Application of TRIZ Methodology in Simultaneous Fermentation/Purification

Thesis Proposal

Application for research grant: 5. Details of the Research, 6. Work Plan and Time Table, 7. Financial Requirements

5. Details of the Research

A. SIGNIFICANCE OF THE STUDY
The world's fossil fuel resources are now at its peak and will eventually be exhausted. This is one of the reasons why countries are looking for renewable energy resources. Energy security and stability of the countrys economy are also reasons why the government is searching for renewable energy resources. Another reason is the climate change issue which encourages all countries to find ways on reducing the emissions carbon dioxide and other pollutants. Majority of the emissions come from the transport sector which utilizes petroleum-based fuels. (Raola, 2007) OPEC is controlling the prices of petroleum products since most of the countries are depending on them. From the historical data on oil prices, it can be observed that oil prices have been very volatile. Oil price increases from US$2.00 per barrel from 1970 to US$59.00 per barrel in 2005. (Raola, 2007) Because of the climate change issue, increasing oil prices and exhaustion of fossil fuel resources, researches on science and technology lean on the developments on renewable energy sources and its utilization. One of the renewable energy sources that are being tapped by most of the countries is biofuel since transport sector has the largest contribution in pollutants emitted in the atmosphere. Bioethanol and biodiesel are now used as substitute or as an additive or blend of diesel and gasoline. Anhydrous ethanol is used for gasoline fueled vehicles which is required to have 99.3% purity or to have a maximum moisture content of 0.5% by volume as stated in the Philippine National Standard. (DOE) Bioethanol is considered as an alternative energy because it can be blended with gasoline up to 10% without changing the design of the engine. It can also fully replace the gasoline as fuel after altering the engine. Climate change issues are addressed since significant reduction of GHG emissions can be observed in the utilization of biofuels. In addition, it is considered renewable since the feedstock used for the production of the fuel is agricultural wastes. (Raola, 2007)The only problem in utilizing biomass as feedstock is the energy intensive processes that are used in ethanol production. This study offers a less

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energy intensive fermentation and purification processes of rice straw to ethanol conversion.

B. BACKGROUND OF THE STUDY

Ethanol derived from lignocellulosic biomass is of emerging interest today. (Souto-Maiora, Runquist, & Hahn-Hgerdal, 2009)Different scientific publications introduced different technologies for derivation of biomass to ethanol. In fermentation of hydrolyzate and purification to produce ethanol, several problems arise: i. INHIBITORY SUBSTANCES Different substances that can be harmful to organisms may be present after the feedstock pre-treatment and hydrolysis. Hemicellulosic components of biomass contain different organic acids. Dehydration of pentoses and hexoses from hemicellulose can produce 2furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (5-hydroxymethylfurfural; HMF) which could inhibit yeast growth and fermentation. (Liu & Blaschek, 2010)Aside from the said component, ethanol can also affect the growth and activity of organisms, particularly yeasts. (Stanley & Hahn-Hagerdal, 2010) Saccharomices cerevisiae exhibits diauxic behavior in aerobic batch culture. Fermentation of glucose to ethanol occurs in the first exponential growth phase while the consumption of ethanol to produce more cells occurs in the second exponential growth phase. (Jones & Kompala, 1999)Because of the diauxic behavior of S. cerevisiae, ethanol must be removed from the reactor before the concentration of glucose drops that will be a signal to the yeast to consume ethanol. Simultaneous fermentation and ethanol separation can solve the problem of ethanol inhibition. ii. LIMITATIONS OF SACCHAROMYCES CEREVISIAE S. cerevisiae cannot utilize pentoses for ethanol formation since xylose is not recognized as a rapidly fermentable carbon source for S. cerevisiae. (Souto-Maiora, Runquist, & HahnHgerdal, 2009) However, attempts have been done to introduce a functional xylose pathway to the yeast strain. (Stanley & Hahn-Hagerdal, 2010)Pentoses should be utilized to increase ethanol yield and to avoid problems in waste water treatment. (Inui, Vertes, & Yukawa, 2010) Thesis proposal for research and thesis grant Page 3

iii.

ENERGY INTENSIVE ETHANOL SEPARATION PROCESS Different ethanol separation processes have been introduced and some are already utilized in ethanol industry. Among the separation processes are: 1. Ordinary Distillation 2. Azeotropic Distillation 3. Extractive Distillation 4. Liquid-liquid Extraction-Fermentation Hybrid (Extractive Fermentation) 5. Adsorption 6. Membrane separation However, most of these processes use significant amount of energy. In ethanol production, least energy intensive process should be utilized. Among these processes, membrane separation, particularly, pervaporation offers a solution to energy intensive separation processes.

