Corn flour with water is saccharificated and liquefied by high pressure jet, and mashing is pumped into plate and frame and then filtered. Residue are used as feed and clean sugar are pulled into the fermenter. By adjusting the concentration of sugar, the fermentation period is about 55~75h, and the conversion rate is not less than 90%().
That lactic acid pathway of glucose metabolism including the way in anaerobic and aerobic conditions. Glucose as raw material technologies is used to produce racemic DL-lactic acid, 0.375% malt,0.25% (NH4)2HPO4 , 74.375% H2O, 10% CaCO3 as buffer, adjusting pH5.8~6, under 49oC, and the period of fermentation is, the concentration of DL lactic acid and the conversion rate is 4~6h, 120~135g/L and 80%~90%().
Figure1.2.2 demonstrates the detail process of lactic acid fermentation. Usually the substrate is glucose in industry (). The process of formation of lactic acid is very complexes. During the process, it will release ADP and some side products.
Lactic acid fermentation of glucose metabolism based on a variety of different ways, lactic acid bacteria can be divided into homo lactic fermentation and heterolactic fermentation.
Figure 1.2.2 process of produce of lactic form glucose.
In recent years, whey is regard as a low cost way to ferment lactic acid(). Among many materials used to produce lactic acid, whey (lactose) has special consideration. Whey is a very clean, wholesome, abundant food-grade material and it is more environmental friendly than the chemical synthesis method. Whey commonly comes from the superstation from milk. The process of lactic acid fermented from whey is as follow:
In general, the main compound of whey is lactose, and protein. However, lactose can only be utilized by a some particular cell which let the lactic acid process need more research(). Whey can be liquid or powder state to participate in the reaction. Whey is very suitable using as a medium for fermentations, because of its special composition inside. The composition of whey is important because during the cheese and milk making procedures, their compositions are not constant. Study on whey indicated that lactose is the only fermentable carbohydrate in whey (). The main composition of sweet whey and acid whey is given in table 1.2.3. It is obvious that the total solids are around 63-70 g/l both in sweet whey and acid whey. The content of lactose and protein are around 46-52 g/l. The calcium, phosphate, lactate and chloride content in sweet whey are all similar to acid whey. All in all, the main compounds in sweet and acid whey are almost same.
Table1.2.3 main composition of whey
During the reaction, a major problem is biological instability of substances during utilizing whey and forming lactic acid production (). The total fee associated with transport and storage. These make the process uneconomical to collect these raw materials (7% solids) from faraway cheese manufacturing plants. In addition, due to whey (lactose)`s low solubility in water, lactose may crystallize at transportation temperatures (low temperature condition). For commercial production of lactic acid from whey, thus, the lactic acid plant should be near to a large cheese manufacture. As a result, this way can save energy and cost less which used for transpiration and temperature maintain. In industry, commercial lactic acid cell are kept in suspension by mixing with agitators in CSTR or the bobble form the airlift reactor. Most of the cell required low oxygen concentrations for growth and production.
Lactobacilli are large group of bacteria, which performance in different shape. Lactic acid bacteria are gram-positive, non-sporing and usually no motile and are categorized as facultative anaerobes, thus making the strict exclusion of air is unnecessary. Lactobacillus casei can ferment homo-fermentative to L(+) lactic acid and do not produce NH3 from bioreactor. Its optimum growth temperature is nearly 37 cent degree. Lactobacillus casei can be found in many food commercial products, as well as in the human body and other natural environments. Thus, it is safety to human and can be used as probiotic. Table 1.2.4 shows different raw material and microorganism form different isomer of lactic acid. This means different cell has great effect on the product and the raw material also plays an important role.
Table 1.2.4 different microorganism and raw material form lactic acid isomer.
1.3 bioreactor
The importance of the bioreactor is recorded in early history. The Babylonians apparently made beer before 5000 BC and wine was produced in wineskins. After microorganisms were discovered, scientists increased their research of the biochemical transformations in bioreactors. Simple fermentations for the production of lactic acid, ethyl alcohol, and butanol were developed. Moreover, aerobic and anaerobic treatment of wastewater became more popular in industry ().
