WO2017037745A1

WO2017037745A1 - An integrated process for production of carbohydratases, ethanol, and xylitol using an isolated candida strain

Info

Publication number: WO2017037745A1
Application number: PCT/IN2016/050292
Authority: WO
Inventors: Harshad Ravindra VELANKAR; Anu Jose MATTAM; Arindam KUILA; Venkata Chalapathi Rao PEDDY; Venkateswarlu Choudary Nettem
Original assignee: Hindustan Petroleum Corp Ltd
Current assignee: Hindustan Petroleum Corp Ltd
Priority date: 2015-09-02
Filing date: 2016-09-02
Publication date: 2017-03-09
Anticipated expiration: 2018-03-02

Abstract

The present disclosure discloses a novel strain of Candida tropicalis (MTCC 25057) which can grown in a wide range of process conditions (temperature, pH), and in the presence of high concentrations of lignocellulosic inhibitors. The said strain has the capacity for simultaneous production of cellulases and xylanses from fermentation of sugars from lignocellulosic mass for production of ethanol, and xylitol.

Description

AN INTEGRATED PROCESS FOR PRODUCTION OF

CARBOHYDRATASES, ETHANOL, AND XYLITOL USING AN ISOLATED

CANDIDA STRAIN

FIELD OF INVENTION

[001] The present disclosure relates to the field of microbiology. In particular the present disclosure relates to an isolated Candida strain HP-CATOl (MTCC 25057) and its use for the production of ethanol, xylitol, and enzymes.

BACKGROUND OF THE IVNENTION [002] Production of renewable fuels, especially bioethanol holds remarkable potential to meet current energy demand (Joshi et al., Biotechnol and Mol Biol Rev, 2011, 6(8): 172-182). The use of bioethanol mitigates greenhouse gas emissions for sustainable environment, contribute to national security by reducing dependency on petroleum imports and reduces the dependency of petroleum the production of which is running low (Bentley RW, Energy Policy, 2002, 30: 189-205). Industrial production of ethanol today employs the use of food materials like sugarcane, tuber, corn which has caused significant stress on food prices and security. (Mitchell D, The World Bank, Washington DC, USA, 2008, pp 1-21). The use of these raw materials makes the production cost for ethanol high (Classen et al., Appl Microbiol Biotechnol, 1999, 52:741-755). A way to reduce the cost of large scale ethanol would be to utilize cheaper and abundant feed stock. Lignocellulosic biomass can be potential substrate for bioethanol production due to its several advantages such as 1) easy and abundant availability 2) higher cellulose and hemicellulose content and 3) don't compete with our food chain. Even though lignocellulose biomass is abundant, the commercialization of the process to produce bioethanol is limited due to insufficient research to minimization of production cost (Joshi et al., Biotechnol and Mol Biol Rev, 2011, 6(8): 172-182). Also the hydrolysis process produces by products which are toxic and interfere with fermentation process (Palmqvist and Hahn-Hagerdal, Bioresour Technol, 2000, 74: 17-24). For commercial production of Hgnocellulosic biomass development of integrated process for complete utilization of all the carbohydrates locked in Hgnocellulosic biomass is needed. The use of pentose fraction remaining after consumption of hexoses by S. cerevisiae for cellulase production by recombinant Aspergillus niger. The produced cellulase was further used for enzymatic hydrolysis of pretreated hgnocellulosic biomass (Alriksson et al., Appl Environ Microbiol, 2009, 75:2366-2374).

SUMMARY OF THE INVENTION [003] In an aspect of the present disclosure, there is provided an isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057.

[004] In an aspect of the present disclosure, there is provided a method of carbohydratases production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one hgnocellulosic biomass; (b) pre-treating said hgnocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7; and (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42°C for 24-96 hours under aerobic conditions to produce said carbohydratases.

[005] In an aspect of the present disclosure, there is provided a method of production of at least one sugar using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one hgnocellulosic biomass; (b) pre-treating said hgnocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6- 7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42° for 24-96 hours under aerobic conditions to produce said carbohydratases; (f) enriching said carbohydratases to obtain concentrated carbohydratases; and (g) incubating said concentrated carbohydratases with said pre- treated solid biomass at a temperature in the range of 48-52°C for 24-96 hours under aerobic conditions to produce said sugar.

[006] In an aspect of the present disclosure, there is provided a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass; (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6- 7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42 °C for 24-96 hours under aerobic conditions to produce said carbohydratases; (f) enriching said carbohydratases to obtain concentrated carbohydratases; (g) incubating said concentrated carbohydratases with said pre-treated solid biomass at a temperature in the range of 48-52°C for 24-96 hours under aerobic conditions to produce said sugars; and (h) contacting said Candida strain with said sugars at 30-42°C for 24-48 hours under aerobic or anaerobic conditions to produce ethanol.

[007] In an aspect of the present disclosure, there is provided a method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass: (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6- 7; and (e) contacting said isolated Candida strain HP-CAT01 having MTCC accession number MTCC 25057 with minimal salts or at least one nitrogen source, and said liquid hydrolysate at a temperature in the range of 30-42°C for 24-48 hours under aerobic conditions to promote xylitol production.

[008] In an aspect of the present disclosure, there is provided an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass; (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% pre- treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1- 5% at a temperature in the range of 30-42°C for 24-96 hours under aerobic conditions to produce said carbohydratases; (f) separating said Candida strain population from (e) to obtain a cell free liquid hydrolysate; (g) contacting said pre-treated solid biomass to said cell free liquid hydrolysate at a temperature in the range of 45-55°C for 24-96 hours under aerobic conditions to promote production of sugars; and (h) contacting said isolated Candida strain HP-CATOl having accession number MTCC 25057 with sugars from step (g) at a temperature in the range of 30-37°C for 24-48 hours in aerobic conditions to promote ethanol and xylitol production.

[009] In an aspect of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of ethanol from lignocellulosic biomass.

[0010] In an aspect of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of xylitol from lignocellulosic biomass.

[0011] In an aspect of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of xylitol and ethanol from lignocellulosic biomass.

[0012] In an aspect of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of carbohydratases from lignocellulosic biomass.

[0013] In an aspect of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of sugars from lignocellulosic biomass.

[0014] In an aspect of the present disclosure, there is provided a bio-plant for production of bioethanol, xylitol, sugars, and enzymes from lignocellulosic biomass using an isolated Candia tropicalis strain HP-CATOl having accession number MTCC 25057.

[0015] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. BRIEF DESCRITPION OF THE ACCOMPANYING DRAWINGS

[0016] The following drawings form part of the present specification and are included to further illustrate aspects of the present disclosure. The disclosure may be better understood by reference to the drawings in combination with the detailed description of the specific embodiments presented herein.

[0017] Figure 1A depicts the effect of different inhibitors (furfural, HMF, acetate) concentration on growth of Candida tropicalis MTCC 25057, in accordance with an embodiment of the present disclosure.

[0018] Figure IB depicts the effect of different temperatures on growth of Candida tropicalis MTCC 25057, in accordance with an embodiment of the present disclosure.

[0019] Figure 1C depicts the effect of different pH on growth of Candida tropicalis MTCC 25057, in accordance with an embodiment of the present disclosure.

[0020] Figure 2A depicts the effect of different substrates at different concentrations on endoglucanase activity, in accordance with an embodiment of the present disclosure.

[0021] Figure 2B depicts the effect of different substrates at different concentrations on exoglucanase activity, in accordance with an embodiment of the present disclosure.

[0022] Figure 2C depicts the effect of different substrates at different concentrations on xylanase activity, in accordance with an embodiment of the present disclosure.

