowed to come to room temperature, then loosened the stopper and carefully removed and discarded the sample holder.

7. The walls of the flask were rinsed with distilled water, and then about 100 ml of mixture was swirled to mix properly. swirling to mix.

8. Blank titrations were prepared by adding 10 ml of acid dichromate solution to a 9. The burette was filled with sodium thiosulfate solution and each flask was conical flask, adding 100 ml of water and 1 ml of potassium iodide solution and titrated with sodium thiosulfate. When the brown iodine colour faded to yellow, 1 ml of starch solution was added and was further titrated until the blue colour

distilled water was added along with 1 ml of potassium iodide solution. The

disappeared. The blank flasks were titrated first, and repeated until concordant results were obtained. Then each of the samples was titrated. If the three need to be prepared.

samples of the beverage do not give concordant results, further samples will

3.3.3 Calculations:

2. The average volumes of sodium thiosulfate used for the blank titration from the concordant blank results are determined.

1. The average volume of sodium thiosulfate used for the sample from the concordant sample results was determined.

3. The volume of the sodium thiosulfate solution used for the sample titration is subtracted from the volume used for the blank titration. This volume of the sodium thiosulfate solution is now used to determine the alcohol concentration.

4. The number of moles of sodium thiosulfate in this volume was calculated.

5. Using the equations, the relationship between the moles of sodium thiosulfate and the moles of ethanol were determined. Using this ratio the moles of alcohol in the sample solution was calculated. as 1 mol of S2O32- is equivalent to 6 mol of Cr2O72and 2 mol of Cr2O72- is equivalent to 3 mol of C2H5OH then 1 mol of S2O32- is equivalent to 0.25 mol of C2H5OH

6. In order to account for the dilution factor, the result was multiplied by 20.

3.4 Fermentation Medium Preparation: The fermentation medium had the following composition. 10% (w/v) Glucose 0.2% (w/v) Yeast Extract 0.1%(w/v) K2HPO4 0.07% (w/v) MgSO4.7H2O 0.4% (w/v) (NH4)2SO4 0.1% (w/v) NaCl

(Note: 1% means 1g made up to 100ml)

The pH of the solution was adjusted to 4.5 with sulphuric acid and then sterilized in an autoclave for 15 min at 15 psi pressure. After which the broth is cooled to room temperature.

3.5 Batch Reactor Setup: The batch reactor consists of a 500 ml double necked round bottom flask fitted with rubber corks. It has three inlets. In the rubber cork covering the second inlet right at the middle a hole was drilled just enough to let a small glass tube pass by. This acts as the outlet for CO2. The glass tube is inserted to about few centimetres into the flask. The top of the glass tube is connected with a silicon tube which is directly sent into a conical flask containing distilled water. The round bottomed flask with the help of a clamp is made to stand on top of magnetic stirrer which is used for agitation. The first inlet is used take out samples at regular intervals. The third inlet can be used for further studies of batch like continuous temperature control or continuous pH control.

4% reactor volume size inoculum is prepared using HiVeg Nutrient Broth. 1ml of concentrated yeast solution is added to every 5ml of inoculum. Concentrated yeast solution is prepared by dissolving bakers yeast in minimal amount of water. The inoculum is grown for 24hrs in a shaker before adding into the reactor. At regular intervals samples are taken in test tubes covered with cotton. Test tubes are stored at 4oC for further analysis.

The reactor is cleaned with ethanol and dried for sterilization purpose. The reactor is then filled with 500ml fermentation medium. The inoculum is then added to the reactor and instantly the magnetic stirrer is switched on. Also a sample is taken immediately after the inoculum has mixed with the fermentation medium. At regular intervals samples are taken in test tubes covered with cotton. Test tubes are stored at 4oC for further analysis.

