This International Application claims priority from a complete patent application filed in India having Patent Application No. 201931026219, filed on July 01, 2019 and titled“TWO-STEP FERMENTATION PROCESS FOR OBTAINING CELLULOSE AND GLUCONIC ACID”

FIELD OF INVENTION

Embodiments of a present disclosure relates to fermentation process of carbon source products by Gluconacetobacter species bacteria, and more particularly to a two-step fermentation process for obtaining cellulose and gluconic acid.

BACKGROUND

Cellulose is a highly abundant natural polymer produced mainly by plants. Apart from plants, many bacteria, especially those belonging to the genus Gluconacetobacter, produce a very peculiar form of the cellulose. Fermentation process enables formation of the cellulose. Here, the cellulose produced may be exploited for numerous applications for the mechanical and structural properties. Similarly, further fermentation leads to production of other metabolites mainly the organic acids like Acetic Acid and Gluconic Acid.

In one approach, a method comprising fermenting a sugar-based carbon source with one or more bacteria from Gluconacetobacter genus is disclosed. The said invention discloses secretion of primary metabolites. Subsequently secretion of a secondary metabolite is also disclosed. The primary metabolites can be bio-organic acids like acetic Acid, Glucuronic Acid, Gluconic Acid, Lactic Acid, and the like. The secondary metabolite comprises cellulose. Here, an efficient approach would be to harvest secreted cellulose, based on the pre-determined required level of the cellulose. Moreover, production of cellulose and gluconic acid should be a continuous process, without an extra need for preparing a fresh culture of bacteria and medium.

Hence, there is a need for an improved two-step fermentation process for obtaining cellulose and gluconic acid and therefore address the aforementioned issues. BRIEF DESCRIPTION

In accordance with one embodiment of the disclosure, a two-step fermentation process for obtaining cellulose and gluconic acid is provided. The process includes a first stage fermentation process for obtaining cellulose and a residual first stage broth. The first stage fermentation process includes preparing of a first culture medium comprising sucrose, Gluconacetobacter species bacteria and yeast extract; pasteurizing the culture medium prepared in above mentioned step; fermenting the pasteurised culture medium for a pre-determined duration while maintaining a predetermined temperature and a pre-determined range of pH and lastly filtering the fermented culture medium, upon reaching a predefined level of cellulose yield, to harvest the cellulose and obtain a residual first stage broth of a predetermined range of pH.

The process also includes a second stage fermentation process for obtaining D- gluconic acid. The second stage fermentation includes preparing a second culture medium by mixing the residual first stage broth and distilled water; adding glucose to the mixture to raise concentration of glucose up to pre-determined range in above mentioned step; fermenting the second culture medium by a bioreactor maintained at a predetermined vvm for a predetermined duration; filtering the second fermented culture medium sequentially by 5 micron filters, 0.45 micron filters and 0.20 micron filters followed by centrifugation to obtain D-Gluconic acid.

To further clarify the advantages and features of the present disclosure, a more particular description of the disclosure will follow by reference to specific embodiments thereof, which are illustrated in the appended figures. It is to be appreciated that these figures depict only typical embodiments of the disclosure and are therefore not to be considered limiting in scope. The disclosure will be described and explained with additional specificity and detail with the appended figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The disclosure will be described and explained with additional specificity and detail with the accompanying figures in which: FIG. 1 is a flowchart representing the steps of a first stage fermentation of a two-step fermentation process for obtaining cellulose and gluconic acid in accordance with an embodiment of the present disclosure; and

FIG. 2 is a flowchart representing the steps of a second stage fermentation of the two- step fermentation process for obtaining cellulose and gluconic acid accordance with an embodiment of the present disclosure;

Further, those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and may not have necessarily been drawn to scale. Furthermore, in terms of the method steps, chemical compounds, and parameters used herein may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the figures with details that will be readily apparent to those skilled in the art having the benefit of the description herein.

DETAILED DESCRIPTION

For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated synthesis of iron nanoparticles coated with biomolecules, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more components, compounds, and ingredients preceded by "comprises... a" does not, without more constraints, preclude the existence of other components or compounds or ingredients or additional components. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”,“an”, and“the” include plural references unless the context clearly dictates otherwise.

Embodiments of the present disclosure relates to a two-step fermentation process for obtaining cellulose and gluconic acid. The present two-step fermentation yields bacterial cellulose in first stage of fermentation and gluconic acid in second stage of fermentation. The produced bacterial cellulose and gluconic acid may be used in many industrial applications.

As used herein, the term“fermentation” refers to the chemical breakdown of a substance by bacteria, yeasts, or other microorganisms, typically involving effervescence and the giving off heat. As used herein, the term“cellulose” refers to a polysaccharide consisting of chains of glucose monomers. Here, the fermentation process provides bacterial cellulose.