[0023] Figure 3A depicts the fermentation of glucose under aerobic conditions by Candida tropicalis MTCC 25057 at 32°C in synthetic media, in accordance with an embodiment of the present disclosure.

[0024] Figure 3B depicts the fermentation of glucose under anaerobic conditions by Candida tropicalis MTCC 25057 at 32°C in synthetic media, in accordance with an embodiment of the present disclosure.

[0025] Figure 3C depicts the fermentation of glucose and xylose under aerobic conditions by Candida tropicalis MTCC 25057 at 32°C in synthetic media, in accordance with an embodiment of the present disclosure. [0026] Figure 4A depicts the fermentation of synthetic media containing glucose and xylose under anaerobic conditions to produce ethanol and xylitol by Candida tropicalis MTCC 25057at 32°C, in accordance with an embodiment of the present disclosure.

[0027] Figure 4B depicts the fermentation of synthetic media containing glucose and xylose under aerobic conditions to produce ethanol and xylitol by Candida tropicalis MTCC 25057at 32°C, in accordance with an embodiment of the present disclosure.

[0028] Figure 5A depicts the fermentation of pretreated hydrolysate under anaerobic conditions to produce ethanol and xylitol by Candida tropicalis MTCC 25057 at 32°C, in accordance with an embodiment of the present disclosure.

[0029] Figure 5B depicts the fermentation of pretreated hydrolysate under aerobic conditions to produce ethanol and xylitol by Candida tropicalis MTCC 25057 at 32°C, in accordance with an embodiment of the present disclosure.

[0030] Figure 6 depicts the schematic representation of a bio plant designed for the production of bio-ethanol, xylitol, value added products, sugars, and enzymes, in accordance with an embodiment of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0031] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.

Definitions:

[0032] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are collected here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0033] The articles "a", "an" and "the" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0034] The terms "comprise" and "comprising" are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as "consists of only".

[0035] Throughout this specification, unless the context requires otherwise the word

"comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0036] The term "including" is used to mean "including but not limited to".

"Including" and "including but not limited to" are used interchangeably.

[0037] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0038] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purposes of exemplification only. Functionally-equivalent products, compositions, and methods are clearly within the scope of the disclosure, as described herein.

Sequences:

[0039] SEQ ID NO: 1 depicts the 18S forward primer sequence:

TCCTCCGCTTATTGATATGC

[0040] SEQ ID NO: 2 depicts the 18S reverse primer sequence:

GAAGTAAAAGTCGTAACAAGG

[0041] SEQ ID NO: 3 depicts the 26S forward primer sequence: GCATATCAATAAGCGGAGGAAAAAG

[0042] SEQ ID NO: 4 depicts the 26S reverse primer sequence:

GGTCCGTGTTTCAAGACG

[0043] SEQ ID NO: 5 depicts the ITS1 forward primer sequence:

TCCGTAGGTGAACCTGCGG

[0044] SEQ ID NO: 6 depicts the ITS1 reverse primer sequence:

TCCTCCGCTTATTGATATGC

[0045] In an embodiment of the present disclosure, there is provided an isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057.

[0046] In an embodiment of the present disclosure, there is provided an isolated Candia tropicalis strain as described herein, wherein said strain is active in a pH range of 4-10, a temperature range of 30-42 ^UC, and presence of inhibitory compounds concentration range of 0.1-3 g/L.

[0047] In an embodiment of the present disclosure, there is provided an isolated Candia tropicalis strain as described herein, wherein said inhibitory compounds can be furfural, HMF, or acetic acid.

[0048] In an embodiment of the present disclosure, there is provided an isolated Candia tropicalis strain as described herein, wherein said inhibitory compounds are furfural, and HMF.

[0049] In an embodiment of the present disclosure, there is provided a method of carbohydratases production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass; (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7; and (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42°C for 24-96 hours under aerobic conditions to produce said carbohydratases.

[0050] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein in step (e), temperature is 32°C, and time is 24-96 hours.

[0051] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein in step (e), temperature is 42°C, and time is 24-96 hours.

[0052] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein in step (e) pre-treated solid biomass w/v percentage is 0.5%.

[0053] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein in step (e) liquid hydrolysate v/v percentage is in the range of 1-3%.

[0054] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein in step (e) liquid hydrolysate v/v percentage is 2%.

[0055] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0056] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0057] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0058] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 150°C for 15 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0059] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

[0060] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said carbohydratases are endoglucanase.

[0061] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said carbohydratases are exoglucanase.

[0062] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said carbohydratases are xylanase.

[0063] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said carbohydratases are a combination of endoglucanase, exoglucanase, and xylanase.

[0064] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein neutralization in step (d) carried out using ammonia.

[0065] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof. [0066] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein lignocellulosic mass is wheat straw.

[0067] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said minimal salts are 0.5 g/L MgS0₄.7H₂0, 0.5 g/L KC1, 1 g/L KH₂P0₄, and 2.5 g/L NaN0₃.

[0068] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057 is active in a pH range of 4-10, a temperature range of 30-42 °C, and inhibitory compounds concentration range of 0.1-3 g/L.

[0069] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said at least one nitrogen source weight percentage is in the range of 0.5-2%.

[0070] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said at least one nitrogen source weight percentage is 1%.

[0071] In an embodiment of the present disclosure, there is provided a method of carbohydratases production as described herein, wherein said at least one nitrogen source is yeast extract.

[0072] In an embodiment of the present disclosure, there is provided a method of carbohydratases production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw; (b) pre-treating wheat straw to obtain a liquid hydrolysate, and a pre- treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said solid loading w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; and (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts, 0.5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 2% at a temperature of 42°C for 24-96 hours under aerobic conditions to produce endoglucanase, exoglucanase, and xylanase.

[0073] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass; (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42° for 24-96 hours under aerobic conditions to produce said carbohydratases; (f) enriching said carbohydratases to obtain concentrated carbohydratases; and (g) incubating said concentrated carbohydratases with said pre-treated solid biomass at a temperature in the range of 48-52°C for 24-96 hours under aerobic conditions to produce said sugar.

[0074] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said sugar is 70-80% hexose, and pentose.

[0075] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

[0076] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said carbohydratases are endoglucanase, exoglucanase, and xylanase. [0077] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145- 155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0078] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0079] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0080] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein in step (e), temperature is 32°C, and time is 24-96 hours.

[0081] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein in step (e), temperature is 42°C, and time is 24-96 hours.

[0082] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0083] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 150°C for 15 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[0084] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said carbohydratases are a combination of endoglucanase, exoglucanase, and xylanase.

[0085] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein neutralization in step (d) is carried out using ammonia.

[0086] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.

[0087] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein lignocellulosic mass is wheat straw.

[0088] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said minimal salts are 0.5 g/L MgS0₄.7H₂0, 0.5 g/L KC1, 1 g/L KH₂P0₄, and 2.5 g/L NaN0₃.

[0089] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057 is active in a pH range of 4-10, a temperature range of 30-42°C, and inhibitory compounds concentration range of 0.1-3 g/L.

[0090] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein in step (e) pre-treated solid biomass w/v percentage is 0.5%.

[0091] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein in step (e) liquid hydrolysate v/v percentage is in the range of 1-3%. [0092] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein in step (e) liquid hydrolysate v/v percentage is 2%.

[0093] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said at least one nitrogen source weight percentage is in the range of 0.5-2%.

[0094] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said at least one nitrogen source weight percentage is 1%.

[0095] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said at least one nitrogen source is yeast extract.