3.6 Procedure to measure dry weight of yeast cells for growth curve:

1. A batch reaction is started with about 250ml medium and inoculum is added into the reactor. At time t=0 the first sample is taken. Then on every 12hrsthe samples are collected in a test tube covered with cotton plug and stored at 4oC for further analysis. 2. 5mL of sample was inserted into the pre weighed dry eppendorf tube. 3. Step (1) was repeated for other samples from different time interval. 4. Samples were centrifuged at 7000 rpm for 15 min. 5. The cell pellet was dried using drier for approximately 15 minutes and dry weight of the sample was calculated in an accurate weighing balance.

3.7 Immobilized Cell Reactor Setup: The reactor was constructed with a glass column with total volume of 2L, 5cm diameter and height of 100 cm. The bottom was fitting with a conical inlet for the feed with a valve. Top of the reactor was closed air tight with a rubber cork. Hole was drilled in the middle of the cork through which a glass rod was inserted one side into the reactor and the other side into a beaker containing distilled water. This side is also used as the outlet when the column gets and filled completely and the overflow product has to be collected. A mesh cloth was inserted at the base of the column so that the immobilized particles will not fall down to stop the flow of feed. Initially for few centimetres the column was packed with glass beads which give good support to the above Ca-Alginate beads. Later the column was filled with Ca-Alginate beads immobilized with yeast to about 65% of reactor volume. The average void volume was found out to be 910 ml. The average packing volume was about 1090 ml. The void volume was found by passing distilled water. Feed was fed through the bottom of the reactor. Feed composition was 50 g/l glucose with 25 g/l Hi Veg Nutrient Broth. 5hrs of retention time was given for the feed to pass through the reactor. Feed was pumped using a peristaltic pump through the bottom of the reactor. The reactor was under anaerobic conditions. The outlet was the overflow on top taken out at the top.

Fig 3.7.1: Experimental Setup for Immobilized Cell Reactor.

CHAPTER 4: RESULTS
4.1 Introduction: The results and analysis chapter is divided into 5 different sections. In the first section, the growth curve of the bakers yeast is discussed in which the specific growth rates at different time intervals are determined. The growth curve shows the bakers yeast activity through which the experimental planning can be optimized. In the second section the standard curve for glucose assay is discussed where known concentration of glucose are reacted with DNS reagent and their Optical Densities (OD) are found. Unknown concentration of glucose can be found using this standard plot. The third section consists of the batch reactor results. Here the glucose concentrations determined at different time intervals and the ethanol concentration determined at the end of the experiment was discussed. In the fourth section the immobilized cell reactors (ICR) results are discussed. A comparative study of batch vs. ICR was also shown. Finally in the fifth section one sample calculation for finding out the ethanol concentration was shown.

4.2 Growth Curve of Baker's Yeast: The growth curve of bakers yeast gives an idea on the activity of the microorganism through which the experimental planning can be made. First the inoculum is prepared and agitated for 24hours, then the fermentation media is prepared and the pH is adjusted to about 4.5 using sulphuric acid or Ammonium hydroxide. The reaction is carried out in the reactor and at regular intervals samples are collected from the reactor into a pre weighed centrifuge vial. Then the sample was centrifuged in an eppendorf centrifuge. The liquid layer is discarded and the vial is dried in an oven at 105 0Cand its weight is determined. The difference in weight gives us the dry weight of the cell. By referring to figure 4.2.1, the growth pattern of bakers yeast under anaerobic condition follows exactly the typical growth curve for batch cell cultivation. The lag phase of the culture is negligible due to high quantity of inoculum (4%) which was added at the beginning of the experiment. The exponential phase was about till 3 days. Later it started decelerating and the growth was close to stationary phase. After 4 days the death phase can be clearly observed, the reduction in yeast cell concentration. The below table 4.2.1 specifies the growth rate at different interval of time.