The basic principles of bioreactor analysis and design are similar to those for chemical reactors. Some of the processes are very complex. Microorganisms obey the laws of chemical thermodynamics. Thus, the chemical energy available for growth and product formation decreases as a result of microbial assimilation of the reactants. Figure 1.3.1 illustrates the CSTR bioreactor. In general, the input is on the top, and outlet is on the bottom. The stirrer could be one or more which depend on the detail of the reaction.
Figure 1.3.1. Process of CSTR bioreactor
The rate of growth and product formation depends on the number of microorganisms and the concentrations of the nutrients. Figure 1.3.2 shows a typical growth curve of batch cultivation of bacterial cells. This curve indicates 5 phases. Phase 1 is the cells adapt themselves from their previous to the new condition. Phase 2 is a acceleration phase, the cells begin to expand in volume and to divide. Phase 3 is a decrees phase. In phase 4, there are no change in cell mass is observed. After that is phase 5, when microbial cells in the culture almost consume of all basic nutrients, cells begin to die a lot. The rate of cell growth decreases to zero. The figure 1.3.2 is the microbial growth curve. Because the cell will die has live time, fresh the cell is necessary.
Figure 1.3.2. Microbial Growth Curve
The important requirements for the growth of any microbial culture are as follows:
- A viable inoculums
- A carbon and energy source
- Essential nutrient for biomass synthesis
- Suitable physicochemical condition
The rate of increase cell concentration when all the above requirements are provided and if cell death can be ignored.
μ is described as the specific growth rate. When the specific growth rate is maximum. (μ=μmax). The relationship between cell concentration and time will as follow:
Figure 1.3.3 states the impact of temperature at a controlled pH of 6.2 on the different bio-kinetic parameters used in the primary model(). The existent of growth assoc is essential to the product. In the mix type, it gains the lowest conversion rate.
Figure 1.3.3. Kinetic patterns of product formation in batch fermentations
Another important parameter is double time (td) of bioprocess. Doubling time is defined as the time the period of time required for a quantity to double in size or value().
Figure 1.3.4 is a schematic diagram of a chemostat at a steady state. The assumption is that the bulk liquid is well mixed. This means the output concentration are the same as in the bioreactor. In an ideal stirred tank reactor, there is no flow bypass. The addition of a substrate through feeding is distributed throughout the reactor. The gas sparging is employed the agitator provides a mixed G-L phase. The stream exiting will have same composition as in the reactor. In a well mixed bioreactor, there is no concentration gradient even in liquid and gas phase.
Figure 1.3.4 . schematic diagram of continuous chemostat.
Among all bioreactor model, Monod equation is the most useful one. The Monod equation empirically fits a wide range of data satisfactorily and is the most commonly applied unstructured, unsegregated model of microbial growth().
Mathematical models are used to predict different operation condition effects on fermentation on cell growth rate, lactic acid production rate and substrate reaction rate. The components of modeling include the Biomass yield, product yield, kinetic parameter and the equations (concentration as a function of time and Monod equation). Assumptions are also very important component of modeling. A good modeling can help engineers to figure out the factors. There are several methods on lactic acid modeling, such as Adomian decomposition method(Biazar, Tango et al. 2003). Figure 1.3.5 present the concentration of different substance in reactor based on modeling. It can be seen that, different form conventional catalyst, cell or microorganism work as a catalyst. However, the concentration of cell is keep growing during the reaction.
Figure 1.3.5 . The experimental and predicted cell, substrate and lactic acid concentrations during batch fermentation of cheese whey()
1.4 Aim of report, problem identification & justification
The aim of this report is to develop a kinetic model for lactic acid production from whey by Lactobacillus casei. Bio-reactor model will be related to be established and discussed. With in this context, fermentation will model on with several different initial substrate concentrations. For any chemical reaction, the reaction from the simple to become a commercial product is very difficult and long road.