[0096] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw; (b) pre-treating wheat straw to obtain a liquid hydrolysate, and a pre- treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said solid loading w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts, 0.5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 2% at a temperature of 42°C for 24-96 hours under aerobic conditions to produce carbohydratases (endoglucanase, exoglucanase, and xylanase); (f) enriching said carbohydratases to obtain concentrated carbohydratases; and (g) incubating said concentrated carbohydratases with said pre- treated solid biomass at a temperature of 50°C for 24-96 hours under aerobic conditions to produce said sugar comprising predominantly 70-80% hexose, and pentose. [0097] In an embodiment of the present disclosure, there is provided a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass; (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6- 7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42° for 24-96 hours under aerobic conditions to produce said carbohydratases; (f) enriching said carbohydratases to obtain concentrated carbohydratases; (g) incubating said concentrated carbohydratases with said pre- treated solid biomass at a temperature in the range of 48-52°C for 24-96 hours under aerobic conditions to produce said sugars; and (h) contacting said Candida strain with said sugars at 30-42°C for 24-48 hours under aerobic or anaerobic conditions to produce ethanol.

[0098] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said sugar is 70-80% hexose, and pentose.

[0099] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

[00100] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said carbohydratases are endoglucanase, exoglucanase, and xylanase. [00101] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00102] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00103] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00104] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein in step (e), temperature is 32°C, and time is 24-96 hours.

[00105] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein in step (e), temperature is 42°C, and time is 24-96 hours.

[00106] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00107] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 150°C for 15 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%. [00108] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said carbohydratases are a combination of endoglucanase, exoglucanase, and xylanase.

[00109] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein neutralization in step carried out using ammonia.

[00110] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.

[00111] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein lignocellulosic mass is wheat straw.

[00112] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said minimal salts are 0.5 g/L MgS0₄.7H₂0, 0.5 g/L KC1, 1 g/L KH₂P0₄, and 2.5 g/L NaN0₃.

[00113] In an embodiment of the present disclosure, there is provided a method of production of at least one sugar as described herein, wherein said isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057 is active in a pH range of 4-10, a temperature range of 30-42°C, and inhibitory compounds concentration range of 0.1-3 g/L.

[00114] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (h) is carried out under aerobic conditions.

[00115] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (h) is carried out under anaerobic conditions.

[00116] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein step (h) is carried out at 32°C. [00117] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein in step (e) pre-treated solid biomass w/v percentage is 0.5%.

[00118] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein in step (e) liquid hydrolysate v/v percentage is in the range of 1-3%.

[00119] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein in step (e) liquid hydrolysate v/v percentage is 2%.

[00120] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said at least one nitrogen source weight percentage is in the range of 0.5-2%.

[00121] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said at least one nitrogen source weight percentage is 1%.

[00122] In an embodiment of the present disclosure, there is provided a method of ethanol production as described herein, wherein said at least one nitrogen source is yeast extract.

[00123] In an embodiment of the present disclosure, there is provided a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw; (b) pre-treating wheat straw to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said solid loading w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts, 0.5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 2% at a temperature of 42°C for 24-96 hours under aerobic conditions to produce carbohydratases (endoglucanase, exoglucanase, and xylanase); (f) enriching said carbohydratases to obtain concentrated carbohydratases; and (g) incubating said concentrated carbohydratases with said pre-treated solid biomass at a temperature of 50°C for 24-96 hours under aerobic conditions to produce said sugar comprising predominantly 70-80% hexose, and pentose; and (h) contacting said Candida strain with hexose and pentose sugars at 32°C for 24-48 hours under aerobic conditions to produce ethanol.

[00124] In an embodiment of the present disclosure, there is provided a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw; (b) pre-treating wheat straw to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said solid loading w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts, 0.5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 2% at a temperature of 42°C for 24-96 hours under aerobic conditions to produce carbohydratases (endoglucanase, exoglucanase, and xylanase); (f) enriching said carbohydratases to obtain concentrated carbohydratases; and (g) incubating said concentrated carbohydratases with said pre-treated solid biomass at a temperature of 50°C for 24-96 hours under aerobic conditions to produce said sugar comprising predominantly 70-80% hexose, and pentose; and (h) contacting said Candida strain with hexose and pentose sugars at 32°C for 24-48 hours under anaerobic conditions to produce ethanol.

[00125] In an embodiment of the present disclosure, there is provided a method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at least one lignocellulosic biomass: (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6- 7; and (e) contacting said isolated Candida strain HP-CAT01 having MTCC accession number MTCC 25057 with minimal salts or at least one nitrogen source, and said liquid hydrolysate at a temperature in the range of 30-42°C for 24-72 hours under aerobic conditions to promote xylitol production.

[00126] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00127] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 150°C for 15 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00128] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.

[00129] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein lignocellulosic mass is wheat straw.

[00130] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein said minimal salts are 0.5 g/L MgS0₄.7H₂0, 0.5 g/L KC1, 1 g/L KH₂P0₄, and 2.5 g/L NaN0₃.

[00131] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein said isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057 is active in a pH range of 4-10, a temperature range of 30-42 C, and inhibitory compounds concentration range of 0.1-3 g/L.

[00132] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein neutralization in step (d) is carried out using ammonia.

[00133] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein step (e) is carried out at 32°C for 24-72 hours.

[00134] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein said minimal salts are 0.5 g/L MgS0₄.7H₂0, 0.5 g/L KC1, 1 g/L KH₂P0₄, and 2.5 g/L NaN0₃.

[00135] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein said at least one nitrogen source weight percentage is in the range of 0.5-2%.

[00136] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein said at least one nitrogen source weight percentage is 1%.

[00137] In an embodiment of the present disclosure, there is provided a method of xylitol production as described herein, wherein said at least one nitrogen source is yeast extract.

[00138] In an embodiment of the present disclosure, there is provided a method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw: (b) pre-treating said wheat straw to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said biomass w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; and (e) contacting said isolated Candida strain HP-CAT01 having MTCC accession number MTCC 25057 with minimal salts, and said liquid hydrolysate at a temperature of 32°C for 24-72 hours under aerobic conditions to promote xylitol production.

[00139] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining at last one lignocellulosic biomass; (b) pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% pre- treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1- 5% at a temperature in the range of 30-42°C for 24-96 hours under aerobic conditions to produce said carbohydratases; (f) separating said Candida strain population from (e) to obtain a cell free liquid hydrolysate; (g) contacting said pre-treated solid biomass to said cell free liquid hydrolysate at a temperature in the range of 45-55°C for 24-96 hours under aerobic conditions to promote production of sugars; and (h) contacting said isolated Candida strain HP-CAT01 having accession number MTCC 25057 with sugars from step (g) at a temperature in the range of 30-37°C for 24-48 hours in aerobic conditions to promote ethanol and xylitol production.

[00140] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein in step (e) pre- treated solid biomass w/v percentage is 0.5%.

[00141] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein in step (e) liquid hydrolysate v/v percentage is in the range of 1-3%. [00142] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein in step (e) liquid hydrolysate v/v percentage is 2%.

[00143] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said sugar is 70- 80% hexose, and pentose.

[00144] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

[00145] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said carbohydratases are endoglucanase, exoglucanase, and xylanase.

[00146] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00147] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00148] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%. [00149] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric acid at 150°C for 15 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

[00150] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein in step (e), temperature is 32°C, and time is 24-96 hours.

[00151] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein in step (e), temperature is 42°C, and time is 24-96 hours.

[00152] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein neutralization in step (d) carried out using ammonia.

[00153] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.

[00154] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein lignocellulosic mass is wheat straw.

[00155] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said minimal salts are 0.5 g/L MgS0₄.7H₂0, 0.5 g/L KC1, 1 g/L KH₂P0₄, and 2.5 g/L NaN0₃.