Specific Time Sample No 1 2 3 4 5 6 7 8 9 10 (hrs) 0 24 36 48 60 72 84 96 108 120 Time (days) 0 1 1.5 2 2.5 3 3.5 4 4.5 5 X (g/L) 0 0.67 3.35 6.73 10.92 12.73 14.18 15.53 14.27 12.53 1/X 1.492537 0.298507 0.148588 0.091575 0.078555 0.070522 0.064392 0.070077 0.079808 dx 0 2.68 6.06 10.25 12.06 13.51 14.86 13.6 11.86 dt 1 1.5 2 2.5 3 3.5 4 4.5 5 dx/dt 0 1.786667 3.03 4.1 4.02 3.86 3.715 3.022222 2.372 Growth Rate (day-1) 0 0.533333333 0.450222883 0.375457875 0.315789474 0.272214386 0.239214424 0.211788523 0.189305666

Table 4.2.1: Yeast cells Growth rate.

18 16 14 Dry Cell Weight X (g/l) 12 10 8 6 4 2 0 0 1 2 3 Time (Days) 4 5 6

Fig 4.2.1 Dry Cell weight X (g/l) vs. Time (days)

4.3 Standard Plot for Glucose Assay: The standard plot is the most important tool for the glucose assay. Using this plot the unknown glucose concentration corresponding to the sample OD value can be found. The procedure for standard plot has been explained in the methodology chapter. It is a must for the DNS solution to be stored in a dark container and kept out of sunlight. The plot has been drawn to best fit. The below table 4.3.1 shows the variation of glucose concentration with OD that were used for the standard plot. The maximum concentration was 1mg/ml. The best fit equation for the above curve is y=0.91x.

Glucose Concentration (mg/ml) 0 0.2 0.4 0.6 0.8 1

OD 0 0.192 0.4 0.522 0.771 0.92

Table 4.3.1 Table for Standard Plot for Glucose Assay

1 0.9 0.8 0.7 0.6 OD 0.5 0.4 0.3 0.2 0.1 0 0 0.2 0.4 0.6 Glucose Concentration (g/L) 0.8 1 1.2

Figure 4.3.1 Standard Plot for Glucose Assay

4.4 Batch Reactor Result: Glucose Concentration: The initial glucose concentration was 100g/L. The pH of the fermentation medium was adjusted to 4.5 using sulphuric acid or ammonium hydroxide. The reaction is carried out at room temperature. The inoculum size is 4% of batch reactor volume. The components of the batch reactor were agitated using a magnetic stirrer for a proper mixing.
At regular intervals samples are collected in test tubes covered with cotton. Test tubes are stored at 4oC for further analysis.

The below table 4.4.1 and figure 4.4.1 gives the summary of the glucose concentration of the batch reactor at different time interval. It is clearly evident that initially the consumption of glucose was relatively less. After about 1 day, the consumption of glucose started increasing substantially which corresponds to the growth curve as well. After 3 days the consumption of glucose decelerated. After 5 days, the glucose concentration fell to 30.6 g/L. Glucose conversion was 69.4%.The glucose concentration was analyzed using the DNS method. All the samples were diluted by a factor of ten for the assay. Later the concentration was multiplied by the dilution factor. The samples were treated with DNS and kept in a water bath at 90oC for 15 minutes. It is then cooled to room temperature and its OD value is measured using a spectrophotometer at 575nm.

Time Sample No 1 2 3 4 5 6 7 8 9 10 (hrs) 0 24 36 48 60 72 84 96 108 120

Time (days) 0 1 1.5 2 2.5 3 3.5 4 4.5 5 Glucose Concentration (g/L) 100 96 83 74.3 58.7 44.4 40.8 37.5 35.1 30.6

Table 4.4.1 Batch reactor- Glucose Concentration (g/L) vs. Time (days)

120 100 80 60 40 20 0 0 1 2 3 Time (days) 4 5 6

Glucose Concentration (g/L)

Figure 4.4.1 Batch Reactor: Plot of Glucose concentration vs. Time

Ethanol Concentration- For the verification of ethanol present the 100 ml sample was taken at end of fermentation and was centrifuged in an eppendorf centrifuge. The clear liquid was then distilled, and 10 ml of distillate was collected. This distillate was poured on the floor and lit with a match stick and it caught fire immediately. This proves the presence of ethanol. After 5 days the sample was tested for ethanol concentration using the wet method mentioned earlier. The concentration was found to be 7.7% (v/v). Pramanik K. (2003) achieved a concentration of about 8.6% for a 500 ml batch reactor after 5 days. The difference could be due to the difference media composition and the operating conditions.