C. STATEMENT OF THE RESEARCH PROBLEM

Based on the Biofuel Act of 2006 (RA9367) which mandates the blending of 2% biodiesel to diesel and 5-10% of bioethanol to gasoline from 2007 to 2015, the country has to produce approximately 559 million liters of bioethanol by 2015. The ethanol demand must be met at the same time it has to be produced efficiently. This study proposes a simultaneous fermentation and purification technology with relatively lower energy requirement. Simultaneous fermentation and purification offers a solution to product inhibition in the fermentation process.

D. OBJECTIVES
General Objectives 1. To develop a simultaneous ethanol fermentation and purification process that employs a solar-assisted pervaporation system. 2. To apply TRIZ methodology in providing a solution to energy intensive fermentation and ethanol separation processes. Thesis proposal for research and thesis grant Page 4

Specific Objectives 1. To develop a kinetic model for rice hydrolyzate fermentation using Saccharomyces cerevisiae. 2. To determine the effects of feed rate, yeast concentration and fermentation temperature in the ethanol yield. 3. To determine the effect of feed temperature of the pervaporation system in the ethanol yield. 4. (ENERGY EFFICIENCY) To determine the energy efficiency of the fermentation and pervaporation system.

E. DISCUSSION OF LITERATURE RELATED TO THE FIELD

FERMENTATION OF RICE STRAW HYDROLYZATE In the bioethanol process previously discussed, only the last two steps which are: fermentation and ethanol separation will be covered in this study. In the fermentation of the hydrolyzate produced in the enzymatic hydrolysis, different species of bacteria and fungi are being utilized. (Stanley & Hahn-Hagerdal, 2010) Inhibitory Substances Different substances that can be harmful to organisms may be present after the feedstock pre-treatment and hydrolysis. Hemicellulosic components of biomass contain different organic acids. Dehydration of pentoses and hexoses from hemicellulose can produce 2furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (5-hydroxymethylfurfural; HMF) which could inhibit yeast growth and fermentation. (Liu and Blaschek, 2010) Aside from the said component, ethanol can also affect the growth and activity of organisms, particularly yeasts. (Stanley & Hahn-Hagerdal, 2010) Saccharomices cerevisiae exhibits diauxic behavior in aerobic batch culture. Fermentation of glucose to ethanol occurs in the first exponential growth phase while the consumption of ethanol to produce more cells occurs in the second exponential growth phase. (Jones & Kompala, 1999)Because of the diauxic behavior of S. cerevisiae, ethanol must be removed from the reactor before the concentration of glucose drops that will be a signal to the yeast to consume ethanol.

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Simultaneous fermentation and ethanol separation can solve the problem of ethanol inhibition. Microorganisms Producing Ethanol In the fermentation of the hydrolyzate produced in the enzymatic hydrolysis, different species of bacteria, microorganisms, yeast and fungi are being utilized.(Stanley & HahnHagerdal, 2010; Lin & Tanaka, 2005) Famous for ethanol fermentation are yeasts, particularly Saccharomyces cerevisiae which can convert up to 18% of the fermentation broth to ethanol. (Lin & Tanaka, 2006) A mini-review of "Ethanol fermentation from biomass resources" by Yan Lin and Shuzo Tanaka (2006) presented the following tables of microorganisms which can produce high ethanol yield.

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Advantages and Disadvantages of Saccharomyces cerevisiae Yeast especially S. cerevisiae (bakers yeast) is commonly utilized to ferment lignocellulose hydrolyzate because it is readily available and can grow on simple sugars like sucrose and on disaccharide sucrose. It is also used in manufacturing alcoholic beverages and for leavening bread and is generally recognized as safe (GRAS). (Lin & Tanaka, 2006) Saccharomyces cerevisiae can assimilate furfural which is known as a growth inhibitor for other microorganisms. (Himmel & McMillan, ) It is one of the most stress-tolerant Thesis proposal for research and thesis grant Page 8

