Isolated bacterial strain of gluconacetobacter oboediens and an optimized economic process for microbial cellulose production therefrom
10053718 ยท 2018-08-21
Assignee
Inventors
Cpc classification
C12P19/04
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides a novel and potent cellulose producing bacterial species, Gluconacetobacter oboediens which was isolated from mixed fruit residue deposited at MTCC, IMTECH, Chandigarh under the deposition number MTCC 5610. The process for the production of microbial cellulose by this bacterium was optimized and thus, an efficient and economic process for producing high titers of microbial cellulose was developed. Further, a novel and improved method for drying of microbial cellulose has been developed wherein the microbial cellulose mats were dried using a wooden plank and porous fabric as a base at room temperature. The microbial cellulose production was successfully scaled up to 5 liters volume of production medium in trays. The present invention also recites the production and optimization of microbial cellulose in different shapes and sizes (gloves and vessels) which will be of great help for burn and injured persons/patients.
Claims
1. A product comprising a culture medium comprising sugar of about 1.0-8.0 (% w/v) and corn steep liquor at about 1.0 to 5.0 (% v/v) and a culture of an isolated, bacterial strain of Gluconacetobacter oboediens having accession number MTCC 5610, wherein the product comprises microbial cellulose of at least 12 grams/liter.
2. The product as claimed in claim 1, wherein the product comprises microbial cellulose of at least 16 grams/liter.
3. A process for the production of microbial cellulose comprising culturing an isolated bacterial strain of Gluconacetobacter oboediens having accession number MTCC 5610 in a culture medium containing sugar of about 1.0-8.0 (% w/v) and corn steep liquor at 1.0 to 5.0 (% v/v) for a period of 4 to 10 days, followed by recovering the cellulose.
4. A process as claimed in claim 3, wherein the sugar is table sugar.
5. A process as claimed in claim 3, wherein during the recovery step placing wet mats of purified cellulose on wooden planks or a porous fabric for air drying.
Description
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
(1) In the drawings accompanying the specification,
(2) In the drawings accompanying the specification,
(3) In the drawings accompanying the specification,
(4) In the drawings accompanying the specification,
(5) In the drawings accompanying the specification,
(6) In the drawings accompanying the specification,
(7) In the drawings accompanying the specification,
DETAILED DESCRIPTION OF THE INVENTION
(8) The present invention provides a process for the isolation of microbial cellulose producing novel bacterial strains isolated from mixed fruit residues. The mixed fruit residue used in the present invention for the isolation of cellulose producing bacteria was collected from a local market of Satya Niketan, New Delhi110021, India. Of the several isolated strains the novel bacterial strain of Gluconacetobacter oboediens was selected for further studies as it was found to be the most potent microbial cellulose producer. This strain was deposited at the Microbial Type Culture Collection, MTCC, Chandigarh, India a depository recognized under the Budapest Treaty and has been accorded the deposit number MTCC 5610.
(9) The detailed morphological, cultural and biochemical characteristics of the isolated strain of Gluconacetobacter oboediens MTCC 5610 are as follows:
(10) TABLE-US-00001 Tests Characteristics Growth on agar medium Small, circular and rough colonies; Pellicle-forming colonies in presence of glucose Growth in liquid medium Not uniform Colour Off-white to cream Pigment production No Gram reaction Negative Morphology Rod shaped Arrangement Singly, in pairs or in short chain Sporulation No Motility Motile Growth on 3% ethanol in the + presence of 5-8% acetic acid Growth at a glucose + concentration of 30% (w/v) Requirement of acetic acid for growth Growth on methanol Acid formation from D-Glucose + Acetic acid production from + ethanol Cellulose formation +
Complete Process of Isolation of the Bacterial Strains from Fruit Residue is Described Herein Below:
(11) For isolation of cellulose producing bacterial strains, each of the collected fruit residue was mixed with sugar and water in the ratio of 1 to 3:0.1 to 0.5:2 to 4, respectively. The mixture was then kept in a wide mouthed plastic container and covered with a piece of cloth. The container was kept at temperature of 25 to 35 degree C. for 10 days undisturbed and observed for the formation of a pellicle (mat like structure) on the top of the fruit residue mixture. The mat like structure obtained was analyzed for the presence of cellulose fibrils by calcofluor staining and electron microscopy. The cellulose producing bacteria from the pellicle was isolated after treatment of pellicle with cellulase enzyme (1 mg/ml) at 50 degree C. for 48 h. Further, the bacterial strain obtained was identified on the basis of its physico-chemical properties.