4.5Immobilization: In order to find the most effective immobilization concentration of Na-Alginate and CaCl2 concentration, various concentrations of broth were tried. At around 4% Na-Alginate and 2% CaCl2 maximum sugar reduction was observed. Change in Na-Alginate concentrations had considerable effects on sugar conversion whereas CaCl2 did not have much considerable effect on sugar conversion. The experiment was carried out under batch conditions, to test the beads; instead of yeast inoculum the immobilized beads were added. The below table 4.5.1 summarized the results obtained. It is to be noted that at 1% Na-Alginate no beads were formed. At 6% Na-Alginate it was extremely viscous therefore difficult to mix with distilled water, moreover the practical difficultly while making beads. The below figure 4.5.1 and 4.5.2 show the final glucose concentration after 3 days with the different percentages of Na-Alginate and CaCl2 used. It is evident that change in Na-Alginate concentrations had considerable effects on sugar conversion whereas CaCl2 did not have much effect. At lower concentrations of Na-Alginate the beads were soft and at higher level of NaAlginate the beads were hard.

Avg Bead %Na-Alaginate %CaCl2 Dia Sugar Concentration at end Of 3days (g/L)

1 2 3 4 5 6

2 2 2 2 2 2 %Na-

None 1.7mm 2.5mm 2.5mm 2.7mm 3mm

None 46 42 36 45 50

%CaCl2 1 2 3 4

Alaginate 4 4 4 4 2.5mm 2.5mm 2.5mm 2.5mm

Sugar Concentration At End Of 3days (g/L) 36 34 36 38

Table 4.5.1 Final Sugar Concentration after 3 days with respect to different % Na-Alginate and % CaCl2

60 Final Glucose Concentration (g/L) 50 40 30 20 10 0 0 1 2 3 4 5 6 7 % Na-Alginate

Fig 4.5.1 Final Glucose Concentration vs. %Na-Alginate after 3 days

60 50 40 30 20 10 0 0 0.5 1 1.5 2 % CaCl2 2.5 3 3.5 4 4.5

Final Glucose Concentration (g/L)

Fig 4.5.2 Final Glucose Concentration vs. % CaCl2after 3 days

4.6 Immobilized Cell Reactor: The below table 4.6.1 summarizes the initial reactor conditions. The feed medium was just glucose and HiVeg Nutrient broth dissolved in distilled water.

Reactor Volume Void Volume Packing Volume Retention Time Initial Glucose Concentration

2000ml 910 ml 1190 ml 5hrs 53 g/l

Table 4.6.1 Initial ICR conditions

After 5hrs retention time the overflow through the top was collected and glucose and ethanol assay were carried out. The glucose concentration fell to about 25 g/L which means the glucose conversion was about 52.8%. The results clearly show the advantage of an immobilized cell reactor over a batch reactor. The below table 4.6.2 summarizes the final concentration obtained in the reactor after 5hrs of retention time.

Final Glucose Concentration Final Ethanol Concentration Glucose Conversion

25 g/l 12.2% (v/v) 52.80%

Table 4.6.2 Final ICR Conditions

The ethanol concentration at the end was around 12.2% after 5hrs which way more than what was achieved in the batch reactor after 5days which was around 8%. The results clearly show that the immobilized bed reactor is much better that that a batch reactor due to both time and concentration at exit. Higher conversion and ethanol concentration can be achieved by higher initial sugar concentration and higher retention time. Also the packing volume can be increased to around 70-75 % for higher conversion. Some modification could be made in the reactor which would increase the efficiency of the reactor. Recycling can be incorporated, and also for better spatial analysis samples should be taken from different points of the reactor. For this the reactor must be improvised with some changes.

