Description:
FORM 2
THE PATENTS ACT, 1970
(39 OF 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
1. TITLE OF THE INVENTION – A NOVEL HIGH TOLERENT MICROORGANISM FOR XYLITOL PRODUCTION
2. Applicant (s)
NAME: CHEM PROCESS SYSTEMS PVT LTD
NATIONALITY: INDIAN
ADDRESS: UNIT 1: 15, NATRAJ INDUSTRIAL ESTATE. IYAVA VASNA,SANAND-VIRMGAM HIGHWAY, TA.SANAND, AHMEDABAD, GUJARAT -382170, INDIA.

3. PREAMBLE TO THE DESCRIPTION

The following specification particularly describes the invention and the manner in which it is to be performed.

A NOVEL HIGH TOLERENT MICROORGANISM FOR XYLITOL PRODUCTION
FIELD OF THE INVENTION
This present invention provide a novel high-tolerant microorganism identified as yeast strain of Candida orthopsilopsis having Accession No. MTCC-25669 which is capable of efficient utilising xylose derived from hemicellulose component of lignocellulosic biomass for xylitol production.

BACKGROUND OF THE INVENTION
Lignocellulosic wastes include agricultural and forest residues which are most promising alternative energy sources and serve as potential low cost raw materials that can be exploited to produce raw materials which act as substrate for many value added products. The components of lignocellulosic bio-waste majorly contain three components cellulose, hemicellulose and lignin which can be hydrolysed to their individual free form by existing chemical, thermochemical, physical, or biological methods.

The hydrolysed hemicellulose fraction of lignocellulosic waste majorly contains pentose sugars like Xylose and arabinose in addition to sugars mentioned organic acids and aldehydes like acetic acid and furfural are also present as side products of hydrolysis reactions. The Xylose sugar present in hemicellulosic hydrolysate can be further processed to produce value added products like xylitol. Xylitol is one of the top 12 renewable added-value chemicals that can be obtained from biomass.

In present world scenario, large scale processing of Xylose sugar is carried out by chemical processes that are based on Xylose hydrogenation, which requires purified Xylose as raw substrate and the process also requires high temperature and pressure that makes the process cost intensive, energy consuming and non-eco-friendly. In addition these chemical processes have limitation of purity and yield due to addition of chemicals and unwanted products produced by side reactions.

Economical and clean processing of Xylose is one of the major bottlenecks for xylitol production on the commercial scale hence alternate methods like biologically processed Xylose are highly desirable. These biological processes are useful processes as they have lowered cost of energy and involves the processing of Xylose by microbes or enzymes secreted by these microbes which render the process environmentaly safe.

With advancement in biotechnology, such as isolation of novel micro organisms, and manipulation of these microorganisms, modifications in the fermentation processes, can enhance the economic feasibility of xylitol production on the large scale. Using micro-organisms for Xylose processing to valuable products like xylitol can be explored.

Xylitol has many interesting applications in the food, pharmaceutical, and odonatological industries, owing to its high sweetening power, its anti-carcinogenic properties, and its insulin-independent metabolism. The bioconversion of detoxified hemicellulosic hydrolysate to xylitol by microorganisms could be a cheaper alternative to the current chemical process, since it is a simple process, with great specificity and low energy requirements.

However, the success of fermentations for xylitol production depends on the productivity of the strain and its tolerance to different toxic or inhibitory compounds existing in the hydrolysates. In addition, a number of optimized upstream process parameters proved to have significant effects on xylitol production in hemicellulose hydrolysate media. An adapted novel Candida species with enhanced resistance to the inhibitors in the hydrolysate can directly ferment the simply detoxified hemicellulose hydrolysate to xylitol.

The microbial strain of the present invention is not found in nature under natural environment, it was acclimatized manually to grow and process Xylose concentrations as high as 120 gpl to 200 gpl preferably 150 gpl, the original wild type strain was only able to process media containing 30 gpl of xylose whereas in laboratory by planning of experiments, studding of various combinations and experimenting with various concentration of Xylose, nitrogen source, pH optimization, temperature optimization , analysing the outcomes of various set experiments and further setting up experiments with researched data and interference, the microbe was screened.