(12) The present invention further describes an optimized economic process for the production of microbial cellulose by the said bacterial strains.
(13) The process of the invention involves the following steps: Isolation of microbial cellulose producer/s was carried using different fruit residues viz. pineapple, apple, orange, pomegranate, sweet lime and mixed fruit. A newer and potent MC producing bacterial species, identified as Gluconacetobacter oboediens was obtained from mixed fruit residue. Process optimization for maximum microbial cellulose production by the said bacterium was carried out by two approaches: 1) One variable at a time approach, 2) Response surface methodology.
(14) Different physiological and nutritional factors were optimized in order to maximize microbial cellulose production viz. agitation, production medium, pH, temperature, inoculum age, inoculum size, incubation period, carbon and nitrogen sources, metal ions, vitamins etc.
(15) Production of microbial cellulose was carried out under shaking and static culture conditions in Hestrin-Schramm medium (containing (%): glucose, 2.0%, peptone, 0.5%, yeast extract, 0.5%, citric acid, 0.115% and disodium hydrogen phosphate, 0.27%). Static culture was found to be more suitable for production of MC giving higher yield (0.45 to 0.75 g/l) as compared to shaking culture (0.08-0.18 g/l).
(16) Microbial cellulose production was carried out using eight different production media. Results showed that CSL-Fructose medium (containing per liter: Corn Steep Liquor 40 ml, Fructose, 70 g, K.sub.2HPO.sub.4, 1 g MgSO.sub.4.7H.sub.2O, 0.25 g, (NH.sub.4).sub.2SO.sub.4, 5.0 g, FeSO4.7H.sub.2O, 3.6 mg, CaCl.sub.2.2H.sub.2O, 14.7 mg, NaMoO.sub.4.2H.sub.2O, 2.42 mg; ZnSO.sub.4.7H.sub.2O, 1.73 mg, MnSO.sub.4.5H.sub.2O, 1.39 mg; CuSO.sub.45H.sub.2O, 0.05 mg Vitamin solution, 10 ml. Vitamin solution consisted of (per 1 L): Inositol, 200 mg; Nicotinic acid, 40 mg: Pyridoxine hydrochloride, 40 mg; Thiamine hydrochloride, 40 mg; Calcium pantothenate, 20 mg, Riboflavin, 20 mg, Folic acid, 0.2 mg; D-biotin, 0.2 mg), supported maximum microbial cellulose production, yielding 1.43 to 2.1 g/l microbial cellulose. Thus, this medium was selected for further optimization studies. Hereinafter, CSL-Fructose medium is also referred to as the production medium.
(17) TABLE-US-00002 Table depicting the yield of Microbial cellulose in different production media Production medium MC (g/l) Hestrin-Schramm medium 0.87 CSL-Fructose medium 1.43-2.1 Y3-3 medium 0.9 Generic medium 0.45 Defined medium 0.41 Coconut water medium 1.1 Pineapple juice medium 1.3 Improved medium 0.56
(18) Production of microbial cellulose by MTCC 5610 was carried out at different pH ranging from 2 to 12 adjusted with different buffers. pH in the range from 3 to 8 was found to be optimum for maximum production yielding 1.9 to 2.5 g/l microbial cellulose.
(19) TABLE-US-00003 Table depicting the Effect of pH on microbial cellulose production pH MC (g/l) 2 nil 3 1.90 4 2.51 5 2.32 6 2.10 7 2.03 8 1.97 9 1.25 10 0.94 11 0.23 12 nil
(20) The bacterium MTCC 5610 was grown at temperature ranging from 10 to 45 degree C. Maximum microbial cellulose production [2.1 to 2.52 g/l] was obtained at temperature ranging from 25 to 35 degree C.
(21) TABLE-US-00004 Table depicting the Effect of temperature on microbial cellulose production Temperature MC (g/l) 10 0.08 15 0.31 20 1.1 25 2.44 30 2.52 35 2.14 40 0.94 45 nil
(22) The microbial cellulose production by MTCC 5610 was carried out for different time periods under the conditions optimized so far. It was observed that the maximum microbial cellulose production (2.3 to 4.1 l was obtained after 4 to 10 days of incubation period.