4.7 Sample Calculation: Below a sample calculation for ethanol concentration is shown: Calculation of ethanol concentration of the ICR is explained in detail below. After procedure followed as given in methodology chapter (3.3) the calculations are done as follows. Blank Titer Value = 20.6 ml Sample Titer Value = 7.3 ml Therefore, Volume of Sodium thiosulphate used for alcohol present = 13.3 ml Concentration of Sodium thiosulphate = 0.03 mol/L = 3x10-5 mol/ml Therefore, Moles of Sodium thiosulphate = 13.3 x 3x10-5 = 3.99x10-4 mol 1 mol Sodium thiosulphate 0.25 mol Ethanol

Therefore, Mol of ethanol present = 3.99x10-4 x 0.25 = 9.975x10-5 mol Multiplying by dilution factor, Actual amount of ethanol present = 9.975x10-5x21 = 2.095x10-3 The sample volume taken is 1ml. Therefore concentration of ethanol present in sample = 2.095x10-3mol/ml In terms of mass of ethanol present in sample = 2.095x10-3x46 (M.Wt. of ethanol = 46)

= 0.0963 g/ml In terms of volume of ethanol present in sample = 0.0963/0.789 (Density of ethanol = 0.789 g/ml) = 0.122 ml/ml In terms of volume % Ethanol = 12.2 %

CHAPTER 5 CONCLUSION
5.1 Discussion: It was very much evident that, an immobilized cell reactor packed with Yeast immobilized Ca-Alginate beads produce higher ethanol concentration at a much higher rate compared to a batch reactor.

5.2 Future Work: For the batch reactor, we can try and optimize all the operating conditions. The pH and temperature keeps changing as the reactions proceeds. A good method to control pH and temperature can be implemented. Also the fermentation medium composition can be varied and tested for higher ethanol yield For the immobilized cell reactor some modification could be made in the reactor which would increase the efficiency of the reactor. Recycling can be incorporated, and also for better spatial analysis samples should be taken from different points of the reactor. For this the reactor must be improvised with some changes. In terms of other reactors that could be used, as per literature survey, fluidized bed reactor will produce higher ethanol concentration. A fluidized bed can be fabricated and tested. If the CaAlginate immobilized beads are heavy therefore making the fluidization hard we can go for alternative immobilizing technique.