Also, Xylose a pentose sugar abundant in the hemicellulose fraction of lignocellulosic biomass, presents substantial challenges in its conversion to valuable products due to microbial intolerance and low conversion rates.

Hence, the inventors of the present invention executed screening of xylose processing organisms which are having high capability for substrate adaptability and high tolerance for product produced without inhibition, moreover it can tolerate high impurities present in thermal and chemically degraded hemicellulose portion of hydrolysed lignocellulosic biomass like acidity, and other aliphatic and aromatic compounds which are known to be toxic and inhibitory for microbial growth and product fermentation in addition the microbe had adapted to tolerance towards high halophilic environment, salt tolerance, acetic acid tolerance, furfural and formic acid tolerance. The novel microorganism of present invention was producing xylitol from pure xylose or xylose containing hemicellulose hydrolysates and displays exceptional attributes suitable for efficient Xylose processing to value added products.

OBJECTIVE OF THE INVENTION
The main objective of the present invention is to provide a novel high-tolerant microorganism for microbial synthesis of xylitol.

Another object of the present invention is to provide a novel high-tolerant microorganism which is having DNA sequence with 98.94% similarity with candida orthopsilopsis.

Another objective of the present invention is to provide a novel high Xylose-tolerant yeast strain of Candida orthopsilosis for microbial synthesis of xylitol with a superior yield in a cost-effective and eco-friendly manner.

One other object of the present invention is to provide a novel high-tolerant microorganism for xylitol production which is able to use pure synthetic xylose or xylose containing hemicellulose hydrolysate component of lignocellulosic biomass for xylitol conversion.

SUMMARY OF THE INVENTION
The main aspect of the present invention is to provide a novel high-tolerant microorganism for xylitol production.

One more aspect of the present invention is to provide a novel high-tolerant microorganism for xylitol production comprising a gene having DNA sequence of SEQ ID No. 1, wherein the said microorganism is yeast strain of Candida orthopsilosis which is having accession number MTCC-25669.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: (a) Microscopic observation of isolated novel Candida showing blastoconidia (b) round to oval cell morphology with absence of pseudohyphae or true hyphae.(c) microscopic observation of Candida cells grown on hemicellulose hydrolysate media
Figure 2: Isolate growth on PDA and YEPD plates depicting smooth glistering white colonies.
Figure 3: Screening of xylose processing microorganisms
Figure 4:HPLC analysis graph of pure synthetic xylose with 110 gpl concentration
Figure 5: HPLC analysis graph of xylitol produced from pure synthetic 110 gpl xylose
Figure 6: HPLC analysis graph of hemicellulose hydrolysate prior to conversion of xylitol
Figure 7: HPLC analysis graph of hemicellulose hydrolysate after conversion of xylose to xylitol
Figure 8: Hemicellulose hydrolysate xylose fermentation to xylitol
Figure 9: Fed batch mode of xylitol fermentation

DESCRIPTION OF THE INVENTION
The main aspect of the present invention is to provide a novel high-tolerant microorganism for xylitol production.

The detailed description set forth below is intended as a description of exemplary embodiments and is not intended to represent the only forms in which the exemplary embodiments may be constructed and/or utilized. The description sets forth the functions and the sequence of steps for constructing and/or operating the exemplary embodiments. However, it is to be understood that the same or equivalent functions and sequences which may be accomplished by different exemplary methods are also intended to be encompassed within the spirit and scope of the invention.

As defined herein, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs.

Although any process and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.
As stated in the present invention herein, the singular forms “a,” “an” and “the” specifically also encompass the plural forms of the terms to which they refer, unless the content clearly dictates otherwise. The term “about” is used herein to means approximately, in the region of, roughly, or around.