(23) In the present invention, pieces of microbial cellulose mat containing Gluconacetobacter oboediens MTCC 5610 were used as inoculum. Inoculum age and size were optimized for microbial cellulose production. Inoculum of 1 to 5 days with a size of 5 to 12 mat pieces of 1012 mm per liter was found to be optimum for maximum cellulose production (3.2 to 6.7 g/l).
(24) Further, different nutritional factors viz., carbon and nitrogen sources, metal ions, vitamins etc. were optimized for maximizing microbial cellulose yield. Different carbon sources (monosaccharides and disaccharides) were used for microbial cellulose production and sucrose was found to be best and cheapest carbon source as compared to fructose (control) producing maximum microbial cellulose (6.5 to 7.2 g/l).
(25) In order to make the production medium more cost effective, three different low cost carbon sources were evaluated for microbial cellulose production, viz. jaggery, cane molasses and table sugar. Among these, table sugar was found to a promising carbon source giving yield equivalent to sucrose. Table sugar is 15-20 times cheaper as compared to sucrose. Thus, the selection of table sugar as the carbon source resulted in an economic medium for microbial cellulose production. The production of microbial cellulose was carried out at different concentrations of table sugar ranging from 0.1 to 20%. Maximum production was obtained at 1.0 to 10.0% concentration of table sugar.
(26) Microbial cellulose production was carried out in the presence of different organic and inorganic nitrogen sources. Corn steep liquor, an agro waste, was found to the best nitrogen source supporting maximum microbial cellulose production. Ammonium sulphate supported microbial cellulose production as an additive. Different concentrations of corn steep liquor ranging from 0.5 to 8.0% were used for producing microbial cellulose. Corn steep liquor at a concentration of 1.0 to 5.0% was found to be optimum for microbial cellulose production yielding 7.1 to 8.7 g/l microbial cellulose.
(27) The basal production medium optimized so far contains a number of metal ions (metal salts) in traces. The effect of these metal ions was evaluated by carrying out microbial cellulose production in the absence and presence of these salts. It was observed that the microbial cellulose production was equal both in the absence and presence of these metal ions. Thus, all these metal salts were omitted from the production medium. This made the production medium more simple and economic. However, it was observed that the other two metal salts i.e. magnesium sulphate and dipotassium hydrogen phosphate significantly affected microbial cellulose production. The production of microbial cellulose decreased in the absence of these two salts.
(28) The basal production medium optimized so far also contained different vitamins. The effect of these vitamins was evaluated on the production medium in the similar manner as for metal ions. The microbial cellulose production was found to be equivalent in the absence and presence of these vitamins. Thus, the vitamins were also omitted from the production medium. This made the cellulose production medium much more simple and economic.
(29) The microbial cellulose production was further optimized by a statistical approach, Response Surface Methodology to enhance the productivity. Results show that the interaction of the most influential parameters (CSL, sugar and inoculum size) obtained after one variable at a time approach resulted in a maximum yield of 12.0 to 16.0 g/l of microbial cellulose after a period of 4 to 10 days of incubation at sugar: 1.0-8.0 (% w/v); CSL: 1.0-5.0 (% v/v) and inoculum size, 1 to 8 (mat pieces/L), whereas the maximum yield by response surface methodology was 18.0 to 20.0 g/l.
(30) The microbial cellulose mats produced were processed and purified by alkali and acid treatment. The mats were further bleached to remove the remaining colour of the medium. The mats were finally washed with water and dried. The microbial cellulose mats were dried by freeze drying and air drying. Freeze dying provides a white paper like sheet of microbial cellulose. This method of drying is quite costly as it consumes a lot of electricity. Thus, in order to make the drying process cost effective the microbial cellulose mats were air dried using a novel, simple and economic method. The mats were dried on a wooden plank and a porous fabric at a temperature of 30 to 40 degree C. It was observed that air dying of microbial cellulose provides a transparent sheet of microbial cellulose.
(31) Scale up of microbial cellulose production was carried out upto 5 liters in trays. It was observed that the production of microbial cellulose was successfully scaled upto 5 liters yielding 60-80 g of microbial cellulose. This proves that microbial cellulose can be successfully produced to any amount and size.
(32) Further, the microbial cellulose was produced in different shapes, viz. gloves and vessels. This explains one of the most important properties of microbial cellulose that it can be molded in any shape, which makes microbial cellulose an important and versatile material for different medical applications.