2. Necessary Data
2.1 lactic acid
Lactic acid has a hydroxyl group adjacent to the carboxyl group. Lactic acid is the simplest 2-hydroxyacid having a chiral centre, and exists as two enantiomers, L(+) lactic acid and D(-) lactic acid. The main compound of whey is lactose. The reaction to produce lactic acid is as follow(). Water plays an important role in this reaction. Thus, choose CSTR reactor can give the reaction enough H20 and stirred rate.
Table 2.1. basic chemical properties of lactic acid()
2.2 rector parameter
It was assumed that the liquid and solid phases within the reactor are completely well mixed. Except the solution, the main part of the reaction is whey as substrate, Lactobacillus casei as cell and lactic acid is the product.
Table 2.2. basic reactor parameter
Table 2.3 shows the kinetic parameters for whey fermentation ()
Table 2.3 the basic parameters of the biochemical reactions from literature
Table shows different initial concentration of substrate and yield of cell and substrate.()
Table2.4 yield coefficient for cell on substrate and yield coefficient for product on substrate
3. Modeling
For a microbial reaction, the relationship of the concentration of the reaction substrate, or cell concentration and time, or metabolite concentration and time are expressed in two forms, one is the relationship of the concentration and the rate of change as a function of time; the other is the relationship between concentration and time. The former can be directly used for differential kinetic equation, so the method is also called differential method; the latter is the integral form of kinetic equations, so the method is also called integral method. In this work, we integrate the existing equation in literature to gain the relationship between concentration and time.
The kinetic model in this study was based on three rate equations: biomass growth, substrate utilization and product formation. The rate equations stated in previous section included many variables and parameters. The state variables were the biomass concentration (Cx), the substrate concentration (Cs), and the product concentration (Cp).
Cell growth rate
The cell growth rate is related to speed of cell growth, cell size and the amount of cell. μ is the growthe rate().
μ is specific substrate rate. We assume that product formation is performed by living cell and hence live biomass concentration is used in the definition of the specific product formation rate.
Substrate consumption rate
Substrate utilization kinetics may be expressed as:
Product growth rate
For growth-associated products, they are produced simultaneously with microbial growth.
For non-associated product, formation takes place during the stationary phase when the growth rate is zero. The specific rate of product formation is constant.
Mixed-growth-associated product formation takes place during the slow growth and stationary phases. In this case, the specific rate of product formation is given by the following equation:
Biomass yield was calculated as the ratio of the weight of biomass produced per weight of substrate utilized and showed as:
Product yield was also defined as the weight of product produced per weight of substrate utilized and the equation was:
Monod equation
Monod model is very useful for growth parameters, and it simplify the process of fermentation(). The relationship between the specific growth rate and limiting sbustrate concentration proposed by monod states that:
3.1 Substrate integration formula
According to Michaelis–Menten kinetics, the relationship between substrate concentration and cell concentration is as follow:
Put the equation into Monod equation:
Simplify the equation:
Assuming some parameter:
Thus, By integrating the above formula:
Thus the relationship between cell concentration and time is as follow:
In order to facilitate the calculation, a simplified model:
3.2 Cell integration formula
According to Michaelis–Menten kinetics,
Put this into monod equation:
In order to simplify the model, assuming:
, ,
Put the equation above into forward one:
→ →
Assume: →
(,)
(,)
Thus:
Integration of the equation:
Thus the equation of the relationship between time and product:
In addition:
Simplify the model:
3.3 Product integration formula
First, assume the product concentration is zero.
In order to calculate convenient, using a, b, c instead of some parameter:
In order to calculate the C1:
Thus the plot of time as a function of cp is as follow:
Table3.1 shows the three equation of bioreactor model.
The four equation need under certain condition. 1. The system under well mixed condition. 2. The temperature and pH value are optimal value. 3. No side product affect on the reaction. 4. The substrate requirement to provide energy for maintenance is negligible
Table 3.1 equation of reaction time with concentration.