The process includes a first stage fermentation process for obtaining cellulose and a residual first stage broth. The first stage fermentation process includes preparing of a first culture medium comprising sucrose, Gluconacetobacter species bacteria and yeast extract. The first stage fermentation process also includes pasteurizing the culture medium prepared in above mentioned step.

The first stage fermentation process also includes fermenting the pasteurised culture medium for a predetermined duration while maintaining a pre-determined temperature and a pre-determined range of pH. The first stage fermentation process also includes filtering the fermented culture medium, upon reaching a predefined level of cellulose yield, to harvest the cellulose and obtain a residual first stage broth of a predetermined range of pH.

The present process also includes a second stage fermentation process for obtaining D-gluconic acid. The second stage fermentation process includes preparing a second culture medium by mixing the residual first stage broth and distilled water.

The second stage fermentation process also includes adding glucose to the mixture to raise concentration of glucose up to a pre-determined range in above mentioned step. The second stage fermentation process also includes fermenting the second culture medium by a bioreactor maintained at a predetermined vvm for a predetermined duration. The second stage fermentation process also includes filtering the second fermented culture medium sequentially by 5 micron filters, 0.45 micron filters and 0.20 micron filters followed by centrifugation to obtain D-Gluconic acid.

FIG. 1 is a flowchart representing the steps of a first stage fermentation of a two-step fermentation process for obtaining cellulose and gluconic acid (10) in accordance with an embodiment of the present disclosure.

The first stage fermentation process (10) includes preparing of a first culture medium comprising sucrose, Gluconacetobacter species bacteria and yeast extract in step 20. As used herein. The term“culture medium” refers to a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plants. A used herein, the term“sucrose” refers to a form of sugar that come from sugar cane or sugar beet. In one embodiment, the sucrose may be of any carbon source product. In one such exemplary embodiment, the sucrose is 20 grams per litre and the yeast extract is of the of 5 grams per litre. In such embodiment, in the first culture medium, the sucrose may be replaced by glucose, xylose, fructose and the like.

Here, Gluconacetobacter species bacteria is characteristically a gram-negative and rod-shaped bacterium. Gluconacetobacter species bacteria is stringently aerobic and may be distinguished by ability to produce multiple poly beta-l,4-glucan chains, chemically identical to plant cellulose called as microfibrils.

In such embodiment, the microfibrils are synthesized at the bacterial surface at sites external to the cell membrane. In one exemplary embodiment, Gluconacetobacter species bacteria used in said fermentation process (10) comprises at least one of a Gluconacetobacter hansenii, Gluconacetobacter oboediens, Gluconacetobacter xylinus and Gluconacetobacter kombuchae.

The first stage fermentation process (10) also includes pasteurizing the culture medium prepared in the step 20. As used herein, the term“pasteurization” means a process in which certain packaged and non-packaged substances are treated with mild heat, usually less than 100 °C, to eliminate pathogens and thereby extends shelf life.

In one embodiment, the pasteurizing is performed to remove any pathogens present in the culture medium. As used herein, the term“pH” refers to a figure expressing the acidity or alkalinity of a solution on a logarithmic scale on which 7 is neutral, lower values are more acid and higher values more alkaline.

Furthermore, the first stage fermentation process (10) also includes fermenting the pasteurised culture medium for a pre-determined duration while maintaining a predetermined temperature and a pre-determined range of pH in step 40. In one exemplary embodiment, fermentation process is carried out in a dark room over a tray apparatus.

In continuation with the step 40, in one embodiment, the fermentation duration may be of 1 week to 3 weeks. In one such specific embodiment, the fermentation duration is for a duration of 15 days. In another embodiment, the temperature may be maintained in the range of between 25 °C to 33°C. In another such specific embodiment, the maintained temperature is of 28°C. Furthermore, in another embodiment, the pH may be maintained in range from 2.0 to 6.5. In another specific embodiment, the maintained pH is of 5.0.

The first stage fermentation process (10) lastly includes filtering the fermented culture medium, upon reaching a predefined level of cellulose yield, to harvest the cellulose and obtain a residual first stage broth of a predetermined range of pH in step 50. In one specific embodiment, the maintained pH is of 3.5. As used herein, the term“broth” refers to a liquid medium containing proteins and other nutrients for the culture of bacteria. Here, in such embodiment, the cellulose yield is 5 to 20 grams per litre of fermentation media (dry weight). In one embodiment, the filtering is performed by a filter press. As used herein, the term“filter press” refers to a tool used in separation processes, specifically to separate solids and liquids.

FIG. 2 is a flowchart representing the steps of a second stage fermentation of the two- step fermentation process for obtaining cellulose and gluconic acid (60) accordance with an embodiment of the present disclosure.

The second stage of fermentation process (60) includes preparing a second culture medium by mixing the residual first stage broth and distilled water in step 70. As used herein, the term“distilled water” refers to water that has been boiled into vapor and condensed back into liquid in a separate container.

