WO2017037745A1 - Procédé intégré de production de carbohydratases, éthanol, et xylitol à l'aide d'une souche de candida isolée - Google Patents

Procédé intégré de production de carbohydratases, éthanol, et xylitol à l'aide d'une souche de candida isolée Download PDF

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WO2017037745A1
WO2017037745A1 PCT/IN2016/050292 IN2016050292W WO2017037745A1 WO 2017037745 A1 WO2017037745 A1 WO 2017037745A1 IN 2016050292 W IN2016050292 W IN 2016050292W WO 2017037745 A1 WO2017037745 A1 WO 2017037745A1
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biomass
production
strain
range
concentration
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Harshad Ravindra VELANKAR
Anu Jose MATTAM
Arindam KUILA
Venkata Chalapathi Rao PEDDY
Venkateswarlu Choudary Nettem
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Hindustan Petroleum Corporation Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/165—Yeast isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/248—Xylanases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/065—Ethanol, i.e. non-beverage with microorganisms other than yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/72—Candida
    • C12R2001/74—Candida tropicalis
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/10—Process efficiency

Definitions

  • the present disclosure relates to the field of microbiology.
  • the present disclosure relates to an isolated Candida strain HP-CATOl (MTCC 25057) and its use for the production of ethanol, xylitol, and enzymes.
  • 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).
  • a method of carbohydratases production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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 method of production of at least one sugar using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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 method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • a method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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
  • an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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
  • Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of ethanol from lignocellulosic biomass.
  • Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of xylitol from lignocellulosic biomass.
  • Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of xylitol and ethanol from lignocellulosic biomass.
  • Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of carbohydratases from lignocellulosic biomass.
  • Candida tropicalis strain HP-CATOl having accession number MTCC 25057 for use in production of sugars from lignocellulosic biomass.
  • 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.
  • 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.
  • Figure IB depicts the effect of different temperatures on growth of Candida tropicalis MTCC 25057, in accordance with an embodiment of the present disclosure.
  • Figure 1C depicts the effect of different pH on growth of Candida tropicalis MTCC 25057, in accordance with an embodiment of the present disclosure.
  • Figure 2A depicts the effect of different substrates at different concentrations on endoglucanase activity, in accordance with an embodiment of the present disclosure.
  • Figure 2B depicts the effect of different substrates at different concentrations on exoglucanase activity, in accordance with an embodiment of the present disclosure.
  • Figure 2C depicts the effect of different substrates at different concentrations on xylanase activity, in accordance with an embodiment of the present disclosure.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • SEQ ID NO: 1 depicts the 18S forward primer sequence:
  • SEQ ID NO: 2 depicts the 18S reverse primer sequence:
  • SEQ ID NO: 3 depicts the 26S forward primer sequence: GCATATCAATAAGCGGAGGAAAAAG
  • SEQ ID NO: 4 depicts the 26S reverse primer sequence:
  • SEQ ID NO: 5 depicts the ITS1 forward primer sequence:
  • SEQ ID NO: 6 depicts the ITS1 reverse primer sequence:
  • 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 U C, and presence of inhibitory compounds concentration range of 0.1-3 g/L.
  • an isolated Candia tropicalis strain as described herein, wherein said inhibitory compounds can be furfural, HMF, or acetic acid.
  • a method of carbohydratases production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • step (e) 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.
  • step (e) 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.
  • step (e) pre-treated solid biomass w/v percentage is 0.5%.
  • step (e) liquid hydrolysate v/v percentage is in the range of 1-3%.
  • step (e) liquid hydrolysate v/v percentage is 2%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • carbohydratases production as described herein, wherein said carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.
  • carbohydratases production as described herein, wherein said carbohydratases are a combination of endoglucanase, exoglucanase, and xylanase.
  • step (d) there is provided a method of carbohydratases production as described herein, wherein neutralization in step (d) carried out using ammonia.
  • lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.
  • CMC carboxymethylcellulose
  • a method of carbohydratases production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • a method of production of at least one sugar using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.
  • 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%.
  • 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%.
  • 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%.
  • step (e) 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.
  • step (e) 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.
  • 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%.
  • 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%.
  • carbohydratases are a combination of endoglucanase, exoglucanase, and xylanase.
  • step (d) 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.
  • lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.
  • lignocellulosic mass is wheat straw.
  • step (e) 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%.
  • step (e) liquid hydrolysate v/v percentage is in the range of 1-3%.
  • step (e) liquid hydrolysate v/v percentage is 2%.
  • a method of production of at least one sugar using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.
  • 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%.
  • 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%.
  • 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%.
  • step (e) temperature is 32°C, and time is 24-96 hours.
  • step (e) temperature is 42°C, and time is 24-96 hours.
  • 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%.
  • 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%.
  • 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%.
  • carbohydratases are a combination of endoglucanase, exoglucanase, and xylanase.
  • lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.
  • lignocellulosic mass is wheat straw.
  • step (h) is carried out under aerobic conditions.
  • step (h) is carried out under anaerobic conditions.
  • step (h) is carried out at 32°C.
  • step (e) pre-treated solid biomass w/v percentage is 0.5%.
  • step (e) liquid hydrolysate v/v percentage is in the range of 1-3%.