4. Calculation & result
4.1 mass transfer
4.1.1 Effect of initial concentration
Growth is the most essential response of microorganisms to their physiochemical environment.
In this part, the substrate concentration 9.0 l/g, 48, 60 and 90 will be discussed. Using the data in the reference written by Biazar().
The maximum specific growth rate (μmax) was determined from the graph of specific growth rate versus initial substrate concentration. Figure 4.2.1 shows the special growth rate change with substrate concentration.
4.1.1 The maximum specific growth rate
Table.4.1.2 Shows the the concentration of substrate, cell and product with the initial rate is 9 kg/m3.
Table4.1.2. the the concentration of substrate, cell and product with the initial rate is 9 kg/m3.
4.1.3 the the concentration of substrate, cell and product with the initial rate is 9 kg/m3.
Table 4.2.4 and figure 4.2.5 shows the concentrarion of substrate, cell and product with the initial rate is 48 kg/m3.
Table 4.1.4 the concentration of substrate, cell and product with the initial rate is 48 kg/m3.
Figure 4.1.5 the concentration of substrate, cell and product with the initial rate is 48 kg/m3.
Table 4.1.6 the concentration of substrate, cell and product with the initial rate is 60 kg/m3.
Table 4.1.7 the concentration of substrate, cell and product with the initial rate is 60 kg/m3.
Table 4.1.8 the concentration of substrate, cell and product with the initial rate is 90 kg/m3.
Figure 4.1.9 the concentration of substrate, cell and product with the initial rate is 90 kg/m3.
During the modeling, lactose is mainly converted into cell, lactic acid. These values are very close to the values in literature such as the relation between the product yield coefficient and initial substrate concentration. Figure 4.1.10, 4.1.11 and 4.1.12 demonstrated the concentration of substrate, cell and product change with time. It is obvious that low initial concentration need longer reaction time to reach high conversion rate. The higher the initial rate is, the shorter the reaction time will be.
Figure 4.1.10 the concentration of substrate with different initial rate
Figure 4.1.11 the concentration of cell with different initial rate
Figure 4.1.12 the concentration of product with different initial concentration
4.1.2 Effect of literature parameter
Parameters play an important in this model. All data in part 4.1.1are based on the parameter from literature and pervious work by (). Because biochemical process is a very complex process, and all work in this paper is total based on the literature work. Thus, using different literature data is very necessary. In this part, the kinetic parameter come from ()
Figure 4.1.13 parameter of bioprocess in ()
Figure 4.1.14 concentration of substrate, cell and product under different literature parameter with initial rate 60 g/l
Figure 4.1.14 shows the concentration of product is very high. In fact, this is no possible in bio process. For biochemical reactions, the kinetic parameters obtained in two ways. The first method is through the experimental data. Then, using the data to make the plot of 1/rs as a function of1/cs. According to the slope and intercept of plot, the value of Ks and μm can be calculated. The second way is to use method of least squares and iterative to. This method is very complex. However, in this paper, all the parameters come from literature work and calculated or experimented. Thus, the modeling has some degree of error. In addition, the data used in this paper all come from experiment based reference. Experimental conditions used, such as Ph, pressure, temperature or the size of the reactor, these factors will affect the concentration. However, in this work, model is purely theoretical point of view, with certain assumptions. Therefore, not all of this makes the experimental parameters in this model can be applied inside.
4.2 Effect of two CSTR reactors
In CSTR, the number of reactors has significant effect on the conversion rate and reaction time. A single reactor and the same reactor also have a certain extent effect on Cf and mass transfer in reactor. Assume that, reactor I only has one reactor, volume is 0.2 m3. Reactor II has two reactors; each reactor volume is 0.1m3. the total volume of reactor I and reactor II are same 0.2 m3. In this part, the impact of reactor size and series effect will be discussed.
Table 4.2.1 shows the necessary data of the tow rectors.
Table 4.2.1 basic parameter of CSTR reactor
Dilute rate:
As steady state, mass balance equation for I are:
Using the constant at D=0.075 h-1, this give that:
For second system, the process is as same as reactor I.