1. Bodalo A. , J. L. Gbmez, E. Gbmez, J. Bastida, and M. F. MBximo, Fluidized bed reactors operating with immobilized enzyme systems (1995) Enzyme and Microbial Technology 17:915-922 2. Carlos A. Cardona, O scar J. Sanchez , Fuel ethanol production: Process design trends and integration opportunities (2007) Bioresource Technology 98, 24152457 3. DI FELICE R., A relationship for the wall effect on the settling velocity of a sphere at any flow regime. (1996) Int. J. Multiphase Flow Vol. 22, No. 3, pp. 527-533 4. Georgi Kostov, Mihail Angelov, Ivan Mihaylov, Denis Poncelet, Mechanical properties of Ca-alginate beads for ethanol fermentation with immobilized yeast ( 2010) Revue de gnie industriel, 5, 25-35 5. Ghasem Najafpour , Habibollah Younesi, Ku Syahidah Ku Ismail, Ethanol fermentation in an immobilized cell reactor using Saccharomyces cerevisiae (2003) Bioresource Technology 92 (2004) 251260 6. Graeme M. Walker, Bioethanol: Science and technology of fuel alcohol (2010) Ventus Publishing ApS ISBN 9788776816810 7. Haiyam Mohammed A. Al-Raheem, Ethanol Bio production in Three-Phase Fluidized Bioreactors (2009) Eng. & Tech .Journal, Vol. 27, No.12 8. JAMUNA R. and S. V. RAMAKRISHNA, High concentration ethanol production using immobilized yeast cells (1992) Biomass and Bioenergy Vol. 3, No. 2, pp. 117-119 9. Kourkoutas Y., A. Bekatorou, I.M. Banat, R. Marchant, A.A. Koutinas, Immobilization technologies and support materials suitable in alcohol beverages production : a review (2003) Food Microbiology 21 (2004) 377397 10. Krishnan M.S , F. Taylor , B.H. Davison , N.P. Nghiem, Economic analysis of fuel ethanol production from corn starch using fluidized-bed bioreactors (2000) Bioresource Technology 7, 99105 11. Lee T.H., J. C. Ahn and Dewey D. Y. Ryu, Performance of an immobilized yeast reactor system for ethanol production (1982) Enzyme Microb. Technol., 1983, Vol. 5 12. Maziar Safaei Asli, A study on some efficient parameters in batch fermentation of ethanol using Saccharomyces cerevisiae SC1 extracted from fermented siahe sardasht pomace (2009) African Journal of Biotechnology Vol. 9 (20), pp. 2906-2912 13. Michael L. Schluer and Fiker Kargi, Bioprocess engineering basic concepts (2009) Second Edition PHI learning Pvt. Ltd. ISBN 9788120321106 14. MICHIMASA KISHIMOTO, J YUKO NITTA, YUYA KAMOSHITA, TAKAHIRO SUZUKI, AND KEN-ICHI SUGA, Ethanol Production in an Immobilized Cell Reactor Coupled with the Recycling of Effluent from the Bottom of a Distillation Column (1997) JOURNALO F FERMENTATIOANN D BIOENCINEERING Vol. 84, No. 5, 449-454. 15. Mustafa Balat, Havva Balat, Recent trends in global production and utilization of bioethanol fuel (2009) Applied Energy 86, 22732282 16. Pilkington P.H., A. Margaritis, N. A. Mensour, and I. Russell, FUNDAMENTALS OF IMMOBILISED YEAST CELLS FOR CONTINUOUS BEER FERMENTATION: A REVIEW (1997) J. Insi. Brew., January-February 1998, Vol. 104, pp. 19-31 17. Pramanik k., Parametric Studies on Batch Alcohol Fermentation Using Saccharomyces Yeast Extracted from Toddy (2003) J. Chin. Inst. Chem. Engrs., Vol. 34, No. 4, 487-492

18. Ranulfo Monte Alegre, Maurcio Rigo and Ins Joekes, Ethanol Fermentation of a Diluted Molasses Medium by Saccharomyces cerevisiae Immobilized on Chrysotile (2003) Brazilian Archives of Biology and Technology Vol.46, n. 4 : pp. 751-757 19. Tri Widjaja, Soeprijanto, Ali Altway, Setiyo Gunawan, R. Darmawan, ETHANOL PRODUCTION FROM MOLASSES USING IMMOBILIZED CELLS CA-ALGINATE AND K-CARRAGEENAN BY MUTATION ZYMOMONAS MOBILIS IN A PACKED BED BIOREACTOR (2010) INTERNATIONAL JOURNAL Of ACADEMIC RESEARCH Vol. 2. No. 6 20. Sinsupha Chuichulcherm, Design and start-up of a fluidized bed bioreactor for ethanol fermentation (2003) Department of Chemical Engineering, Srinakharinwirot University, Thailand 21. Tyagi R.D., S. K. Guptab & Subhash Chand, Process Engineering Studies on Continuous Ethanol Production by Immobilized S. cerevisiae (1992) Process Biochemistry 23-32 22. Yekta G.KSUNGUR, Nebe ZORLU, Production of Ethanol from Beet Molasses by Ca-Alginate Immobilized Yeast Cells in a Packed-Bed Bioreactor (2000) Turk J Biol 265-275.