Patent Publication Number: US-2019169658-A1

Title: Bacillus aerolacticus for producing l-lactic acid or its salts from various carbon sources

Description:
SUMMARY OF THE INVENTION 
     This invention relates to a novel  Bacillus aerolacticus  BC-001 species and a method for producing lactic acid or its salts using said bacteria, wherein said novel bacteria is deposited to NITE Patent Microorganisms Depositary (NPMD), Japan, Accession Number NITE BP-01943. Said  Bacillus aerolacticus  can produce L-lactic acid or its salts from various carbon sources. 
     Said bacteria can grow well under an aerobic condition, be tolerant to high temperature at more than 45° C., and can produce L-lactic acid or its salts with high optical purity. 
     Moreover, the invention relates to a method for producing L-lactic acid or its salts, comprising the following steps: 
     (1) cultivating the  Bacillus aerolacticus  BC-001 species to obtain a seed culture; and 
     (2) fermenting the seed culture obtained from step (1) in a carbon source. 
     FIELD OF THE INVENTION 
     Biotechnology relates to bacteria that can produce lactic acid. 
     BACKGROUND OF THE INVENTION 
     It is well known that lactic acid has been widely used in plastic, food, pharmaceutical, and cosmetic industries. 
     Lactic acid is a chiral molecule which its polarization property results in a categorization of lactic acid to 3 isomers that are L-lactic acid, D-lactic acid, and racemic lactic acid. For plastic industry, L-lactic acid is widely used, especially for production of polyester such as polylactic acid or poly(lactic-co-glycolic acid). Polymer produced from lactic acid has its advantage that it is biodegradable and biocompatible. Said polymer can be used in many applications such as fiber in textile, film, packaging, catgut, and scaffold in medical field. 
     At present, there are several production processes of lactic acid, such as chemical synthesis and biotechnology. Biotechnology possesses several advantages including the utilization of renewable resources for microbial fermentation such as tapioca, corn, wheat, or sugarcane. Moreover, the microbial fermentation is capable of producing lactic acid with high optical purity. 
     Most of industrial lactic acid productions are the fermentation of sugar such as glucose, sucrose, maltose, or other carbohydrates such as starch or cellulose, wherein microorganisms that can produce lactic acid are bacteria and fungi. 
     Bacteria in genus  Lactobacillus, Leuconostoc , and  Streptococcus  are well known in the production of lactic acid from sugar under anaerobic condition, leading to low energy consumption and providing product with higher titer than from fungi. However, said bacteria group is fastidious bacteria which need vitamin and essential amino acids on its growth. Moreover, said bacteria group cannot produce enzyme to convert starch into sugar, leading to the need of pretreatment step prior to fermentation, which increases production cost. 
     Persson et al., (Biotechnol. Bioeng., 2001, 72, 269-277) and Soccol et al., (Biochem. Eng. J., 2003, 13, 205-218) disclosed the use of Lactobacilli bacteria in lactic acid production. However, said bacteria yielded the mixture of L- and D-lactic acids, thus additional purification process is required to separate such isomers before using them in the polylactic production, which was the limitation. Min Young Jung et al., (IJSEM, 2009, 59, 2226-2231) reported the new species of  Bacillus  that can produce lactic acid and named said species as  Bacillus acidiproducens . However, as disclosed,  Bacillus acidiproducens  could not produce lactic acid from some carbon sources such as starch, and the optical purity of produced lactic acid was not discussed. To date, the efficiency of lactic acid production from said  Bacillus acidiproducens  has not been studied and reported yet. 
     One problem of polymer production from lactic acid is its high production cost. It is necessary to develop the lactic acid production process with lower production cost, increase yield and provide high optical purity isomer product. Therefore, there are several attempts to study and develop robust microorganisms that have ability to grow and proliferate with high rate, and can utilize low cost carbon sources as raw materials in lactic acid production. 
     One attempt to reduce lactic acid production cost is to use complex carbon sources derived from plant biomass, agricultural residues and industrial waste rather than an expensive monosaccharide in fermentation. However, most of lactic acid bacteria naturally found could not digest and utilize complex carbon sources. Thus, a pretreatment process is required prior to lactic acid fermentation. Several pretreatments can be used including mechanical treatment, heat treatment, chemical treatment, or enzyme treatment, depending on the physical, chemical, and nutritional properties of such carbon sources. Said pretreatment processes are generally performed at high temperature in a range from about 50 to about 60° C. Generally, bacteria cannot grow at said temperature, additional step is therefore needed prior to fermentation so as to reduce the temperature to a room temperature, resulting in a complexity of production process and increasing of production cost. 