Replace cx2 by cs2 in . A quadratic equation in cs2 is obtained as follows:
Taking (is not possible), so .
Table 4.2.2 final concentration of reactor I and reactor II
Thus, the maximum cell productivity (xD) is obtained from reactor II.
4.3 Effect of pH and temperature
Although the model in this paper did not cover the effect of ph and temperature, they still play an important role during the bioreactor process. Next section we will introduce some simple pH and temperature on lactic acid fermentation reaction. The data are described from the literature().
Figure 4.3.1. Lactic acid fermentation experiments at pH 6, performed at 30 °C (A), 33.5 °C (B), 37 °C (C), and 40 °C (D).
Figure 4.3.1 shows the effect of different temperature on the concentration. It is obvious, the fermentation deceased. At a high temperature, it will form more D-lactic acid and not the L-lactic acid. All cells have a optimal reaction temperature. If the deviation from the optimal reaction temperature is too far, may lead to form the side-product or reduce the yield. The worst case may lead to cell death.
Figure 4.3.2Concentration of lactic acid at pH 4.0 (♦), pH 5.0 (■), pH 5.5 (□), pH 6.0 (○), pH 6.5 (♢), pH 7.0 (●), S0 = 50 g/l, and T = 39 °C().
It is obvious in figure 4.3.2, there exist a optimal pH level for lactic acid fermentation. Yuowono and Kokugan present that pH value has significant effect on product concentration, but no effect on fermentation time. According to the figure, basically, all concentrations are reached the maximum concentration within 20 hours. Furthermore, when the pH is 5.5, the reaction has maximum product concentration.
4.4 energy transfer
The total power input to reactor (eT) may be determined from the contribution of each phase in energy consumption().
(1) Isothermal expansion of gas as it moves up the vessel
(2) The kinetic energy of the sparged gas
(3) The kinetic energy of the liquid
(4) Energy lost in the spargers
Because fail to find any parameter in lactic acid, here is no detail calculation of lactic acid fermentation. The equation above is a general principle of energy transfer in bioreactor.
Conclusion
In this part, the effect of initial concentration, model parameter, temperature and pH value on the production concentration have been discussed.
5. Start-up & shut-down
A common requirement during the startup of bioreactors is that startup features reduce reaction time, waste material, or consumption(). The process will remain operating under this region for a certain time period. At the start up process, the reactor need fill of enough nutrient solution. The agitator starts working and adjusts the speed in case to hurt the cell. it also need to adjust the ph level and temperature to form a living condition for cell.
During fermentation process, the CSTR reactor is filled with substrate and microorganism. The culture is allowed cell to grow until no more of the product is formed after which the reactor is harvested and cleaned out for another run.
Fresh substrate medium is keeping supplying to a well-stirred reactor and products and cells are simultaneously withdrawn. After a certain period, the system usually reaches a steady state. At the end, the concentration of substrate, cell and product will be a constant.
At the shut down process, first, it need to turn off the agitator and temperature control, as a result, there are no reaction keep going in the reactor. Because the fermentation is under atmospheric pressure, it can close the cooling water valve. Then shut down the power supply. Remove the solution from the reactor.
6. Scale-Up
The amplification of the production equipment of fermentation industry, particularly the scale-up design of fermenter is very important for fermentation and biochemical engineering. The production of fermenter has been serialized and standardized by more and more professional company, and the volume of laboratory-scale is mostly 5~100L, and the pilot plant with the volume of 50~10000L, production plants with the volume of more than 5000L. In general, the small fermenters of 10L are mostly made of glass, and they can be operated on the test stand. The fermenter used in the small scale projects is always equipped with automatic detection and control systems, and automatic test items including temperature、pH、dissolved oxygen and oxygen and carbon dioxide gas, liquid and foam of inward and outward air. Large fermentation is costly, and difficult and costly to dispose().