microorganisms thus possessing an important characteristic for industrial application. (Stanley & Hahn-Hagerdal, 2010) On the other hand, S. cerevisiae cannot utilize pentoses for ethanol formation. However, attempts have been done to introduce a functional xylose pathway to the yeast strain. (Stanley & Hahn-Hagerdal, 2010) Pentoses should be utilized to increase ethanol yield and to avoid problems in waste water treatment. (Inui, Vertes, & Yukawa, 2010) Industrial fermentation processes are categorized as batch, fed-batch, continuous operation and immobilized cell systems. In determination of which fermentation process should be used, properties of raw materials, technological complexity and productivity should be considered. The most commonly used process in large-scale ethanol production today is the fed-batch process. (Inui, Vertes, & Yukawa, 2010) In batch fermentation processes, the substrate and cells which are separately grown are fed into the reactor together with the nutrients and additional enzymes needed by the cells. The concentrations of substrates, cells and products changed with time. At the end of cultivation, the fermented material is pumped to a distillation column where separation of ethanol takes place. This process has been used by the beverage industries for centuries. On the other hand, fed-batch process is considered as a combination of batch and continuous fermentation processes. The start-up is relatively similar to the batch process where substrates, cells, and nutrients are being fed to the reactor. The introduction of necessary nutrients, cells and substrates can be done at the start or during the middle of cultivation period. Effluent is removed at regular intervals while maintaining the substrate concentration by substrate feeding. In continuous processes, the substrates, nutrients and enzymes needed is pumped continuously to the reactor equipped with constant agitation. The fermentation cells are already present and active inside the reactor at the start of the process. High cell growth rate is maintained through constant aeration of the reactor. The fermented material is collected at the top of the reactor while maintaining the composition of the solution in the bioreactor. Lastly, immobilization processes are done by restricting the cell mobility within a defined space. Generally, four types of immobilization technique are recognized based on physical mechanism of cell localization and nature of support mechanisms (Inui, Vertes, & Yukawa, 2010):

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i. ii.

Surface attachment - cells are allowed to attach to a solid support using a wide variety of various carrier materials. Entrapment - various matrices used for active cell immobilization includes porous polymers, porous metal screens, polyurethane, silica-gel, polystyrene, and cellulose triacetate. The matrix around the cells should be porous to allow transport of substrates and products in and out of the bead.

iii. iv.

Containment behind a barrier - achieved either by the use of microporous membrane filters, or by entrapment of cell in microcapsules. Self-aggregation - referred to as 'flocculation.' Cells adhere to form flocks consisting of thousand of cells.

SEPARATION OF ETHANOL In separation of ethanol, there are several processes or techniques that can be implemented: 1. Ordinary Distillation 2. Azeotropic Distillation 3. Extractive Distillation 4. Liquid-liquid Extraction-Fermentation Hybrid (Extractive Fermentation) 5. Adsorption 6. Membrane separation This study will utilize the ethanol separation process which operates with low energy requirement which is membrane separation, particularly pervaporation. Membrane separation or membrane pervaporation is one of the most energy efficient or low energy separation processes of azeotropic mixtures (Huang, 2008) and will be the focus of this study. Membrane separation processes being used in ethanol recovery today are ultrafiltration, nanofiltration, or pervaporation. Microfiltration, microfiltration,

ultrafiltration and nanofiltration are a pressure-driven and membrane-based separation process which only differs in membrane pore sizes. The first one retains matter as small as 0.02 m but larger than 0.1 m. Ultrafiltration, on the other hand, separate components based on the molecular weight cut-off size and can retain components with molecular

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weight greater than 500 Da. Nanofiltration can separate components with molecular weights ranging from 200 to 1000 Da. (Ezeji & Li, 2010) This study will apply a solar-assisted pervaporation on simultaneous fermentation and purification of ethanol. Solar energy will be needed to pre-heat the feed to the pervaporation system, thus utilizing a free energy. Pre-heating will help to increase the permeation flux thus increasing the efficiency of the pervaporation module. However, increasing feed temperature may decrease the separation factor of ethanol and water. (Jiraratananon, Chanachai, Huangb, & Uttapap, 2002)

F. THEORETICAL FRAMEWORK
This study will use the continuous fermentation technology coupled with pervaporation. The simultaneous fermentation and purification process solves the problem of ethanol inhibition in the fermentation process. Saccharomyces cerevisiae will be used to ferment the rice straw hydrolyzate because of several advantages previously discussed in the literature review. This yeast exhibits two growth regimes in continuous cultures. The first exponential growth phase is the utilization of glucose by the fermenting microorganism. Under aerobic conditions and low dilution rate, glucose oxidation prevail producing biomass and carbon dioxide. At high dilution rate, glucose oxidation is inhibited and fermentation of glucose producing ethanol and carbon dioxide occurs. This phenomenon is known as the Crabtree effect. (Jones & Kompala, 1999) (Lei, Rotbll, & Jrgensen, 2001) Second exponential growth phase occurs when the glucose concentration dropped significantly. This time, the microorganisms will consume ethanol to produce more cells. Determination of critical dilution rates is needed to ensure that glucose oxidation is inhibited and ethanol production is maximized. Ethanol should be removed from the fermentation system as soon as it is formed to ensure that the microorganisms will not consume it.