  • step (e) liquid hydrolysate v/v percentage is 2%.
  • a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • a method of ethanol production using an isolated Candia strain HP-CAT01 having accession number MTCC 25057 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
  • a method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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
  • 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%.
  • 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%.
  • lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.
  • lignocellulosic mass is wheat straw.
  • step (d) there is provided a method of xylitol production as described herein, wherein neutralization in step (d) is carried out using ammonia.
  • step (e) is carried out at 32°C for 24-72 hours.
  • a method of xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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.
  • an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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
  • step (e) pre- treated solid biomass w/v percentage is 0.5%.
  • step (e) liquid hydrolysate v/v percentage is in the range of 1-3%.
  • step (e) liquid hydrolysate v/v percentage is 2%.
  • carbohydratases are selected from the group consisting of endoglucanase, exoglucanase, beta-glucosidase, xylanase, and combinations thereof.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • step (e) 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.
  • step (e) 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.
  • step (d) there is provided an integrated method of ethanol and xylitol production as described herein, wherein neutralization in step (d) carried out using ammonia.
  • lignocellulosic mass is selected from the group consisting of wheat straw, rice straw, sorghum straw, agricultural feedstock, carboxymethylcellulose (CMC), and combinations thereof.
  • 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%.
  • an integrated method of ethanol and xylitol production as described herein wherein said at least one nitrogen source weight percentage is 1%.
  • step (h) is carried out at 32°C for 24-48 hours.
  • 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.
  • an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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
  • an integrated method of ethanol and xylitol production using an isolated Candida strain HP-CAT01 having accession number MTCC 25057 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
  • Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of ethanol from lignocellulosic biomass.
  • 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.
  • Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol from lignocellulosic biomass.
  • 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.
  • Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of xylitol and ethanol from lignocellulosic biomass.
  • 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.
  • an isolated Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of carbohydratases from lignocellulosic biomass.
  • 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.
  • Candida tropicalis strain HP-CAT01 having accession number MTCC 25057 for use in production of sugars from lignocellulosic biomass.
  • 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.
  • 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.
  • 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.
  • 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 _1 ds 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.
  • 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.
  • 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 _1 ds .
  • IU International units
  • FPU Filter Paper Unit
  • 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.
  • 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.
  • furfural, 5 - hydroxymethylfurfural or acetic acid 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.
  • 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.
  • 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 H 2 SO 4 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.
  • 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.
  • PARR pressurized reactor
  • 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).
  • inhibitory compounds such as furfural, HMF and acetic acid that are usually generated during biomass pretreatment.
  • 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).
  • 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.
  • 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
  • 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.
  • yeast 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.
  • 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).
  • 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.
  • lignocellulosic hydrolysate contains a mixture of pentose and hexose sugars
  • 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.
  • 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.
  • 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.
  • 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.
  • the yeast strain also exhibits significant inhibitor tolerance and produces xylitol from hemicellulosic sugars.
  • Figure 6 outlines the steps involved for the integrated production of xylitol, ethanol, enzymes and value added products at an industrial scale.
  • the Candida tropicalis strain described here is versatile and capable of producing carbohydratases to degrade lignocellulosic biomass as well as produce ethanol and xylitol.
  • 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.
  • 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.
  • 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.
  • 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.
  • the neutralized pretreated hydrolysate is routed to either the xylitol production reactor or the enzyme production reactor.
  • 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.
  • 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.
  • the enzymes are allowed to hydrolyse the solid biomass at 50 °C for 24 - 72 hours for the production of soluble sugars.
  • the glucose rich liquid hydrolysate is sent to the ethanol production reactor while the solid treated biomass is conveyed to the burner/turbogenerator unit.
  • 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.
  • CBP consolidated bioprocessing
  • 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).
  • CMC carboxymethylcellulose
  • WS wheat straw
  • 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.

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Abstract

La présente divulgation concerne une nouvelle souche de Candida tropicalis (MTCC 25057) qui peut être cultivée dans une large plage de conditions de procédé (température, pH), et en présence de concentrations élevées d'inhibiteurs lignocellulosiques. Ladite souche a la capacité de produire simultanément des cellulases et des xylanases à partir de la fermentation des sucres issus d'une masse lignocellulosique destinée à la production d'éthanol, et de xylitol.
PCT/IN2016/050292 2015-09-02 2016-09-02 Procédé intégré de production de carbohydratases, éthanol, et xylitol à l'aide d'une souche de candida isolée WO2017037745A1 (fr)

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