     From the reasons mentioned above, there is a need for microorganisms that tolerate to high temperature, provide high lactic acid yield with high optical purity, and can utilize various carbon sources. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows nucleotide sequence of 16S rRNA gene of  Bacillus aerolacticus  BC-001. 
         FIG. 2  shows phylogenetic tree of  Bacillus aerolacticus  BC-001. Horizontal solid line shows differences in phylogenetic of BC-001 compared to the closest related type strain,  Bacillus acidiproducens  SL213 T  strain (from IJSEM, 2009, 59, 2226-2231). 
         FIG. 3  shows micrograph from SEM of  Bacillus aerolacticus  BC-001 obtained from cultivation for 3 hours at temperature of 50° C., and at shaking speed of 250 rpm. 
         FIG. 4  shows optical density of  Bacillus aerolacticus  BC-001 at various initial concentrations of BC-001 and different cultivation periods. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Definition 
     Technical terms or scientific terms used herein, have definitions as understood by those having an ordinary skill in the art, unless stated otherwise. 
     Equipment, apparatus, methods, or chemicals mentioned here means equipment, apparatus, methods or chemicals commonly operated or used by those skilled in the art, unless explicitly stated otherwise that they are equipment, apparatus, methods, or chemicals specifically used in this invention. 
     The use of the singular or plural nouns with the term “comprising” in the claims or in the specification refers to “one” and also “one or more”, “at least one”, and “one or more than one”. 
     Throughout this application, the term “about” is used to indicate that any value presented herein may potentially vary or deviate. Such variation or deviation may result from errors of apparatus, methods used in calculation or from individual operator implementing apparatus or methods. These include variations or deviations caused by the changes of physical properties. 
     “Starch” means purified starch, raw starch, liquefied starch, or any material that comprises starch or liquefied starch. Example of starch in this invention includes but not limited to tapioca starch, corn starch, wheat starch, or potato starch. 
     “Liquefied starch” means starch that is obtained from liquefaction process. Said process includes but not limited to the breakdown of starch structure by using physical method and/or chemical method such as heating, heating under pressure, chemical and enzyme treatments. 
     “Microaerobic condition” means condition that air has been controlled to be limited without adding additional air during fermentation or growth of the microorganisms. 
     Hereafter, invention embodiments are shown without any purpose to limit any scope of the invention. 
     The present invention relates to a novel thermotolerant  Bacillus aerolacticus  BC-001 species that can produce lactic acid from various carbon sources, and a method for producing L-lactic acid using said bacteria. 
       Bacillus aerolacticus  BC-001 of this invention can grow well under an aerobic condition, be tolerant to high temperature at more than 45° C., and can produce L-lactic acid or its salts with high optical purity. 
       Bacillus aerolacticus  BC-001 of this invention is deposited to NITE Patent Microorganisms Depositary (NPMD), Japan, under Budapest Treaty. The accession number given by NPMD is NITE BP-01943. 
       Bacillus aerolacticus  BC-001 is a gram-positive bacteria with the nucleotide sequence of 16S rRNA as shown in  FIG. 1 . 
       Bacillus aerolacticus  BC-001 has the morphology as shown in  FIG. 3 . 
       Bacillus aerolacticus  BC-001 is isolated from leaf and bark of tamarind in Lopburi, Thailand. 
     In one embodiment, said  Bacillus aerolacticus  BC-001 can grow well under an aerobic condition, be tolerant to a temperature in a range of around 45 to 60° C. Preferably, said  Bacillus aerolacticus  BC-001 can grow well under the aerobic condition, be tolerant to the temperature at around 50° C. 
       Bacillus aerolacticus  BC-001 can produce L-lactic acid and its salts with optical purity more than 95%, preferably more than 99%. 
     In another embodiment, this invention relates to the method for producing L-lactic acid or its salts using  Bacillus aerolacticus  as described above. 
     The method for producing L-lactic acid or its salts, comprising the following steps: 
     (1) cultivating the  Bacillus aerolacticus  BC-001 species to obtain a seed culture; and 
     (2) fermenting the seed culture obtained from step (1) in a carbon source. 