[00156] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said at least one nitrogen source weight percentage is in the range of 0.5-2%. [00157] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said at least one nitrogen source weight percentage is 1%.

[00158] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said at least one nitrogen source is yeast extract.

[00159] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057 is active in a pH range of 4-10, a temperature range of 30^-2 °C, and inhibitory compounds concentration range of 0.1-3 g/L.

[00160] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein step (h) is carried out at 32°C for 24-48 hours.

[00161] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production as described herein, wherein said method further comprises the step of fractional distillation to separate ethanol from xylitol.

[00162] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw; (b) pre-treating wheat straw to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said solid loading w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts, 0.5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 2% at a temperature of 42°C for 24-96 hours under aerobic conditions to produce carbohydratases (endoglucanase, exoglucanase, and xylanase); (f) separating said Candida strain population from step (e) to obtain a cell free liquid hydrolysate; (g) contacting said pre-treated biomass to said cell free liquid hydrolysate at a temperature of 50°C for 24-96 hours under aerobic conditions to promote production of sugars comprising predominantly 70-80% hexose, and pentose; and (h) contacting said isolated Candida strain with sugars from step (g) at a temperature of 32 °C for 24-48 hours in aerobic conditions to promote ethanol and xylitol production.

[00163] In an embodiment of the present disclosure, there is provided an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of: (a) obtaining wheat straw; (b) pre-treating wheat straw to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with phosphoric acid having v/v concentration of 0.5%, and said solid loading w/v concentration is 10% at 150°C for 15 minutes; (c) separating said liquid hydrolysate, and said pre-treated solid biomass; (d) neutralizing said liquid hydrolysate by adjusting the pH with ammonia in the range of 6-7; (e) contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts, 0.5% w/v pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 2% at a temperature of 42°C for 24-96 hours under aerobic conditions to produce carbohydratases (endoglucanase, exoglucanase, and xylanase); (f) separating said Candida strain population from step (e) to obtain a cell free liquid hydrolysate; (g) contacting said pre-treated biomass to said cell free liquid hydrolysate at a temperature of 50°C for 24-96 hours under aerobic conditions to promote production of sugars comprising predominantly 70-80% hexose, and pentose; (h) contacting said isolated Candida strain with sugars from step (g) at a temperature of 32 °C for 24-48 hours in aerobic conditions to promote ethanol and xylitol production; and (i) separating ethanol from xylitol by fractional distillation. [00164] In an embodiment of the present disclosure, there is provided a method as described herein, wherein said carbohydratases can be produced by any other microorganism.

[00165] In an embodiment of the present disclosure, there is provided a method as described herein, wherein said sugars can be fermented by carbohydratases produced by Candida strain MTCC 25057, any other microbial strain, or by combination of strains.

[00166] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of ethanol from lignocellulosic biomass.

[00167] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of ethanol from lignocellulosic biomass by a process as described herein.

[00168] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol from lignocellulosic biomass.

[00169] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol from lignocellulosic biomass by a process as described herein.

[00170] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol and ethanol from lignocellulosic biomass.

[00171] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol and ethanol from lignocellulosic biomass by a method as described herein.

[00172] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of carbohydratases from lignocellulosic biomass. [00173] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of carbohydratases from lignocellulosic biomass by a method as described herein.

[00174] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of sugars from lignocellulosic biomass.

[00175] In an embodiment of the present disclosure, there is provided an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of sugars from lignocellulosic biomass by a method as described herein.

[00176] In an embodiment of the present disclosure, there is provided a bio-plant for production of bioethanol, xylitol, value added products, sugars, and enzymes from lignocellulosic biomass using an isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057.

[00177] In an embodiment of the present disclosure, there is provided a bio-plant as described herein, schematic representation of which is as set forth in Figure 6.

[00178] Although the subject matter has been described in considerable detail with reference to certain preferred embodiments thereof, other embodiments are possible.

EXAMPLES [00179] The disclosure will now be illustrated with working examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.

Example 1

Materials and methods

[00180] Microorganism and culture conditions: Soil samples were collected from Sri Chamarajendra Zoological Gardens, Mysore, India and stored in sterile containers at 4°C until subsequent use. Approximately 1 g of soil sample was inoculated into yeast extract peptone dextrose broth (YEPD) and incubated at 32 °C for 48 hr. On obtaining growth, the liquid culture was serially diluted in sterile saline and plated onto agar plates with 1% CMC which were incubated at 32 °C for 48 hr. The colonies obtained were sub-cultured till pure isolates were obtained. Upon microscopic observations, one of the isolated colonies was found to be a yeast. The isolate was maintained on YEPD agar slants at 4 °C. Liquid cultures of the isolate were obtained by inoculating a loopful of culture from YEPD slants into YEPD broth and incubating at 32 °C for 24 h at 150 rpm.

[00181] Identification of new strain: Strain identification of the new yeast isolate was done by sequencing the 18S, 5.8S and 26S rRNA and the isolate was found to be Candida tropicalis. The primers used for strain identification spanned the 18S rRNA, the variable D 1 -D2 domain of the 26S rRNA and the internal transcribed spacer region of the 5.8S rRNA. The primer sequences were as follows 18S_forward primer (SEQ ID NO: 1), 18S_reverse primer (SEQ ID NO: 2), 26S_forward primer (SEQ ID NO: 3), 26S_reverse primer (SEQ ID NO: 4), ITS1 forward primer (SEQ ID NO: 5), and ITS4 reverse primer (SEQ ID NO: 6). The PCR conditions used were: initial denaturation at 95 °C for 5 minutes, followed by thirty cycles of denaturation, annealing and extension at 95 °C, 52 °C and 72 °C, respectively for 30 seconds or 1 minute. A final product extension at 72 °C for ten minutes was also done before analyzing the PCR products by gel electrophoresis. The PCR products were purified using the GeneJET gel extraction kit (Thermo Fisher Scientific) before sequencing. The strain was submitted to Microbial Type Culture Collection (MTCC), India and assigned the accession number -MTCC 25057.

[00182] Biomass pretreatment and enzymatic hydrolysis: Wheat straw procured from local sources around Bangalore was used in the present disclosure. Wheat straw was subjected to phosphoric acid (0.2% v/v) pretreatment as described previously (Prabu et al., RSC Adv 2015, 5: 51642). The cellulose, hemicellulose and lignin composition of the wheat straw was determined using standard NREL procedures (Sluiter et al., Laboratory analytical procedure. NREL/TP-510-42618 2011). The liquid hydrolysate obtained was neutralized to pH 6 - 7 using ammonia and used for cellulolytic enzyme production. The solid biomass obtained after phosphoric acid pretreatment was washed with distilled water, dried at 60°C and used for enzymatic hydrolysis. Briefly, 5 g of pretreated biomass, 20 Ug^_1ds enzyme and 50 mL sodium citrate buffer (50 mM, pH 4.8) was taken in 250 mL Erlenmeyer flasks and incubated at 50 °C and 150 rpm shaking for 24 - 48 h. The sugar hydrolysate was separated by centrifugation at 15,000 rpm for 10 min and the reducing sugar content determined by uHPLC.

[00183] Cellulase and xylanase production under submerged fermentation: Submerged fermentation was carried out by inoculating 0.2 ml of the exponential phase MTCC 25057 culture to 250 mL Erlenmeyer flasks containing 20 mL of wheat straw hydrolysate supplemented with minimal salts. The composition of minimal salts (gL ¹) was - NaN0₃: 2.5, KH₂P0₄: 1, MgS0₄.7H₂0: 0.5, KC1: 0.5. To these flasks, cellulosic substrates (CMC or pretreated WS) at different concentrations (0.5% w/v, 1% w/v and 2% w/v) was added. The cultures were incubated on a rotary shaker (150 rpm) at different temperatures (32 °C and 42 °C) and for varying periods (24 h, 48 h, 72 h and 96 h). The crude supernatant obtained by centrifuging the culture broth at 8000 rpm for 10 min was used for determining enzyme activity.