The purpose of the fermentation tank to enlarge the industrialization of fermentation products is divided into three phases:
laboratory test phase →In a pilot → factory production
The design principle of general fermenter is based on whether the fermenter is suitable for the requirement of enlarging the production process, and whether it has the maximum production efficiency. When determine the maximum production capacity of the fermenter, it is necessary to consider two main factors:
1. the microbial productivity and conversion rates of a product;
2. The transfer performance of the fermenter including the mass transfer efficiency and heat transfer efficiency and mixing.
If the intrinsic value of the microbial growth rate and conversion rate is low, then the equipment meets the requirements. Obtaining yielding strains by screening strain further more can improve equipment utilization and give full play to equipment. If the eigenvalues of the microbial growth rate and the rate of makeup is high, and the effect of the strain characteristics is bad than small fermenter, then it shows that the maximum design capacity of the fermenter is low to meet the production requirements. The productive capacity of the fermenter need be improved and the appropriate amplification of fermenter is used to meet the need of microbial growth. Increasing the maximum production capacity of the fermenter is to solve the problem of transmission performance degradation during the amplification, that is, to focus on improving the fermenter mass transfer, heat transfer, mixing and other effects.
Scale up method
In this paper, the reactor is assumption of CSTR. Thus, geometry similar can be used as the amplification method(). The calculation is as follow:
V2 is the reactor volume after amplification. V1 is the lab-scale volume. M is the amplification time. H is the high of reactor, same as D is the diameter. This method is very simple, just to enlarge the size of the reactor.
=m
After scale up, other parameters such as d, B, s, W can be calculated by H and D. as a result; the size of scale up reactor can be calculated. However, Stirring rate n and other parameters need to be scale up. Assume using the same stirring rate. Power consumption per unit volume is equal(). If the power ratio to the volume is a constant, d is proportional to the stirring power. As a result, we can calculate the stirring power.
Thus:
7. Commercial Using of lactic acid and further concern
Pharmaceutical
Lactic acid is a raw material to produce biodegradable poly (actic acid). A biodegradable polymer has a growing demand, and it can be serve as both traditional and new materials, such as drug delivery(). This poly is a good surgical suture, seam stitches do not healing, automatically broken down into lactic acid is absorbed by the body, without adverse consequences. In particular, surgical sutures in vivo, eliminating the second surgery stitches trouble. Such polymers can be made into adhesive organ transplantation and bone in the application. In the wards, operating rooms, laboratories and other places in the steam sterilization using lactic acid, which can effectively kill airborne bacteria, decrease the risk of disease, to improve health purposes().
Recovery of lactic acid
Lactic acid can be produced by the fermentation of biomass. The pH level is very important for lactic acid fermentation. However, during the process, the pH level will drop because the accumulation of lactic acid. Thus, the lactic acid accumulation inhibits the product forming. In conventional methods, lactic acid has been recovered from precipitation of calcium lactate. This method is quite expensive and not friendly to environment. Thus, Kailas() presents that reactive extraction could be a efficient way to recovery of lactic acid. The advantage of extraction is that lactic acid can be easily removed from the fermentation.
Notation
μm = specific rate
α= cell growth coefficient
β = cell growth coefficient
Cx= cell growth
cs = substrate growth
cp = rate of product formation
Ks= kinetic parameter g/L
Yx/s= yield coefficient for cell on substrate (g/g)
Yp/s= yield coefficient for product on substrate (g/g)
D = dilution rate
F = volumetric feed flow rate
V = volume of reactor
t = time, h
S = substrate
C = cell
P = product
0 = initial condition
eT = total power input per unit volume (W/m3)
H = height of bioreactor (m)
HL = unaerated liquid height (m)
ΔPs = differential pressure between inlet and outlet of sparger (Pa)
QL = volumetric liquid flow rate (m3/h)
Qm = molar gas flow rate (kmol/s)
R = universal gas constant (J/kmol K)
S = separator to bioreactor volume ratio
ρL = liquid density (kg/m3)
Ω= efficiency factor
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