     In one embodiment, the cultivation in step (1) may be performed for a period of about 2 to 10 hours, preferably from about 3 to 5 hours and most preferably about 5 hours. In one embodiment, a concentration of  Bacillus aerolacticus  species in the cultivation step is about 0.5 to 5% by volume, preferably about 0.5 to 2% by volume and most preferably about 1% by volume. 
     In one embodiment, the fermentation of seed culture in step (2) may be performed at a temperature in a range of about 45 to 60° C., preferably at about 50° C. In one embodiment, the fermentation of seed culture in step (2) may be performed under a microaerobic condition. 
     Carbon sources for the fermentation may be selected from, but not limited to, fermentable sugar, starch, liquefied starch, or a mixture thereof. 
     Fermentable sugar is any sugar that can be found in nature or any sugar derived from a substance comprising sugar. Said sugar may be modified or unmodified. 
     In one embodiment, the fermentable sugar is monosaccharide that may be selected from glucose, fructose, galactose, or a mixture thereof. 
     In one embodiment, the fermentable sugar is disaccharide that may be selected from sucrose, lactose, maltose, cellobiose, or a mixture thereof. 
     In one embodiment, the fermentable sugar is trisaccharide that may be selected from raffinose, isomaltotriose, maltotriose, nigerotriose, kestose, or a mixture thereof. 
     Preferably, the fermentable sugar is selected from glucose, sucrose, or a mixture thereof. 
     In one embodiment, starch is selected from tapioca starch, corn starch, wheat starch, potato starch, or a mixture thereof. 
     The liquefied starch is starch that is contacted with amylase enzyme. 
     More preferably, the concentration of carbon source in the fermentation of seed culture is in a range of about 50 to 200 g/L. 
     In one embodiment, the fermentation of seed culture in step (2) may further comprise the step of adding glucoamylase enzyme during the fermentation of seed culture. The following is property tests according to the invention, wherein the methods and equipment used in the tests are commonly used and are not intended to limit the scope of the invention. 
     Glucose, lactic acid, and by-product are analyzed by high performance liquid chromatography using a Shimadzu equipped with a Biorad, Aminex HPX-87H ion exclusion organic acid 300 mm×7.8 mm column, at a temperature around 45° C., and reflective index detector Shimadzu-RID-10A for detecting a signal comparing to a standard signal. 
     Optical purity of L-lactic acid is analyzed by a chiral column Sumipack Sumichiral OA5000 at a temperature of 40° C. Copper sulfate (CuSO 4 ) is used as an eluent with a flow rate of about 1 ml/min. The signals are detected by using a UV Detector at a wavelength of 254 nm. 
     Optical density (OD) of BC-001 during the cultivation or fermentation is analyzed by Spectrophotometry at a wavelength of 600 nm. 
     Yield is calculated from a ratio of an amount of produced lactic acid to an amount of carbon sources used during fermentation. 
     The following examples are presented to illustrate the present invention without limiting the scope of the invention. 
     Isolation and Characterization of Bacteria According to the Invention 
       Bacillus aerolacticus  BC-001 is isolated from leaf and bark of tamarind, Lopburi, Thailand. A soil sample is added into a test tube filled with a medium for microorganism isolation, wherein said medium contains glucose in a concentration of around 10 to 15 g/L. The isolation is performed at the temperature of around 50° C. The colonies that can acidify the medium or give a clear zone are picked up. After that, a catalase test of obtained colonies is conducted to select colonies that can grow under an aerobic condition. After said method,  Bacillus aerolacticus  BC-001 that can produce lactic acid, grow under an aerobic condition and be tolerant to a high temperature is isolated from other bacteria strains. 
       Bacillus aerolacticus  BC-001 isolated from above method is then analyzed for a nucleotide sequence of 16s rRNA, a phylogenetic tree, bacterial species identification by DNA-DNA hybridization, and a morphology. Results are showed in  FIG. 1, 2 , Table 1 and  FIG. 3  respectively. 
     The phylogenetic tree in  FIG. 2  shows that  Bacillus aerolacticus  BC-001 is closest to  Bacillus acidiproducens  SL213 T  strain. The 16S rRNA gene sequence of BC-001 in  FIG. 1  indicates 98.85% similarity to the SL213 T  strain. Therefore, DNA-DNA hybridization of BC-001 is further conducted to determine the species of BC-001, wherein BC-001 and  Bacillus acidiproducens  13078 type strains are used as DNA probes and  Bacillus coagulans  6326 type strain is used as a negative control. The DNA-DNA hybridization result is shown in Table 1. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 DNA-DNA hybridization result of BC-001 
               