The second stage of fermentation process (60) includes adding glucose to the mixture to raise concentration of glucose up to a pre-determined range in above mentioned step 70. In one specific embodiment, the glucose to the mixture to raise the concentration of the mixture up to 500 grams per litre. In step 80, raising of concentration enables maximum production of gluconic acid in last step 110.

The second stage of fermentation process (60) includes fermenting the second culture medium by a bioreactor maintained at a predetermined vvm for a predetermined duration in step 90. As used herein, the term“bioreactor” refers to a vessel in which a chemical process is carried out which involves organisms or biochemically active substances derived from such organisms. In one specific embodiment, the bioreactor is maintained at lvvm. Moreover, in another specific embodiment, the bioreactor may be a fermenter.

In one embodiment, the fermentation duration may be of range of 8 hours to 72 hours. In such specific embodiment, the fermentation duration is of 24 hours. In another embodiment, the temperature may be maintained in the range from 25 °C to 33°C. In another such embodiment, the pH may be maintained in the range from 2.0 to 6.5.

The second stage of fermentation process (60) also includes filtering the second fermented culture medium sequentially by 5 micron filters, 0.45 micron filters and 0.20 micron filters followed by centrifugation in step 100. As used herein, the term “centrifugation” is a technique which involves the application of centrifugal force to separate particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. In such embodiment, the centrifugal speed is between 10000 Revolutions per minute (RPM) to 15000 Revolutions per minute (RPM). Here, filter cartridges for sediment removal are rated in microns.

Furthermore, in one embodiment, purifying the filtered second culture medium is achieved by a downstream processing. In such embodiment, the purification is done to remove bacterial cells and cellular debris. As used herein, the term“downstream processing” refers to the recovery and purification of biosynthetic products, particularly pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including the recycling of salvageable components and the proper treatment and disposal of waste.

The second stage of fermentation process (60) lastly provides D-gluconic acid in step 110. In one exemplary embodiment, the D-gluconic acid is present in range of 400 to 450 gram per litre of the second culture medium. In such embodiment, Gluconic Acid is the carboxylic acid formed by the oxidation of the first carbon of glucose with antiseptic and chelating properties.

Examples

Example 1: Two-step fermentation process Primary Fermentation

A culture medium was prepared which had primarily sucrose as the carbon source (20 grams/litre) along with yeast extract (5 grams/litre) as nitrogen source. Sodium Citrate and Acetic Acid was added for pH adjustment. A total of 5 litres of fermentation media was prepared and the media was pasteurised. The temperature of the media was set at 28°C at the time of introduction of seed culture along with a pH of 5.0. The culture media was mixed and the culture media was poured in a sterilised food grade tray. The opening of the tray was cover with a sterilised cotton fabric. The sterilised cotton fabric allows the gaseous exchange in the media. CO ² produced by the bacteria is exchanged with the atmospheric O ² . The fermentation was allowed to continue for 15 days without disturbing the tray in a dark room. The ambient temperature of the room was set for 28°C. Upon completion of 15 days, the bacterial cellulose formation on top of the medium was harvested. As the cellulose is highly hydrophilic, large volumes of the medium was trapped in the microfibrils network. Using a filter press, the trapped media was extracted and collected along with the remaining broth. A total of 4 litres of broth was collected with a pH of 3.5.

Secondary Fermentation

The collected media broth of 4 litres was transferred in a sterile bioreactor and added with 500 grams per litre of Glucose. The total volume of the media was increased to 10 litres by adding distilled water. The pH was set to 4.5 and temperature of 28°C. The aeration in the bioreactor was set to 1 VVM and allowed for secondary fermentation to take place. The duration of the secondary fermentation was set for 24 hours. The bioreactor’s pressure was set at 1 bar. Upon the completed duration, the media was harvested and downstream processing was initiated. The media was subjected to 5- micron filtration followed by high speed centrifugation. A subsequent filtration was carried out with 0.45 micron and 0.20 micron filters. The obtained broth was subjected to analysis for finding the D-Gluconic acid concentration. The total concentration of D-Gluconic acid in the broth was approximately between 400-450 ml per litre of the broth.

The present disclosure provides two-step fermentation process for production of bacterial cellulose and gluconic acid. Here, the major advantage is that both bacterial cellulose and D-Gluconic acid are produced in a continuous process without a need to prepare a fresh culture of bacteria and media, therefore two different products/outputs are achieved at a higher yield. This greatly minimises the cost of operation, requirement of raw material and human intervention. As a result, market price of the bio-cellulose and D-gluconic acid can be brought down which is beneficial to various dependent industries. It is to be noted that in normal fermentation process as the concentration of the bio-organic acids (gluconic acids etc.) is very minimal comprising only 5-10% of the fermentation broth, more focus was given on commercialization of extraction of bacterial cellulose only. However, with the help of present invention even the D-gluconic acid can be harvested at a very high yield from the used broth.

While specific language has been used to describe the disclosure, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.

The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependant on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.