(33) Thus, it can be inferred that the microbial cellulose produced by the novel isolated strain of Gluconacetobacter oboediens MTCC 5610 has immense importance in different sectors, especially in the medical field. The important applications of microbial cellulose are presented in the following table:
(34) TABLE-US-00005 INDUSTRIAL SECTORS APPLICATIONS Health care 1. Wound care dressings 2. Drug delivery agent, either oral or dermal 3. Artificial skin substrate 4. Component of dental and arterial implants Cosmetics and Beauty 1. Skin creams 2. Astringents 3. Base for artificial nails 4. Thickener and strengthener for fingernail polish 5. Tonics 6. Nail conditioners Food 1. Desserts (Nata de Coco, low calorie ice creams chips, snacks, candies) 2. Thickners (ice cream and salad dressing) 3. Base for weight reduction 4. Sausage and meat casings 5. Serum cholesterol reduction 6. Kombucha elixir or Manchurian tea Cellulose derived Production of cellophane, carboxymethyl products cellulose and cellulose acetate Clothing and shoe 1. Artificial leather products 2. One piece textiles 3. Highly adsorptive materials Petroleum and mining 1. Mineral and oil recovery 2. Recycling of minerals and oils Papers 1. Archival document repair 2. Paper base for long lived currency 3. Specialty papers 4. Napkins Forest products 1. Artificial wood strengthener (plywood laminates) 2. Filler for paper 3. High strength containers 4. Multilayer plywood 5. Heavy duty containers Audio products Superior audio speaker diaphragms Outdoor sports 1. Disposable tents and camping gear 2. Sport clothes Public utilities 1. Water purification via ultra filters and reverse osmosis membranes Babycare products 1. Disposable recyclable diapers Automotive and aircraft 1. Car bodies 2. Airplane structural elements 3. Sealing of cracks in rocket casings
EXAMPLES
(35) The following examples are given by way of illustration and therefore should not be construed to limit the scope of the present invention.
Example 1: Isolation of Cellulose Producer/s from Fruit Residues
(36) The isolation of cellulose producer/s was carried out using six different fruit residues (apple, pineapple, orange, sweet lime, pomegranate and mixed). Here, each fruit residue was mixed with sugar and water in the ratio of 1:0.2:3, respectively. The mixture was kept in a wide mouthed plastic container and covered with a piece of cloth. The container was kept at temperature of 30 degree C. for 10 days undisturbed and observed for MC production. After 10 days, it was observed that at the top of the pineapple, orange, sweetlime and mixed fruit residue mixtures, a mat like structure was deposited. This mat like structure was analyzed for the presence of cellulose fibrils by calcofluor staining and electron microscopy. The results showed that the mat like structure was composed of a network of ultrafine cellulose fibrils and it also contained rod shaped bacterial cells producing cellulose [
Example 2: Screening of the Bacterial Isolates Obtained from Fruit Residues for Microbial Cellulose Production
(37) All the isolates obtained from different fruit residues were evaluated for their potential to produce microbial cellulose. These isolates were inoculated individually in 250 ml Erlenmeyer flasks containing 50 ml cellulose production medium (Hestrin-Schramm medium) containing (g/l) glucose, 20; peptone, 5; yeast extract, 5; disodium hydrogen phosphate, 2.7 and citric acid, 1.15; and incubated for 15 days at 30 degree C. under static conditions for cellulose production. A compact mat was formed on the air-liquid interface of the medium by all the isolates. The mat was removed from the medium and examined for the presence of cellulose fibrils by calcoflour staining and SEM observation [
(38) TABLE-US-00006 SEQIDNo.1:16SrRNAsequenceof Gluconacetobacteroboediens TTTTTTTCCCCCCCGGAACGTCACGCGGCATCCTGATCCGCGATTACTAG CGATTCCACCTTCATGCACTCGAGTTGCAGAGTGCAATCCGAACTGAGAC GGCTTTTTGAGATCGGCTCGGTGTCACCACCTGGCTTCCCACTGTCACCG CCATTGTAGCACGTGTGTAGCCCAGGACATAAGGGCCATGAGGACTTGAC GTCATCCCCACCTTCCTCCGGCTTGTCACCGGCAGTTCCTTTAGAGTGCC CACCCAGACGTGATGGCAACTAAAGGCGAGGGTTGCGCTCGTTGCGGGAC TTAACCCAACATCTCACGACACGAGCTGACGACAGCCATGCAGCACCTGT GCTGGAGGTCTCTTGCGAGAAATGCCCATCTCTGGACACGGCCTCCGCAT GTCAAGCCCTGGTAAGGTTCTGCGCGTTGCTTCGAATTAAACCACATGCT CCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGG CCGTACTCCCCAGGCGGTGTGCTTATCGCGTTAACTACGACACTGAATGA CAAAGTCACCCAACATCCAGCACACATCGTTTACAGCGTGGACTACCAGG GTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTCAT GAGCCAGGTTGCCGCCTTCGCCACCGGTGTTCTTCCCAATATCTACGAAT TTCACCTCTACACTGGGAATTCCACAACCCTCTCTCACACTCTAGTCGCC ACGTATCAAATGCAGCCCCCAGGTTAAGCCCGGGAATTTCACATCTGACT GTGTCAACCGCCTACGCGCCCTTTACGCCCAGTCATTCCGAGCAACGCTT GCCCCCTTCGTACTACAGCGCTGCGCGCGCGCACACAAAG