            
           
           
               
               
               
            
               
                   
                   
                 % 
               
               
                   
                   
                 DNA-DNA 
               
               
                 Isolate 
                 DNA probe 
                 hybridization 
               
               
                   
               
            
           
           
               
               
               
            
               
                 BC-001 
                 BC-001 
                 100 
               
               
                 
                   Bacillus acidiproducens 
                 
                 BC-001 
                 58.7 
               
               
                 13078 
               
               
                   Bacillus coagulans  6326 
                 BC-001 
                 48.1 
               
               
                 BC-001 
                   Bacillus acidiproducens  13078 
                 30.7 
               
               
                 
                   Bacillus acidiproducens 
                 
                   Bacillus acidiproducens  13078 
                 100 
               
               
                 13078 
               
               
                   Bacillus coagulans  6326 
                   Bacillus acidiproducens  13078 
                 34.7 
               
               
                   
               
            
           
         
       
     
     From Table 1, when using BC-001 as DNA probe for  Bacillus acidiproducens  13078 and  Bacillus coagulans  6326 and using  Bacillus acidiproducens  13078 as DNA probe for BC-001, % DNA-DNA hybridization is less than 70%. Therefore, it indicates that the BC-001 belongs to distinct species in  Bacillus  genus and is deposited at NITE Patent Microorganisms Depositary (NPMD), Japan, accession number NITE BP-01943 with the scientific name of  Bacillus aerolacticus.    
     Concentration of BC-001 in Cultivation Step 
       Bacillus aerolacticus  BC-001 is added to the cultivation medium containing the following compositions per liter: about 10 g of glucose, about 15 g of yeast extract, about 4 g of ammonium chloride (NH 4 Cl), about 5 g of calcium hydroxide, and about 20 ml of salt solution. Various concentrations of BC-001 are studied, including 0.5%, 1%, and 2% by volume. Thereafter, the mixture is shaken at around 250 rpm and the temperature about 50° C. The optical density (OD) of BC-001 is analyzed at different time. The result is shown in  FIG. 4 . 
     Study of Various Conditions in Cultivation and Fermentation Steps 
     To study the effect of cultivation period and aeration condition during fermentation on the ability to produce lactic acid of BC-001, various conditions in Table 1 are studied for lactic acid production. 
     The production of lactic acid is carried out by adding  Bacillus aerolacticus  BC-001 with a concentration of about 1% by volume in a culture medium containing the following composition per liter: about 10 g of glucose, about 15 g of yeast extract, about 4 g of ammonium chloride (NH 4 Cl), about 5 g of calcium hydroxide and about 20 ml of salt solution. Thereafter, said mixture is shaken at around 250 rpm and a temperature of about 50° C. to obtain a seed culture. Then, about 25 ml of said seed culture is added into a flask filled with 25 ml of 200 g/L glucose solution by using calcium carbonate (Ca(CO) 3 ) to control pH to be about 6.5 to 6.8. After that, the fermentation of obtained seed culture is performed for 24 hours at the temperature about 50° C. with various aeration and shaking conditions. At the end of fermentation, the products are centrifuged at about 10,000 rpm for about 5 minutes. The obtained products are analyzed for the optical density of bacteria and amount of lactic acid produced. The results are shown in Table 2. 
     From Table 2, it is found that an increase of cultivation period, shaking speed during fermentation, and microaerobic condition during fermentation result in an increase of the optical density of bacteria, final concentration of lactic acid and the productivity of lactic acid. Therefore, according to above results, it can be summarized that the microareobic condition and shaking during fermentation of  Bacillus aerolacticus  can enhance the efficiency of lactic acid production. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Lactic acid production of  Bacillus aerolacticus  BC-001 at various cultivation and fermentation conditions 
               