As stated herein, that it follows in a transitional phrase or in the body of a claim, the terms “comprise(s)” and “comprising” are to be interpreted as having an open-ended meaning. That is, the terms are to be interpreted synonymously with the phrases “having at least” or “including at least”. When used in the context of a process, the term “comprising” means that the process includes at least the recited steps but may include additional steps. When used in the context of a composition, the term “comprising” means that the composition includes at least the recited features or components but may also include additional features or components.

Nothing in this disclosure is to be construed as an admission that the embodiments described in this disclosure are not entitled to antedate such disclosure by virtue of prior invention. As used in this document, the term "comprising" means "including, but not limited to.
The term used herein, “agar and broth” refers to types of growth medium used to grow microorganisms.

The term used herein, “microorganism” refers to a a very small living thing that you can only see with a special piece of equipment (a microscope).

The term used herein, “isolate CPS YSY 2301” and “CPS YSY 2301” (which is novel microorganism of present inventon with SEQ ID No.1), can be interchanged with each other and convey the same meaning.

As per one main embodiment of the present invention, a novel high-tolerant microorganism for xylitol production comprising a gene having DNA sequence of SEQ ID No. 1.

As per one embodiment of the present invention, a novel high-tolerant microorganism is yeast strain of Candida orthopsilosis species.

As per one embodiment of the present invention, a novel high-tolerant microorganism is deposited to Microbial Type Culture Collection and Gene Bank having accession number MTCC-25669.

As per one embodiment of the present invention, the DNA sequence of novel microorganism of present invention is having 98.94% similarity with Candida orthopsilopsis.

As per one embodiment, the novel microorganism of present invention showed yeast like morphology with smooth and homogeneous cell surface on PDA, YEPD and MGYP agar, during microscopic observation round to oval cells could be seen with budding as major mode of reproduction few conjugating cells could be seen. Absence of pseudohyphae or true hyphae observed, germ tube formation not detected, also health and viable cells with similar morphology observed in hemicellulose hydrolysate media (figure 1).

As per one embodiment, the novel microorganism of present invention showed cream white colonies on PDA and YEPD agar (figure 2) and Smooth glistering white colonies and also having moist looking pin point colonies. Spherical to oval cells and reproduce mainly by budding or conjugation.

As per one embodiment, yeast cell blastoconidia is observed in the novel microorganism of the present invention.The blastoconidia yeast is less virulent to humans because the conditions required for growth do not occur in humans, but the hyphal form is virulent because it thrives in the environment a human provides as a host. So, when microorganism converts to the hyphal form,it exhibits tissue invasive properties causing more infections compared to microbial strains which lack hyphal formation exhibiting lower ability to invade tissues.

As per one embodiment, the novel microorganism of the present invention was not able to grow at temperatures above 37ºC and Eliminating the possibility for human infection if it is inhaled by humanor exposed to humans, the true hyphae were also not observed in isolated novel microorganism when grown on serum agar also germ tube or biofilm formation were not detected. The isolated microorganism was susceptible to major antifungal agents at concentrations =2 µg/ml by disk diffusion protocol M44-A2 when compared with quality control strain Candida psilopsis ATCC 22019, Therefore, microorganism of the present invention is non virulent.

As per one embodiment, the novel microorganism is having diameter of 2-10 µm (micron).

As per one embodiment, the novel microorganism of present invention gives xylose reductase activity.

As per one embodiment of the present invention, the Optical density (O.D600.) of novel microorganism of present invention is 2.2 to 2.5 when fully grown in xylose or hemicellulose media with cell count corresponding to 1.2 to 1.3 x 108 cells /ml and 30 to 40 gpl dry biomass weight.

As per one embodiment of the present invention,the temperature for xylose processing is preferably between 30ºC to 33ºC for the novel isolate. 32ºC is more prefered temperature for the xylitol production.

As per one embodiment , the microorganism prefers agitation speed of 150 to 250 rpm, more preferably 200 rpm for xylose processing.