Example 3: Air Drying (Drying at Room Temperature) of Microbial Cellulose
(39) The air drying method of microbial cellulose has one drawback i.e it sticks to the base on which it is kept for drying and it becomes difficult to recover it. For solving this problem, two bases were discovered and used for the air drying of the microbial cellulose mats. These were: a wooden plank and a porous fabric. The wet mats of purified microbial cellulose were placed on these two bases and left for 45 hours at a temperature of 35 degree C. After this time period, the mats were fully dried. It was observed that the mats did not sticked on the bases used and were easily recovered [
(40) The reason behind the success of these two bases is that both the materials are porous and the air passes through them, while in all the other cases where the mat sticks on the base, a vacuum is created because the bases used were not porous but rigid and do not allow any air to pass through them. Therefore, the microbial cellulose sticks on these bases and cannot be removed.
Example 4: Scale Up of Microbial Cellulose Production Up to 5 L in Static Culture in Different Tray Sizes
(41) Trays of four different sizes viz. 18145 cm.sup.3, 28235 cm.sup.3, 33.5284.5 cm.sup.3 and 42347 cm.sup.3 were used for scale up of production of microbial cellulose upto 5 L in static culture. The trays were sterilized and the sterilized production medium was poured aseptically in trays with different volumes i.e. 200, 500, 1000, 2000, 3000, 4000 and 5000 ml. These trays were inoculated with mat pieces (2 to 8 mat pieces of 1012 mm per liter) and incubated at a temperature of 30 degree C. for 10 days under static conditions [
(42) After incubation it was observed that a compact and rigid microbial cellulose mat having considerable strength and dimension as the respective tray size and depth of the medium was produced successfully upto 5 L. The dimension of the 5 L microbial cellulose mat was 42342.7 cm.sup.3 with a cellulose yield of 60 to 80 g.
Example 5: Production of Microbial Cellulose in the Shape of Gloves and Vessels
(43) In this experiment, latex gloves and silicon tubes (30 cm long) of different diameters viz. 3 and 6 mm (inner diameter) were used for producing microbial cellulose in their respective shapes. These materials were sterilized at 15 psi for 15 min. Before sterilization, both mouth ends of the silicon tubes were closed with a piece of klin wrap.
(44) Cellulose production medium was prepared and sterilized. Now, the sterilized medium was poured aseptically in the gloves (200 ml) and tubes (10-40 ml capacity). The gloves were hanged with the help of a support in a big glass container. They were incubated at a temperature of 35 degree C. for 5 days under static conditions. It was observed that the microbial cellulose was successfully produced in the shape of gloves and tubes/vessels [
(45) Advantages
(46) The main advantages of the present invention are: The bacterial species used in the present invention, Gluconacetobacter oboediens, is a new microbial cellulose producer. The production of microbial cellulose by this species of Gluconacetobacter is not reported earlier. This is the first report of microbial cellulose production by this bacterial culture. Thus, the present invention relates to the production of microbial cellulose by a novel microorganism. The optimized production medium i.e. CSL-Fructose medium used for microbial cellulose production is simple and economic containing low cost carbon and nitrogen sources, viz. table sugar & corn steep liquor (agro waste), respectively and only few salts in traces. It provides an optimized, efficient and cost effective process for the production of high titers of microbial cellulose and further, its successful scale up in static culture in trays. All the optimization experiments of microbial cellulose production conducted in 1 liter volume have the potential to be scaled up in all sets of experiments. The present invention also provides a novel and economic method for air drying of microbial cellulose mats using a wooden plank and porous fabric as a base. This step is very important as after drying only, the final dry weight of the microbial cellulose can be taken.