            
           
           
               
               
            
               
                 Cultivation step 
                 Fermentation step 
               
            
           
           
               
               
               
               
               
               
               
               
            
               
                   
                 Initial optical 
                 Final optical 
                   
                 Final optical 
                 Final lactic acid 
                   
                   
               
               
                 Time 
                 density 
                 density 
                   
                 density 
                 concentration 
                 Yield 
                 Productivity 
               
               
                 (hour) 
                 (OD) 
                 (OD) 
                 Shaking:Aeration 
                 (OD) 
                 (g/L) 
                 (g/g) 
                 (g/L/hr) 
               
               
                   
               
               
                 3 
                 0.4 
                 3.1 
                 wo:an 
                 13.2 
                 53.7 
                 1.13 
                 2.2 
               
               
                   
                   
                   
                 wo:micro 
                 19.7 
                 73.1 
                 1.06 
                 3.0 
               
               
                   
                   
                   
                 w:an 
                 13.2 
                 59.3 
                 1.11 
                 2.5 
               
               
                   
                   
                   
                 w:micro 
                 22.6 
                 92.4 
                 0.93 
                 3.9 
               
               
                 4 
                 0.4 
                 4.1 
                 wo:an 
                 13.8 
                 54.4 
                 1.24 
                 2.3 
               
               
                   
                   
                   
                 wo:micro 
                 21.3 
                 75.1 
                 1.06 
                 3.1 
               
               
                   
                   
                   
                 w:an 
                 13.2 
                 58.9 
                 1.21 
                 2.5 
               
               
                   
                   
                   
                 w:micro 
                 25.3 
                 92.2 
                 0.93 
                 3.8 
               
               
                 5 
                 0.4 
                 4.6 
                 wo:an 
                 13.8 
                 51.2 
                 1.22 
                 2.1 
               
               
                   
                   
                   
                 wo:micro 
                 20.2 
                 71.1 
                 1.06 
                 3.0 
               
               
                   
                   
                   
                 w:an 
                 15.0 
                 67.5 
                 1.10 
                 2.8 
               
               
                   
                   
                   
                 w:micro 
                 24.6 
                 95.5 
                 0.96 
                 4.0 
               
               
                   
               
               
                 Remarks: 
               
               
                 “wo” refers to fermentation without shaking 
               
               
                 “w” refers to fermentation with shaking at about 250 rpm 
               
               
                 “an” refers to fermentation under an anaerobic condition by using a flask with T-type silicone stopper and placed in an AnaeroPack 
               
               
                 “micro” refers to fermentation under a microaerobic condition by using a flak with C-type silicone stopper 
               
            
           
         
       