As per one embodiment of the present invention the microorganism exhibits cell growth from 10 gpl dry biomass initially to 32 gpl dry biomass in the xylose processing media when processing either pure xylose or hemicellulose hydrolysate component of lignocellulosic biomass exhibiting healthy growth without inhibition of known inhibitory compounds present in hemicellulose hydrolysates.

As per one embodiment, present invention is related to novel high-tolerant microorganism, capable of efficient utilizing Xylose derived from hemicellulose component of lignocellulosic biomass such as cotton stalk, corn cobs, bagasses, mustard stalks, juliflora etc. In the preffered embodiment, cotton stalk hemicellulose hydrolysate is used in the present invention for utilizing xylose.

As per one embodiment, the microorganism exhibits exceptional tolerance to environmental stress conditions, such as high salt concentrations, acetic acid, furfural, and impurities present in Xylose streams. This resilience enables the microorganism to thrive and efficiently utilize Xylose as a carbon source for fermentation processes. This unique tolerance also enables the microorganism to serve as a highly efficient catalyst for fermentation processes, resulting in significant yields of xylitol.

As per one embodiment, the isolated novel microorganism of present invention displays exceptional attributes suitable for efficient Xylose processing to value added products such as xylitol.

As per one embodiment, the present invention encompasses batch and continuous fermentation methods, achieving remarkable xylitol yields of up to 0.65 to 0.70gm/gm Xylose derived from agrowastes.
As per one embodiment, Both batch, fed batch and continuous fermentation methods can be employed with CPS YSY 23 01 Candida orthopsilopsis as the microbial catalyst. In either mode of fermentation, the microorganism demonstrates remarkable performance, achieving xylitol yields of up to 0.65 to 0.70gm/gm from Xylose substrates allowing for consistent and sustainable production of this valuable compound.

As per one embodiment, the utilization of microorganism as a high-tolerant microorganism highlights its suitability as an efficient catalyst for the conversion of Xylose derived from lignocellulosic biomass. The microorganism's exceptional tolerance to Xylose and its ability to convert this substrate into xylitol enhance the overall efficiency and yield of fermentation processes, resulting in improved economic viability.

The invention is further illustrated by the following examples, which are provided to be exemplary of the invention and do not limit the scope of the invention. While the present invention has been described in terms of its specific embodiments, certain modifications and equivalents will be apparent to those skilled in the art and are intended to be included within the scope of the present invention.

EXAMPLE 1: SAMPLE COLLECTION
The sludge sample containing yeast strain of candida orthopsilosis of present invention was collected from production unit of CHEM PROCESS SYSTEMS PVT LTD located at 15, natraj Industrial Estate. Iyava Vasna, Sanand-Virmgam Highway, Ta.Sanand, Ahmedabad, Gujarat 382170, India

EXAMPLE 2: SCREENING OF XYLOSE PROCESSING MICROORGANISMS
The sludge samples were dispensed in sterile distilled water and streaked on nutrient agar plates containing Dextrose 1gpl, Xylosesugar 5 gpl, peptone 5 gpl and yeast extract 3 gpl enough to make 5000 ppm concentration of Xylosein agar. Plates were incubated at 33ºC for two to three days.

Bacterial and yeast Colonies appeared on 3rd day were further grown on YEPX (Yeast Extract 3gpl, Peptone 3 gpl, xylose 5 gpl) agar plates and broth with increased concentration of Xyloseup till 10 gpl and being only sole carbon source with 3 gpl yeast extract and 5 gpl peptone, pH of the broth was maintained 5.5 to 6.2 and flasks were incubated at 32ºC at 200 rpm environmental shakers. Regular Samples were drawn from the flask and were analyzed for xylitol production.