[00184] Enzyme assays: Crude endoglucanase activity was determined by the CMC method (Ghose TK., Pure Appl Chem 1987, 69: 257-266). The substrate was prepared by solubilizing carboxymethyl cellulose (Sigma) (2 % w/v) in 0.05 M sodium citrate buffer at pH 4.8. Briefly, 0.5 ml of the crude enzyme supernatant (diluted appropriately) was added to 0.5 mL of the substrate and incubated at 50 °C for 30 minutes. The reducing sugars liberated were estimated by the DNS method (Miller GL., Anal Chem 1959, 39: 426 - 428). Crude exoglucanase activity was determined by the filter paper assay method (Mendels et al., Biotechnol Bioeng Symp., 1976, 6: 21- 33). The substrate used was 50 mg of Whatman No. 1 filter paper in 0.05 M sodium citrate buffer at pH 4.8. The crude enzyme supernatant (0.5 mL) was added to 1 mL of the buffer having 50 mg filter paper and incubated at 50 °C for 60 min and the reducing sugars liberated were estimated by the DNS method. Xylanase activity was determined by the beechwood xylan assay method (Ghose TK., Pure Appl Chem 1987, 69: 257-266). Substrate preparation was done by adding beechwood xylan (Sigma) (1 % w/v) to 0.05 M sodium citrate buffer, pH 4.8. The appropriately diluted crude enzyme supernatant (0.1 mL) was added to 0.5 mL of the substrate and 0.4 mL of buffer and incubated at 50 °C for 15 min. The reducing sugars released were measured by the DNS method. All the estimations were carried out in triplicates. The enzyme activity was expressed in terms of International units (IU) for endoglucanase and xylanase and Filter Paper Unit (FPU) for exoglucanase, respectively, or as units per gram of dry substrate (gds) i.e. Ug^_1ds . One IU or FPU is the defined as the amount of enzyme activity required to release one micromole of reducing sugar from a cellulosic substrate (CMC, xylan, filter paper etc.) per mL in a minute under the suitable assay conditions.

[00185] Fermentation conditions: The C. tropicalis MTCC 25057 strain was grown in 50 mL of yeast extract - peptone broth (20 gL^-1 peptone and 10 gL^-1 yeast extract) supplemented with either 10% glucose, 10% xylose or a mixture of 5% glucose and 5% xylose in 250 mL Erlenmeyer flasks in an incubator shaker maintained at 32 °C and 150 rpm. Samples were collected at appropriate intervals and the metabolites produced were analysed using UHPLC. Anerobic condition was maintained by flushing nitrogen initially through the headspace of the flask and then incubating at 32 °C as mentioned previously. For inhibitor tolerance studies, furfural, 5 - hydroxymethylfurfural or acetic acid (Himedia Laboratories) were added to yeast extract - peptone broth with 1.5% xylose and 0.5% glucose (to simulate pretreated hydrolysate), in concentrations ranging from 0.5 - 3 gL^"1 and grown at 32 °C for 24 h. The yeast was grown in YEPD i.e. yeast extract peptone broth with 20 gL^"1 glucose medium maintained at different pH (4, 5, 6, 7, 8, 9, 10) and at different temperatures (32, 37, 42, 45, 50 °C) for 24 h for determining the pH and temperature tolerance of the strain.

[00186] The pretreated and sugar hydrolysates were supplemented either with minimal salts (as mentioned previously) or with 10 gL^"1 yeast extract for ethanol/xylitol production studies. The yeast was inoculated from a fresh plate into 50 mL hydrolysate and incubated at 150 rpm at 32 °C in 250 mL Erlenmeyer flasks. The samples were collected at suitable intervals and analysed.

[00187] Analytical methods: The soluble sugars or metabolites content in the culture samples were determined using a 1290 Infinity series UHPLC system (Agilent) equipped with a Hiplex H anion exchange column (Agilent). Filtered and degassed 5mM H₂SO₄ was used as the mobile phase at a flow rate of 0.4 mL/min. The column was maintained at 45°C in a thermostat chamber while the refractive index (RI) detector was maintained at 45°C. The concentrations of glucose, xylose, ethanol and xylitol were estimated using appropriate calibration curves. The biomass or cell density of the culture samples was estimated by measuring the optical density at 600 nm using a Perkin Elmer Lambda 35 UV/visible spectrophotometer.

[00188] Li nocellulose biomass and its pre treatment: Wheat straw was shredded using a pulveriser to achieve particle size less than 2mm. To 10% (w/v) of wheat straw, phosphoric acid to a final concentration of 0.5% was added. The sample was heated at 150°C for 15 minutes in a pressurized reactor (PARR) for pre treatment. Example 2

Tolerance of Candida tropicalis MTCC 25057 to inhibitory compounds

[00189] For utilizing sugars in lignocellulosic hydrolysates, the biocatalyst has to withstand the presence of inhibitory compounds such as furfural, HMF and acetic acid that are usually generated during biomass pretreatment (Almeida et al., Appl Microbiol Biotechnol 2009, 82: 625-638). In the instant disclosure, wheat straw procured from local sources was subjected to pretreatment with 0.2 % (v/v) phosphoric acid as optimized previously (Huila et al., Environ Prog Sus Energ 2015, 34, 1243-1248). The lignocellulosic hydrolysate contained glucose (~ 0.5 % w/v) and xylose (~ 1.5 % w/v) as the major components along with inhibitory compounds such as furfural and HMF (~ 0.01 % w/v). Several reports indicate the formation of acetic acid during biomass pretreatment, although the same was not detected in the present instant. Preliminary studies for determining the tolerance of the MTCC 25057 yeast strain to higher concentrations of furfural, HMF as well as acetic acid, which might get generated during harsher pretreatments indicate that the Candida isolate tolerates furfural, HMF and acetate concentrations of 1.5 gL^"1, 2 gL^"1 and 2 gL^"1, respectively (Fig 1 a). Further increase in the levels of furfural, HMF and acetic acid resulted in a gradual lowering of biomass densities (Fig la). It has been previously reported that fermentative capacities of Candida tropicalis isolate to produce ethanol and xylitol in hydrolysate could not function beyond increased furfural, HMF and acetate levels of 0.23 gL^"1, 0.15 gL^"1 and 1.37 gL^"1 respectively (Cheng et al., Biotechnol Biofuel 2014, 7: 166). In comparison, quite unexpectedly and surprisingly, the new yeast isolate (MTCC 25057) was found to be more tolerant to commonly occurring lignocellulosic inhibitors.

[00190] In addition to ethanol toxicity, osmotic stress and pH variations (Zhao et al., J Biotechnol., 2009, 144, 23-30; Arino J., OMICS J Integrative Biol., 2010, 14, 517- 523), commercial scale ethanol bioprocesses also encounter increased temperatures during summer seasons during which the yeast is supposed to perform as normally as under optimal conditions (Daquinag et al., Mol Cell Biol., 2007, 27, 633-650). It was observed that cell growth of the new yeast isolate was not affected over a wide range of temperatures (32 °C - 42 °C) and pH (4 - 10) (Fig lb, lc). Also, the cellulase and xylanase production by the yeast isolate remained largely unaffected over a wide range of temperatures (32 °C - 42 °C).