     
     Ability to Produce Lactic Acid of BC-001 from Various Carbon Sources 
     In order to demonstrate that  Bacillus aerolacticus  BC-001 is able to produce lactic acid from various carbon sources, the carbon sources including glucose, sucrose and liquefied tapioca starch, are used in the production of lactic acid of BC-001. These carbon sources are intended to be examples selected to illustrate carbon sources previously described and do not limit the scope of the invention. 
     The production of lactic acid could be carried out by adding  Bacillus aerolacticus  BC-001 with a concentration of about 1% by volume into a culture medium containing the following composition per liter: about 10 g of glucose, about 15 g of yeast extract, about 4 g of ammonium chloride (NH 4 Cl), about 5 g of calcium hydroxide, and about 20 ml of salt solution. The mixture is shaken at around 250 rpm and a temperature about 50° C. for about 5 hours to obtain seed culture. Then, 25 ml of said seed culture is added into a flask filled with the following carbon sources. 
     Example 1 
     Carbon source is 25 ml of glucose solution with a concentration of 240 g/L by using calcium carbonate (Ca(CO) 3 ) to control pH to be in a range of about 6.5 to 6.8. After that, the fermentation of obtained seed culture is performed for about 48 hours at about 50° C. under a microaerobic condition and shaking speed around 250 rpm. 
     Example 2 
     Carbon source is 25 ml of glucose solution with an initial concentration of 200 g/L by using calcium carbonate (Ca(CO) 3 ) to control pH to be in a range of about 6.5 to 6.8. After that, the fermentation of obtained seed culture is performed for about 24 hours at about 50° C. under a microaerobic condition and shaking speed around 250 rpm. Then, the glucose solution is added to adjust the concentration to be 150 g/L. Fermentation is further carried out for about 24 hours. 
     Example 3 
     Carbon source is 25 ml of sucrose solution with a concentration of 300 g/L by using calcium carbonate (Ca(CO) 3 ) to control pH to be in a range of about 6.5 to 6.8. After that, the fermentation of obtained seed culture is performed for 48 hours at about 50° C. under a microaerobic condition and shaking speed around 250 rpm. 
     Example 4 
     Carbon source is 25 ml of liquefied tapioca starch with a concentration of 300 g/L by using calcium carbonate (Ca(CO) 3 ) to control pH to be in a range of about 6.5 to 6.8. After that, the fermentation of obtained seed culture is performed for 48 hours at about 50° C. under a microaerobic condition and shaking speed around 250 rpm. 
     The liquefied tapioca starch is obtained from adding alpha-amylase enzyme into tapioca starch solution at a temperature around 100° C. for about 1.5 hours with pH controlled to be around 5.8. 
     Example 5 
     Carbon source is 25 ml of tapioca starch obtained from the method as described in example 4. Calcium carbonate (Ca(CO) 3 ) is used to control pH to be in a range of about 6.5 to 6.8. After that, the fermentation of obtained seed culture is performed for 48 hours at about 50° C. under a microaerobic condition and shaking speed around 250 rpm. 200 μL of glucoamylase enzyme with a concentration of 37 g/L is added during the fermentation after 2 and 24 hours. 
     At the end of fermentation, the products are centrifuged at 10,000 rpm for about 5 minutes. The obtained products are analyzed for remaining glucose, an amount of lactic acid produced and optical purity of lactic acid. The results are shown in Table 3. 
     As seen from Table 3,  Bacillus aerolacticus  BC-001 could produce lactic acid from various carbon sources including monosaccharide, disaccharide and liquefied starch, provide L-lactic acid with high optical purity that is more than 99%, and high productivity of lactic acid. 
     The liquefied tapioca starch with the addition of glucoamylase enzyme during fermentation yields the highest productivity of lactic acid. 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                 Lactic acid production of  Bacillus aerolacticus  BC-001 from various carbon sources 
               
            
           
           
               
               
               
            
               
                   
                 Lactic acid 
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                 Final 
                   
                   
                 Optical purity of 
                 Remaining 
               
               
                   
                 Type of carbon 
                 concentration 
                 Yield 
                 Productivity 
                 L-lactic acid 
                 glucose 
               
               
                 Example 
                 sources (g/L) 
                 (g/L) 
                 (g/g) 
                 (g/L/hr) 
                 (% ee) 
                 (g/L) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
            
               
                 1 
                 Glucose (120) 
                 174.72 
                 0.96 
                 3.7 
                 100.0 
                 34.2 
               
               
                 2 
                 Glucose (100 + 50) 
                 181.87 
                 0.96 
                 3.8 
                 100.0 
                 3.9 
               
               
                 3 
                 Sucrose (150) 
                 173.94 
                 0.89 
                 3.6 
                 100.0 
                 ND 
               
               
                 4 
                 Liquefied tapioca 
                 21.58 
                 0.10 
                 0.5 
                 100.0 
                 ND 
               
               
                   
                 starch (150) 
               
               
                 5 
                 Liquefied tapioca starch 
                 195.13 
                 0.98 
                 4.2 
                 99.7 
                 ND 
               
               
                   
                 with the addition of 
               
               
                   
                 glucoamylase enzyme 
               
               
                   
                 during fermentation (150) 
               
               
                   
               
               
                 Remarks: 
               
               
                 For sucrose, remaining glucose is reported as remaining sucrose 
               
               
                 “ND” refers that the remaining glucose cannot be detected. 
               
            
           
         
       
     
     BEST MODE OF THE INVENTION 
     Best mode of the invention is as disclosed in the detailed description.