The results are shown below:
Innoculated microorganism
Parameters Growth on Day 1 Growth on Day 2 Growth on Day 3 Growth on Day 4
Yeast (C7) Glucose (g/l) 0 0 0 0
Xylose (g/l) 3.48 2.56 1.48 0.16
Arabinose (g/l) 0 0 0 0
Acetic acid (g/l) 0 0 0 0
Ethanol (g/l) 0 0 0 0
Xylitol (g/l) 0.22 0.33 3.04 3.35
Glycerol (g/l) 0 0 0.76 0.29
lactic acid (g/l) 0 0 0 0
HMF (g/l) 0 0 0 0
Furfural (g/l) 0 0 0 0
Mucoid bacteria (C1) Glucose (g/l) 0 0 0 0
xylose 5.06 4.93 3.56 2.89
Arabinose 0 0 0 0
Acetic acid 0 0.01 0 0
Ethanol 0 0 0 0
Xylitol 0 0 0 0
lactic acid 0 0 0 0
HMF 0 0 0 0
Furfural 0 0 0 0
Red pigmented bacteria (C4) Glucose 0 0 - -
xylose 4.96 4.43 - -
Arabinose 0 0 - -
Acetic acid 0.04 0 - -
Ethanol 0 0 - -
Xylitol 0 0 - -
lactic acid 0 0 - -
HMF 0 0 - -
Furfural 0 0 - -

The table 1 shows that, the samples drawn from flasks with bacterial inoculation had no traces of xylitol, while the flasks with yeast inoculation showed xylitol production (Refer Figure 3).

Yeast isolates could grow at 150 gpl. Xylose concentration processing it and producing xylitol. The isolate which were able to tolerate and process high concentration of xylose in pure synthetic as well as hemicellulose hydrolysate media were isolated and these strains were further subjected to toxicity like acetic acid ( 5.0 and 10 gpl), furfural (5.0 and 10.0 gpl) and also grown on media containing high concentration of salt (NaCl), the isolates actively growing on these adverse unnatural conditions were screened and isolated. This strain was designated as CPS YSY 2301 and selected for further study for industrial use. Also, this strain was considered novel and was deposited at MTCC and assigned Accession number MTCC-25669 and identified as Candida orthopsilopsis species.

EXAMPLE 3: MORPHOLOGICAL, BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF NOVEL MICROORGANISM
Characterization of the selected microbe was carried out on the basis of techniques described Gram’s staining. Microscopic observation of novel microorganism of present invention showed yeast like morphology smooth and homogeneous cell surface with budding scar. Isolate CPS YSY 2301 was grown on PDA (Potato dextrose agar) , MGYP ( Malt extract glucose yeast extract agar) and YEPD (Yeast extract peptone dextrose agar) selective agar for fungus growth the positive growth confirms the fungus yeasts). Slants of pure cultures of isolate was sent to Microbial Type Culture Collection and Gene bank (MTCC-25669), Chandigarh for identification by sequencing of ITS/D1-D2 gene sequencing and use of.EzTaxon server 2.1 Software and Genebank database for identification. The said isolate or microorganism is having Accession number MTCC-25669.

EXAMPLE 4: OPTIMIZATION OF INCUBATION PARAMETERS FOR NOVEL MICROORGANISM
The isolated microorganism was adapted for growth on Xylose(present in hemicellulose hydrolysate component of lignocellulosic biomass) by gradually increasing its concentration up to 30 gpl, in MXU (medium xylose 30 gpl and urea 1 gpl, yeast extract 1 gpl and K2HPO4 1 gpl) media. Optimum growth was observed under aerobic condition at 30ºC to 33ºC and pH 6.0 to 6.2 with xylitol production up till 21 gpl under aerobic condition at 200 rpm.