Example 3 Cellulase and xylanase production by Candida tropicalis MTCC 25057

[00191] It has been previously reported that temperature of incubation and the nature of substrates mainly affect cellulolytic enzyme production (Tanskul et al., Carbohyd Polym., 2013, 92: 421 - 428; Berger et al., FEMS Microbiol Lett., 2007, 268: 194- 201). In order to ascertain the effect of temperature, and pH on enzyme production by the strain of the instant disclosure, cellulase (endoglucanase and exoglucanase) and xylanase production at different temperatures (32 °C and 42 °C), on different cellulosic substrates (CMC and WS) at different concentrations (w/v) of 0.5 %, 1% and 2 % in pretreated lignocellulosic hydrolysate was ascertained (Fig. 2). Maximum endoglucanase activity of 98 ± 2 Ug^_1ds was obtained using 0.5 % WS at 42°C (Fig 2a). Similarly, the exoglucanase production in hydrolysate containing 0.5 % wheat straw was maximum (114 ± 3 Ug^_1ds) at 42°C (Fig 2b). At lower incubation temperature of 32°C, the endo and exoglucanase levels in hydrolysate were lower than their corresponding levels at 42°C with both the substrates and at all the concentrations tested (Fig 2a, 2b).

[00192] Previously, it has also been reported that cellulolytic enzyme blends with approximately equal exoglucanase and endoglucanase activities are more suitable for optimal biomass hydrolysis (Lambertz et al., Biotechnol Biofuel., 2014, 7: 135). In the present instance it was observed that amongst the two temperatures tested, maximum endoglucanase (Fig 2a) and exoglucanase (Fig 2b) activities were obtained at 42°C while 0.5% WS was found to be a more suitable cellulosic substrate. The ratios of endo and exo glucanases at 42°C were proportional and their activities were comparable to the previously reported cellulase expression levels by well-known enzyme producer strains such as Trichoderma and Aspergillus sp. (Sohail et al., New Biotechnol., 2009 6: 437^141; Castro et al., Appl Biochem Biotechnol., 2010, 162: 2111-2122; Lee et al., Composition of herbaceous biomass feedstocks - North Central Centre, South Dakota State University 2007).

[00193] Regarding xylanases, the activity obtained at 32°C was marginally higher (825 ± 14 Ug^_1ds) than that obtained at 42°C (689 ± 17 Ug^_1ds) with 0.5 % WS (Fig 2c) in hydrolysate. The expression of xylanases with wheat straw could be attributed to the presence of xylan (Park et al., Appl Microbiol Biotechnol., 2002, 58: 761-766); however it is interesting and surprising that xylanase production is observed even in presence of CMC, which is apparently xylan-free.

Example 4

Wheat straw hydrolysis

[00194] Different acids have been used to carry out biomass pretreatments (Kim et al., Bioresour Technol., 2005, 96, 1249-1255; Sun et al., Bioresour Technol., 2002, 83, 1-11) and the use of phosphoric acid is known to generate lesser inhibitory compounds in the hydrolysate (Vasconselos et al., Bioresour Technol., 2013, 135, 46- 52). The quantity of inhibitory compounds generated during biomass pretreatment is mainly dependent on pretreatment severity. In the present disclosure, wheat straw pretreatment was done by using 0.2 % phosphoric acid at 150°C for 15 minutes (Kuila et al., Environ Prog Sus Energ., 2015, 34, 1243-1248). The cellulose, hemicellulose and lignin content of biomass before and after phosphoric acid pretreatment was determined (Table 1). Phosphoric acid pretreatment of wheat straw caused approx. 65 % lignin removal and 64 % hemicellulose hydrolysis. After separating the hydrolysate and neutralizing the biomass, the cellulosic biomass was subjected to hydrolysis with the enzymes produced by the novel yeast strain at an approximate enzyme loading of 20 U per gram of wheat straw for 48 hours at 50 °C. The sugar hydrolysate generated contained about 500 mg glucose and 30 mg xylose per gram of dry wheat straw used. Table 1

Example 5 Pentose and hexose fermentation Candida tropicalis MTCC 25057 in synthetic media

[00195] Biomass consists of approx. 20-40 % of hemicellulose (Bruinenberg et al., J Gen Microbiol., 1983, 129: 965-971), which upon hydrolysis, releases xylose as the main component. The naturally occurring industrial ethanol producing yeast, Saccharomyces cerevisae, cannot utilize xylose and hence a hexose and pentose fermenting microorganism is needed. Candida sp. is a distinctive class of yeast that can ferment glucose into ethanol and xylose into xylitol and ethanol (Cheng et al., Biotechnol Biofuel., 2014, 7: 166; Qu et al., Biotechnol J., 2006, 1,1235-1240; Bruinenberg et al., Gen Microbiol 1983, 129: 965-971; Yokoyama et al., J Ferment Bioeng., 1995, 79: 217-223). Studies have shown that different strains of Candida sp. produce optimum levels of xylitol (from xylose) and ethanol (from glucose and xylose) under aerobic and anaerobic conditions respectively (Cheng et al., Biotechnol Biofuel., 2014, 7: 166).

[00196] It was presently observed that Candida (MTCC 25057) cell growth and ethanol production patterns were similar under both, aerobic and anaerobic conditions (Fig 3a, b). Ethanol production using Candida strains is essentially an anaerobic process. However, it was observed that the Candida isolate of the instant disclosure could produce comparable concentrations of ethanol under aerobic conditions although the productivities were decreased. On culturing the yeast strain in synthetic YEPD media under anaerobic conditions, maximum ethanol levels of ~ 36 gL^"1 were obtained within 24 h; whereas similar ethanol concentrations were attained under aerobic conditions after 48 h of incubation (Fig 3a, b). Under anaerobic and aerobic conditions, the corresponding glucose consumption of 100 % and 80 % was observed within 24 hours. Under aerobic conditions, the complete consumption of the remaining 20 % glucose required extended incubation for up to 48 hours (Fig 3a). Therefore, the ethanol yields (0.36 gg^"1 i.e. ~ 72% conversion) were similar while the ethanol productivities under anaerobic conditions (1.5 gL^h^"1) were higher than under aerobic conditions (0.75 gL^"1]!^"1). Under both the conditions, approx. 3-4 gL^"1 glycerol was detected in the medium.

[00197] The Candida tropicalis isolate could also utilize xylose under aerobic conditions and produce xylitol (68 ± 2 gL^"1) within 96 h of fermentation (Fig 3c) when cultured in YEPD media. The xylitol yields and productivities obtained during aerobic fermentations were 0.67 gg^"1 (67% conversion) and 0.70 gL^h^"1 respectively. No consumption of xylose was observed under anaerobic conditions even after extended incubation up to 120 hours.

[00198] Since lignocellulosic hydrolysate contains a mixture of pentose and hexose sugars, it was next determined of the yeast isolate could ferment both simultaneously. Therefore, in order to simulate fermentation of lignocellulosic hydrolysate containing mixed sugars (hexose and pentose), the strain of Candida tropicalis was cultured in synthetic lab media in which 50 gL^"1 each of xylose and glucose had been added. The anaerobic cultivation of C. tropicalis in glucose - xylose medium led to complete utilization of glucose within 24 h whereas xylose remained unutilized in the medium (Fig 4a). The maximum ethanol concentrations obtained under anaerobic (18.8 ± 0.8 gL^"1) and aerobic (20.4 ± 1.3 gL^"1) conditions as well as the ethanol yields of 0.37 gg^"1 and 0.4 gg^"1 were found to be similar. However, no xylitol was detected during anaerobic fermentations of Candida tropicalis even till the end of fermentation (Fig 4a). Under aerobic cultivation, xylose utilization started 48 h after complete utilization of glucose (Fig 4b) and a xylitol yield and productivity of 0.93 gg^"1 and 0.28 gL^h^"1, respectively was obtained. The xylitol yield obtained in case of mixed sugar fermentation was higher than the previously reported values in the range of 0.49 - 0.84 gg^"1 produced by other Candida strains (Miura et al., Wood Sci Technol., 2013, 47: 515-522; Ping et al., Biochem Eng J., 2013, 75, 86-91). It was also observed that the yield of xylitol obtained when xylose was the sole carbon source was lower (0.67 gg^"1) than the yield (0.93 gg^"1) obtained on using xylose and glucose in the media.