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Figure 2. Alternative routes of pyruvate metabolism in S. cerevisiae. Numbers refer to the enzyme, which catalyses the corresponding reaction. (1) Pyruvate dehydrogenase complex (Pdh). (2) Pyruvate decarboxylase (Pdc). (3) Acetaldehyde dehydrogenase (Acdh). (4) Acetyl-CoA synthetase (Acs). (5) Alcohol dehydrogenase (Adh). The anaplerotic pathway from pyruvate to oxaloacetate is not included in the figure.

Figure 2. The metabolic and biomass structured model. The model reactions can be divided into catabolic and anabolic reactions as indicated by the two circles. Glucose and acetate participate in both catabolic and anabolic reactions

The figures above are presented in a published journal: A biochemically structured model for Saccharomyces cerevisiae by Frede Lei, Morten Rotbll, Sten Bay Jrgensen (2001) which shows the metabolic pathways of S. cerevisiae. In figure 2, ethanol is shown to be consumed by reaction r6 to form acetaldehyde producing more carbon dioxide. To prevent

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this ethanol consumption, ethanol must be removed from the fermentation system simultaneously using pervaporation. There are six phases of growth and death of a unicellular organism: 1. Lag phase 2. Accelerated growth phase 3. Exponential growth phase 4. Decelerated growth phase 5. Stationary phase 6. Death phase The lag phase is when the organisms adapt to its environment; in accelerated growth phase is where the cell number starts to grow and the division rate increases to reach a maximum; exponential growth phase is when the cell number increases exponentially as the cells start to divide; decelerated growth phase occurs when growth rate reaches its maximum and finally decelerate; and lastly death phase occurs when the cells start to die due to lack of nutrients. (Lee, 1992) In the exponential growth rate, the rate of cell population is proportional to the number density (Cn) of bacteria present at that time:

where is known as the specific growth rate (h-1). There are different factors affecting the specific growth rate: 1. Substrate Concentration the effect of substrate concentration is given by the Monod equation which shows that a further increase in the nutrient concentration after reaches max does not affect the specific growth rate.

where Cs is the concentration of limiting substrate in the medium and Ks is a system coefficient. Ks is equal to the concentration of nutrient when the specific growth rate is half of its maximum value. Maintenance of cells can be added to the equation (ke).

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2. Product Concentration the growth of the microorganisms is usually inhibited by the products. Their effect can be added to Monod Equation:

Where Cp is the concentration of the product and Kp is another system coefficient. The specific growth rate may also be affected by temperature, pH and oxygen supply. (Lee, 1992) Pervaporation Pervaporation is based on the diffusion of the components. The change in chemical potential between the permeate side of the membrane and the feed serves as the driving force for the diffusion mechanism. The partial pressure of the permeate side is lowered through the use of a vacuum pump. Membranes that are used in pervaporation may be hydrophobic or hydrophilic. (Huang, 2008) Hydrophilic membranes are used to remove water in organic solutions, often from azeotropes while hydrophobic membranes are used to remove organic compounds from water and when solvent concentrations is 6% or less. (Matsuura, 1994) Hydrophilic membranes are usually used due to lower molecular size of water relative to ethanol. (Huang, 2008) The mass transport of a component in pervaporation involves three steps: 1. absorption of the desired components from the liquid phase at the membrane-feed solution interface; 2. diffusion of the absorbed components through the polymer matrix to the vapor membrane interface; 3. desorption of the components into the vapor phase (Scott & Hughes, 2006)

Among the factors that should be considered in choosing a membrane is the permeation rate which is determined by getting the flow rate of liquid permeate. Permeation rate is expressed as flux, (L/m2-hr). Another consideration is selectivity which is the measure of the preferential permeation of a component in a binary mixture in terms of the separation factor, and expressed as Thesis proposal for research and thesis grant Page 14