EXAMPLE 5: HEMICELLULOSE ANALYSIS BEFORE AND AFTER MICROORGANISM AUGMENTATION

Characteristics of Hemicellulose portion of hydrolysed product of lignocellulose average values from cotton stalk thermochemical hydrolysis
Constituents Range
pH 2.5 to 3.0
D-Glucose 5.0 to 10.0 gpl
D-xylose 15.0 to 25.0 gpl
Arabinose 2.0 to 4.0 gpl
Acetic acid 3.0 to 7.0 gpl
Furfural 5.0 to 8.0 gpl
Sodium 1.0 to 3.0 gpl
Chloride 3.0 to 9.0 gpl

Hemicellulose portion of hydrolyzed lignocellulosic biomass was analyzed for the composition present in it, pH was maintained 6.0 to 6.2 with calcium oxide, 1% activated carbon was added to it to remove the impurities if present. The media was stirred for half an hour under room temperature and then filtered out from ordinary filter paper, the residue was discarded and the filtrate was supplemented with 1 ppm urea was subjected to fermentation by isolate CPS YSY 2301. The flask bioreactor was kept under aerobic condition at 200 rpm at 32 deg temperature.

Periodic samples were drawn up till 120 hrs and the substrate xylose consumed and xylitol produced were anlysed.
The results are shown below.
Innoculated microorganism
Parameters Growth on Day 1 Growth on Day 4 Growth on Day 6 Growth on Day 8
Yeast isolate CPS YSY 2301 Glucose (g/l) 4.38 3.41 0.00 0
Xylose (g/l) 26.36 24.33 21.22 0.58
Arabinose (g/l) 2.75 2.05 2.24 1.57
Acetic acid (g/l) 5.29 0.37 1.91 1.29
Ethanol (g/l) 0.00 0.00 1.45 1.54
Xylitol (g/l) 0.00 0.02 2.95 10.31
Glycerol (g/l) 0.00 0.00 0.00 0
lactic acid (g/l) 4.38 3.41 0.00 0.58
HMF (g/l) 0 0 0 0
Furfural (g/l) 0 0 0 0

The table 2 shows that, the Xylose with yeast CPS YSY 2301 inoculation showed xylitol production.

The novel microorganism of present invention can tolerate high impurities present in thermal and chemically degraded hemicellulose portion of hydrolysed lignocellulosic biomass like furfural, acetic acid and other aliphatic and aromatic compounds which are known to be toxic and inhibitory for microbial growth and product fermentation. The novel microorganism converts xylose from hemicellulose to xylitol without being affected by addition components of hemicellulose portion. The results are illustrated in figure 4 to 7. The figure 4 to 7 represents graphs for pure synthetic xylose, xylitol produced from pure synthetic xylose, hemicellulose prior to conversion and xylitol produced from hemicellulose respectively.

The results were analyzed by using High performance liquid chromatography (HPLC), Agilent 1260 with RI Detector and Hi-Plex H Column (300 X 7.7 mm) operated with 5mM H2SO4 as mobile phase at a flow rate of 0.6 ml/min.

EXAMPLE 6: BIOPROCESSING STUDIES
Bioprocessing studies were carried out in two phases:

In first phase, 2 L bioreactor with pure commercial grade Xylose concentration up till 120 gpl in 2 L flask bioreactor, 2 ppm urea and 1 ppm K2HPO4 were added as nutrients in deionized water was used, 100 ml of laboratory grown (0.5 OD) CPS YSY 2301 microorganism was added to it, aeration was done continuously using environmental shaker at 200 rpm, pH was maintained around 6.0 to 6.2 throughout for a period of 5 days at 32ºC. Samples were withdrawn every 24 hrs and analysed for Xylose consumed, xylitol produced by HPLC and cell mass generated by measuring OD. A control was kept in the same way without inoculum and it is not showing xylitol production.

The results of xylitol production from pure commercial grade synthetic Xylose with yeast CPS YSY 2301 inoculation are shown below. The results shows 51% carbon utilization efficiency i.e. conversion of xylitol from xylose.