Example 6

Pentose and hexose fermentation Candida tropicalis MTCC 25057 in hydrolysate [00199] Although wheat straw pretreatment with 0.2 % phosphoric acid has been optimized for minimal inhibitor generation as mentioned previously, in the instant disclosure harsher pretreatment conditions (4% phosphoric acid) have been used so as to achieve maximal xylose generation in the hydrolysate as the yeast isolate (MTCC 25057) has the capability to withstand high inhibitor concentrations. Interestingly, even after the use of such highly acidic conditions for pretreatment, the amount of inhibitors produced was minimal, 1 gL^"1 furfural and 0.075 gL^"1 HMF, reaffirming the use of phosphoric acid as an effective yet gentle pretreatment agent. The pretreated hydrolysate generated was fermented initially using the new yeast isolate after supplementation with minimal salts only. However, no ethanol or xylitol production was observed. Subsequently, in order to meet the nutritional deficiencies, an additional nitrogen source (1 % yeast extract) was added to the hydrolysate which resulted in increased xylose utilization. Almost 60% of the xylose was consumed within the first twelve hours of fermentation itself and no residual xylose was observed beyond 72 h of fermentation (Fig 5a). Xylitol production reached maximum levels of -15 ± 1 gL^"1 in 72 h with an average yield of ~ 32%. The hydrolysate also contained some amount of glucose (10 gL^"1) which was converted into ethanol (~ 4.5 gL^"1) in the initial 24 hours.

[00200] In a similar study reported earlier, fermentation of concentrated corn cob hydrolysate (without nutritional supplementation) using a hydrolysate adapted Candida strain resulted in a xylitol yield of approx. 14.81 % with most of the xylose being diverted for biomass formation which affected the final yields (Li et al., Chem Eng J., 2015, 263, 249-256). In the present experiments, a xylitol yield of ~ 32% was achieved using a wild type Candida isolate although here too, most of the sugar was being used for cell growth instead of xylitol production.

[00201] The sugar hydrolysate obtained post enzymatic saccharification of the pretreated wheat straw was also fermented using the same yeast strain. This hydrolysate had more hexoses than pentoses as it is derived from the cellulose rich biomass. In this case also, supplementation of the hydrolysate with additional nitrogen source led to greater diversion of the sugars to cell growth than to product formation. The yeast strain was able to convert ~ 25 gL^"1 glucose to 7.3 ± 1.1 gL^"1 ethanol with a net yield of 0.28 gg^"1 (Fig 5b), which was slightly lower than the ethanol yield observed in case of mixed sugar fermentation in synthetic medium. The sugar hydrolysate also contained ~ 7.5 gL^"1 of xylose that was converted to xylitol in 24 h with a yield of 0.31 gg^"1. The ethanol yield and productivity could be enhanced further by fine-tuning the addition of nutritional supplements so as to ensure maximum sugar fermentation into ethanol production. The strain of the present disclosure shows cellulolytic as well as ethanol fermenting abilities. In addition, the yeast strain also exhibits significant inhibitor tolerance and produces xylitol from hemicellulosic sugars.

Example 7

Steps for integrated production of ethanol, xylitol, enzymes, sugars, and value added products

[00202] Figure 6 outlines the steps involved for the integrated production of xylitol, ethanol, enzymes and value added products at an industrial scale.

[00203] The Candida tropicalis strain described here is versatile and capable of producing carbohydratases to degrade lignocellulosic biomass as well as produce ethanol and xylitol. In addition, the theremostability and high inhibitor tolerance of this strain enable its cultivation at higher temperatures and inhibitor concentrations, thus minimizing the risk of contamination from other microorganisms. In addition, this strain can be grown directly in pretreated hydrolysate without conventional detoxification steps like liming, thus simplifying the overall process as well as minimizing waste generation. This strain can therefore be used in an integrated biorefinery approach for the simultaneous production of carbohydratases, ethanol, xylitol etc. and the process flow scheme for the same is described below.

[00204] Lignocellulosic biomass like wheat straw, rice straw, sorghum straw or any other agricultural feedstock is mechanically ground in a pulverizer so that the average particle size is reduced to 3-4 mm. This fine biomass is fed to the Pretreatment Reactor by means of a conveyor belt. The biomass is then subjected to acid or alkali pretreatment and/or steam explosion in high temperature and high pressure conditions, so that the lignin and hemicellulose component can be degraded so as to expose the cellulose part. The pretreatment process results in the production of a xylose rich liquid hydrolysate and a glucose rich solid biomass, which are sent to two separate tanks, namely the neutralization tank and the saccharification tank, respectively.

[00205] The acidic hydrolysate is neutralized in the Neutralization Tank using either caustic soda or ammonia to achieve a final pH of 6 - 7. If ammonia is used to neutralize phosphoric acid pretreated hydrolysate, a white precipitate of ammonium phosphate is obtained which can be used as a nitrogen source for growing the Candida tropicalis MTCC 25057 strain needed in the subsequent steps. Additionally, it can be used as a cheap and effective fertilizer in agriculture. The precipitate obtained in case of sulphuric acid neutralization with caustic soda, will have to be washed off the neutralization tank.

[00206] The neutralized pretreated hydrolysate is routed to either the xylitol production reactor or the enzyme production reactor. In the former, the hydrolysate is mixed with minimal salts and inoculated 1 % (v/v) with Candida tropicalis MTCC 25057 and incubated at a temperature ranging from 30 - 42 °C with stirring for 24 - 48 hours for the production of xylitol. In the enzyme production reactor, the hydrolysate along with minimal salts and pretreated solid biomass is inoculated 1 % (v/v) with Candida tropicalis MTCC 25057 and incubated at 30 - 42 °C with stirring for 24 - 48 hours for the production of carbohydratases like endoglucanase, exoglucanase, xylanase, beta glucosidase etc. The enzyme rich supernatant is concentrated almost 30 fold using a tangential flow diafiltration system and the concentrated suspension is sent to the saccharification tank. Here, the enzymes are allowed to hydrolyse the solid biomass at 50 °C for 24 - 72 hours for the production of soluble sugars. After the separation of the solid and liquid phases, the glucose rich liquid hydrolysate is sent to the ethanol production reactor while the solid treated biomass is conveyed to the burner/turbogenerator unit. In the ethanol reactor, the liquid hydrolysate along with minimal salts is inoculated 1% (v/v) with Candida tropicalis MTCC 25057 and incubated in the temperature range of 30 - 42 °C with stirring for 24 - 48 hours for the production of ethanol. The broth from this reactor is then diverted to a wash settling tank and the liquid is sent to a distillation/ethanol purification unit from where the ethanol extracted is deposited in a storage tank while the water is sent directly for wastewater treatment. Thus in this integrated approach, a single organism is used to produce various high value products like carbohydratases, ethanol, xylitol, ammonium phosphate etc. Moreover, all components of the lignocellulosic biomass are used gainfully, with minimal waste generation.