Where Y is the weight fraction of selected component in the permeate X is the weight fraction of selected component in the feed solution. The higher the separation factor, the better the performance of the membrane. (Green, 1999) In this study, ethanol-water mixture will be separated. The membrane that should be used should be hydrophobic and ethanol selective since the desired product is ethanol. Feed temperature and flux affected flux and selectivity in pervaporation following the Arrhenius equation:

where Ji is the permeation flux of i (g/m2 s), Ai the pre-exponential parameter (g/m2 s), R the gas constant, T the absolute temperature (K) and Ea is the activation energy for permeation (kJ/mol). The Arrhenius relationship shows that increasing the temperature will increase the permeation flux in the pervaporation system. The schematic diagram of the pervaporation process is shown in the figure below:
feed

liquid membrane vapor

Liquid permeate

retentate

condenser permeate

Vacuum pump

Figure 3. Schematic Diagram of Membrane Separation Process

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There are different kinds of membranes that could be used in pervaporation depending on the operating conditions and the desired product. Different studies tested the efficiencies of several membranes that could be used in separation of ethanol-water mixtures (Burshe, Sawant, Joshi, Pangarkar, 1997, Chen et.al., 1995, Dong, Zhang, Sheng, Song, and Chen, 2006, and Ruckenstein and Liang, 1996). Among the membranes that were tested are cellophane, PTFE-PVP (PTFE-N-vinylpyrrolidone), polyvinyl alcohol-polyacrylonitrile (PVA-PAN), cellulose-acetate, cellulose acetate-polyphosphate (CAPPN), polyethylene, polypropylene (Scott and Hughes, 1996) and polydimethyl-siloxane (PDMS) (Wu, Xiao, Huang, & Zhong, 2005). PDMS will be used in this study since it is known to be suitable for separating ethanol at low concentrations with reported ethanolwater separation factor of which ranges from 4.4 to 10.8 (Ezeji & Li, 2010) and known to produce clean aqueous ethanol solution. (Wu, Xiao, Huang, & Zhong, 2005)

G. METHODOLOGY The methodology that will be used in this study is adapted from a scientific journal by Wu, Y., Xiao, Z., Huang, W., & Zhong, Y. (2005) Mass transfer in pervaporation of active
fermentation broth with a composite PDMS membrane Cultivation of Saccharomyces Cerevisiae _______ Preparation of composite membrane PDMS An organophilic polymer with pore size of 0.58 m will be used as a supporting layer in the composite membrane. The substrate will be pre-wetted by a solvent saturating its pores. To form the skin layer, a dilute solution of polydimethylsiloxane (PDMS) pre-polymer, a cross linking agent and solvent was coated on the substrate surface. Then the composite membrane will be treated for 2 hours in a vacuum oven and dried flat-plate composite membrane with a dense skin layer of silicon will be formed. The thickness of the formed skin layer will be determined by means of SEM pictures. The PDMS membrane that will be used will be 128 m with 120 m support layer and 8m dense silicon rubber layer. Experimentation Thesis proposal for research and thesis grant Page 16

The schematic diagram of the set-up that will be used in the experiment is shown in the figure below. The rice straw hydrolyzate will be feed to the fermentation reactor at different rates (______ L/s) and with varying yeast concentration (_____). The fermentation temperature will be also varied to determine the optimum parameters of fermenting rice straw hydrolyzate with Saccharomyces cerevisiae. Continuous fermentation will be utilized in this study. Product gases will be removed from the fermentation reactor while the fermentation broth will be pumped out from the reactor to the solar pre-heater and then to the pervaporation module. Temperature of the feed to the pervaporation module will be varied by adjusting the flow rate of the fermentation broth in the solar pre-heater. Permeate will be fed in the condenser to recover the ethanol in liquid form while the retentate will be recycled back to the reactor.
CO2

Vacuum pump retentate Coolant out Fermentation broth biomass Pump Solar pre-heater permeate Pervaporation module condenser Fermentation Reactor ethanol

Coolant in

Figure 3. Schematic diagram of experimental set-up adapted from Figure 16.2 of Biomass to Biofuels: Strategies for Global Industries (Ezeji & Li, 2010)with added solar pre-heater.

Data Analysis Ethanol yield will be optimized using the Box-Behnken experiment design. By varying different parameters from time to time, the effect of the parameters will be determined. However, their overall effect to the ethanol yield is to be determined to maximize ethanol production. Analysis of the obtained data in the experiment will be done using Minitab 15.

6. Work Plan and Time Table

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Activity Membrane and yeast sourcing Yeast cultivation Experimental set-up preparation design fabrication Experiment data analysis thesis writing defense

August September October November December January February March 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

7. Financial Requirements

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