Parameters Initial Growth on Day 1 Growth on Day 3 Growth on Day 5 Growth on Day 6
Glucose (g/l) 0.04 0.00 0.00 0.00 0.00
Xylose (g/l) 45.89 29.94 20.06 17.06 13.87
Arabinose (g/l) 0.24 0.78 0.00 0.00 0.00
Acetic acid (g/l) 0.00 0.72 0.96 0.95 0.94
Ethanol (g/l) 0.00 0.36 0.00 0.00 0.00
Xylitol (g/l) 0.33 7.40 9.24 10.40 16.57
Glycerol (g/l) 0.03 0.00 0.00 0.00 0.00
lactic acid (g/l) 0.06 0.56 0.96 1.02 1.13
Glycerol (g/l) 0.04 0.00 0.00 0.00 0.00

The second phase of the study included the investigation to degrade hemicellulose portion of hydrolyzed biomass, the hemicellulose hydrolysate was taken as such and was also concentrated from 25 gpl to 110 gpl by slowly evaporating at low temperature in the 2 L flask bioreactor. 2 ppm urea and 1 ppm K2HPO4 were added as nutrients in deionized water was used, 100 ml of laboratory grown (0.5 OD) CPS YSY 2301 was added to it, aeration was done continuously using environmental shaker at 200 rpm, pH was maintained around 6.0 to 6.2 throughout for a period of 5 days at 32 0C. Samples were withdrawn every 24 hrs and analysed for Xylose consumed, xylitol produced, acetic acid, furfural consumed. A control was kept in the same way without inoculum it is not showing xylitol production.

The results of xylitol production from hemicellulose containing Xylose with yeast CPS YSY 2301 inoculation are shown below. The results shows 53% conversion of xylitol from xylose.
Parameters Initial Growth on Day 1 Growth on Day 3 Growth on Day 5 Growth on Day 6
Glucose (g/l) 5.89 0.00 0.00 0 0
Xylose (g/l) 29.43 22.40 0.99 0.23 0.24
Arabinose (g/l) 2.85 3.02 2.17 1.76 1.42
Acetic acid (g/l) 0.72 0.71 4.52 4.44 3.69
Ethanol (g/l) - 4.39 5.32 4.9 3.54
Xylitol (g/l) - 3.03 16.26 16.57 15.61
Glycerol (g/l) - 0.00 0.00 0 0
lactic acid (g/l) - 0.34 0.17 0.1 0
Glycerol (g/l) - 0.00 0.00 0 0

The results of xylitol production from hemicellulose containing high Xylose concentration with yeast CPS YSY 2301 inoculation are shown below. The results shows 0.61 gms/gm of xylitol from xylose.

Parameters Initial Growth on Day 4 Growth on Day 5 Growth on Day 6
Glucose (g/l) 7.80 0.05 0.00 0.00
Xylose (g/l) 108.0 36.75 31.06 7.73
Arabinose (g/l) 5.5 5.5 5.23 4.50
Acetic acid (g/l) 0.0 0.38 0.27 0.27
Ethanol (g/l) 0.0 16.53 8.58 9.85
Xylitol (g/l) 0.0 28.13 49.36 62.21
Glycerol (g/l) 0.0 0.00 0.00 2.54
lactic acid (g/l) 0.0 0.00 0.00 0.00
Glycerol (g/l) 0.00 0.00 0.00 1.69

Scale up of the bioreactor up till 5 L with concentrted hemicellulose up till 110 gpl xylose concentration was carried same way in two phase under batch mode. For fed batch and continuous mode xylose containing media was added daily through a funnel in the bioreactor, daily 30 gpl of Xylose was added for 5 days and xylitol production ,xylose consumed was analyzed by HPLC, biomass produced was analyzed by OD measurement at 600 nm. Results (not shown) obtained showed a yield of 0.57 to 0.65 gms/gms xylitol produced from xylose in pure as well as hemicellulose hydrolysate xylose containing media.

• Comparison for efficiency in Batch vs fed batch mode
In batch mode Glucose and xylose were found completely consumed from the hemilellulose component by the organism within 36 to 90 hrs respectively producing cell biomass up till 30 gpl Indicating high cell growth of candida in xylose rich media. The results shows 62% conversion of xylitol from xylose.