[00207] An ideal consolidated bioprocessing (CBP) operation for lignocellulosic bioethanol production would depend upon higher expression of hydrolytic enzymes for optimum biomass hydrolysis and the rapid fermentation of released sugars, all occurring within a relatively narrow range of process parameters. In actuality, since the temperature optima for enzyme production (30-32°C), biomass hydrolysis (50°C) and fermentation (30-32°C) vary, the processes are still carried out in a successive manner. In the present disclosure, the inhibitor tolerant strain of Candida exhibits maximal enzyme production (42°C and pH 6-7) and biomass hydrolysis (at 50°C and pH 5) within a relatively narrower range of process parameters; thereby indicating its potential for CBP applications.

[00208] A single microorganism that expresses cellulolytic enzymes for hydrolysis of biomass and ferments the released sugars into ethanol and other value added products such as xylitol is of potential interest to the industry. The present disclosure describes a yeast strain isolated from soil samples and identified as Candida tropicalis MTCC 25057, which expresses cellulases and xylanases over a wide range of temperatures (32 °C, and 42 °C as tested) and in the presence of different cellulosic substrates such as carboxymethylcellulose (CMC), and wheat straw (WS). Further, cultivation of the novel yeast isolate at 42 C in pre-treated hydrolysate containing 0.5 % WS results in a proportional expression of cellulases (exo and endoglucanases) at concentrations of 114.1 Ug^_1ds and 97.8 Ug^_1ds, respectively. A high xylanase activity (689.3 Ug^_1ds) is also exhibited by the yeast isolate under similar growth conditions. Maximum expression of cellulolytic enzymes by the yeast isolate occurred within 24 hours of incubation. Of the sugars released from biomass after pretreatment, 49 gL^"1 xylose was aerobically converted into 15.8 gL^"1 of xylitol. In addition, 25.4 gL^"1 glucose released after the enzymatic hydrolysis of biomass was fermented by the same yeast isolate to obtain an ethanol titre of 7.3 gL^"1. Interestingly, the yeast strain of the instant disclosure exhibits enhanced xylitol, and ethanol production even in the presence of lignocellulosic inhibitors such as furfural, HMF, and acetic acid.

Claims

I/We claim:

1. An isolated Candia tropicalis strain HP-CATOl having accession number MTCC 25057.

2. A method of carbohydratases production using an isolated Candia strain HP- CATOl having accession number MTCC 25057, said method comprising the steps of:

a. obtaining at least one Hgnocellulosic biomass;

b. pre-treating said Hgnocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%;

c. separating said liquid hydrolysate, and said pre-treated solid biomass; d. neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7; and

e. contacting Candia strain HP-CATOl having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre- treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42°C for 24-96 hours under aerobic conditions to produce said carbohydratases.

3. The method as claimed in claim 2, wherein step (b) comprises contacting said hgnocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

4. The method as claimed in claim 2, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

5. A method of production of at least one sugar using an isolated Candia strain HP-CATOl having accession number MTCC 25057, said method comprising the steps of: a. obtaining at least one lignocellulosic biomass;

b. pre-treating said lignocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%;

c. separating said liquid hydrolysate, and said pre-treated solid biomass; d. neutralizing said liquid hydrolysate by adjusting the pH in the range of 6-7;

e. contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% w/v pre- treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1-5% at a temperature in the range of 30-42° for 24-96 hours under aerobic conditions to produce said carbohydratases;

f. enriching said carbohydratases to obtain concentrated carbohydratases; and

g. incubating said concentrated carbohydratases with said pre-treated solid biomass at a temperature in the range of 48-52°C for 24-96 hours under aerobic conditions to produce said sugar.

6. The method as claimed in claim 5, wherein said sugar is 70-80% hexose.

7. The method as claimed in claim 5, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

8. The method as claimed in claim 5, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

9. A method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of:

a. obtaining at least one lignocellulosic biomass; b. pre-treating said Hgnocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%;

e. contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, 0.5-5% pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1 - 5% at a temperature in the range of 30-42° for 24-96 hours under aerobic conditions to produce said carbohydratases;

f. enriching said carbohydratases to obtain concentrated carbohydratases; g. incubating said concentrated carbohydratases with said pre-treated solid biomass at a temperature in the range of 48-52°C for 24-96 hours under aerobic conditions to produce said sugar; and

h. contacting said Candida strain with said sugar at 30-42°C for 24 -48 hours under aerobic or anaerobic conditions to produce ethanol.

10. The method as claimed in claim 9, wherein said sugar is predominantly hexose.

11. The method as claimed in claim 9, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

12. The method as claimed in claim 9, wherein step (b) comprises contacting said hgnocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

13. A method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of:

a. obtaining at least one Hgnocellulosic biomass; b. pre-treating said Hgnocellulosic biomass to obtain a liquid hydrolysate, and a pre-treated solid biomass, wherein said pre-treating is with acid or alkali having v/v concentration of 0.2-5%, and said biomass w/v concentration is in the range of 10-20%;

e. contacting said isolated Candida strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at least one nitrogen source, and said liquid hydrolysate at a temperature in the range of 30-42°C for 24-72 hours under aerobic conditions to promote xylitol production.

14. The method as claimed in claim 13, wherein step (b) comprises contacting said hgnocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

15. An integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057, said method comprising the steps of:

a. obtaining at least one hgnocellulosic biomass;

e. contacting Candia strain HP-CAT01 having accession number MTCC 25057 with minimal salts or at last one nitrogen source 0.5-5% pre-treated solid biomass, and said liquid hydrolysate of step (d) in a v/v percentage of 1 - 5% at a temperature in the range of 30-42° for 24-96 hours under aerobic conditions to produce said carbohydratases;

f. separating said Candida strain population from (e) to obtain a cell free liquid hydrolysate;

g. contacting said pre-treated solid biomass to said cell free liquid hydrolysate at a temperature in the range of 45-55°C for 24-96 hours under aerobic conditions to promote production of sugars; and

h. contacting said isolated Candida strain HP-CAT01 having accession number MTCC 25057 with sugars from step (g) at a temperature in the range of 30-37°C for 24^1-8 hours in aerobic conditions to promote ethanol and xylitol production.

16. The method as claimed in claim 15, further comprising the step of fractional distillation to separate ethanol, and xylitol.

17. The method as claimed in claim 15, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.

18. The method as claimed in claim 15, wherein step (b) comprises contacting said lignocellulosic biomass with phosphoric or sulphuric acid at 145-155°C for 10-20 minutes, wherein the acid concentration is 0.5% and solid loading w/v concentration is 10%.

19. The method as claimed in any of the claims 2-18, wherein said minimal salts are selected from the group consisting of 0.5 g/L MgS0₄.7H₂0, 0.5 g/: KCl, 1 g/L KH₂PO₄, 2.5 g/L NaN0₃, and combinations thereof.

20. The method as claimed in any of the claims 2-18, wherein said at least one nitrogen source concentration is in the range of 0.5-2%.

21. The method as claimed in any of the claims 2-18, wherein said isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057 is active in a pH range of 4-10, a temperature range of 30-42 °C, and inhibitory compounds concentration range of 0.1-3 g/L.

22. The method as claimed in any of the claims 2-18, wherein said Hgnocellulosic biomass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.

23. An isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of ethanol from hgnocellulosic biomass.

24. An isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol from Hgnocellulosic biomass.

25. An isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol and ethanol from hgnocellulosic biomass.

26. An isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of carbohydratases from Hgnocellulosic biomass.

27. An isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of sugars from Hgnocellulosic biomass.

28. A bio-plant for production of bioethanol, xylitol, value added products, sugars, and enzymes from hgnocellulosic biomass using an isolated Candia tropicalis strain HP-CAT01 having accession number MTCC 25057.

Dated this 2^nd September 2015