In hemicellulose media containing initial 107.79 gpl of xylose, 58.04 gpl of xylitol was produced giving yield between 0.65 to 0.70 gm/gm of substrate in addition to xylitol 5.94 gpl of ethanol was also produced which may be due to glucose present in the hemicellulose hydrolysate.(Fig 8)

Hrs Xylose g/l Xylitol g/l Ethanol Xylitol yield
0 107.79 0.00 0.00 0.00
6 100.01 1.79 0.68 23.01
22 93.26 9.96 1.59 68.55
28 84.37 16.97 1.77 72.46
46 58.18 32.94 2.60 66.40
52 53.29 38.56 2.75 70.75
70 36.75 48.99 4.01 68.96
71 32.72 49.36 4.36 65.75
115 7.73 58.07 5.94 58.04

In fed batch mode in flask bioreactor with 2 L capacity, the initial concentration of Xylose was 40 gpl with daily addition of 50.0 gpl of xylose for 5 days, final concentration of Xylose added up was 116 gpl the isolate was able to consume xylose as added in the flask reactor and at the end of 6 days only 7.73 gpl of residual xylose left making 97.4% xylose consumed by the organism.

The microorganism was found to tolerate up till 120 gpl of xylose substrate in the medium and also produce 62.21 gpl of xylitol product in the media without any toxicity.

? Results of Fed batch system Xylitol production by the isolate in Pure xylose medium 2 L flask bioreactor (Fig 9)
Hrs Xylose addition gms Xylose in broth g/l Xylitol g/l Ethanol g/l Xylitolyeild %
0 4.00 16.00 0.00 0.00 0.00
24 0.00 10.29 5.68 0.00 99.48
48 0.00 0.84 9.05 0.60 59.70
72 5.00 7.11 16.12 0.00 55.80
96 10.00 21.99 27.20 2.20 50.36
120 10.00 26.44 49.81 2.63 55.62
144 0.00 12.95 58.65 2.60 56.91
168 0.00 3.01 62.21 2.65 55.06

The isolate was also found to tolerate high salt concentrations up till 150 gpl in the hemicellulose media and acidity tolerance was 5 gpl. Also the microorganism is able to tolerate high concentrations of xylitol produced in fermentation broths up till 60 to 80 gms/litre with no product inhibition observed.

Summary
The utilization of CPS YSY 23 01 Candida orthopsilopsis of present invention as a high-tolerant microorganism highlights its suitability as an efficient catalyst for the conversion of xylose derived from lignocellulosic biomass. The microorganism's exceptional tolerance to xylose and its ability to convert this substrate into xylitol enhance the overall efficiency and yield of fermentation processes, resulting in improved economic viability.
, C , C , Claims:CLAIMS:
We claim;
1. A novel high tolerent microorganism for xylitol production comprising a gene having DNA sequence of SEQ ID No. 1.
2. The novel high tolerent microorganism for xylitol production as claimed in claim 1, wherein the said microorganism is yeast strain of Candida orthopsilosis.
3. The novel high tolerent microorganism for xylitol production as claimed in claim 1 and 2, wherein the said yeast strain of Candida orthopsilosis is deposited to Microbial Type Culture Collection and Gene Bank having accession number MTCC-25669.
4. The novel high tolerent microorganism for xylitol production as claimed in claim 1, wherein the DNA sequence of said microorganism is having 98.94% similarity with candida orthopsilopsis.
5. The novel high tolerent microorganism for xylitol production as claimed in claim 1, wherein the said microorganism is able to use xylose as carbon source for fermentation process.
6. The novel high tolerent microorganism for xylitol production as claimed in claim 1, wherein the said microorganism gives xylose reductase activity.
7. The novel high tolerent microorganism for xylitol production as claimed in claim 1, wherein the said microorganism is able to use xylose from the hemicellulose component of lignocellulosic biomass for xylitol conversion.

Dated this 22nd Sep 2023