Process For the Detoxification of Lignocellulosic Hydrolysate And Its Use In Synthesizing Xylitol

Abstract

The present invention realtes to a process for the detoxification and concentration of xylose rich biomass derived liquid hydrolysate and its application as a substrate for the synthesis of crystalline and pure xylitol.

Claims

1. A process for the simultaneous detoxification and concentration of xylose rich biomass derived liquid hydrolysate using a custom-designed glass chamber, wherein said process comprises the steps of: i. hydrolysing the biomass for the preatreatment by using dilute sulphuric acid, wherein the ratio of biomass and sulphuric acid is 1:8 at a temperature in the range of 138 C.-140 C. with a holding time period in the range of 85-90 minutes at 100-120 rpm to obtain a biomass slurry; ii. passing the discharged biomass slurry as obtained in step i) through a squeezer for solid-liquid separation to obtain a xylose rich liquid solution; iii. keeping the xylose rich liquid solution as obtained in step ii) in a solar concentrator and exposing to sunlight at a temperature in the range of 25 C. to 45 C. for a period in the range of 47-48 hours to obtain the substantially furfural free xylose rich acid hydrolysed detoxified hydrolysate, wherein, the said detoxification and concentration process resulted in removal of 95% of furfural in the acid hydrolysed xylose rich lignocellulosic composition.

2. The process as claimed in claim 1, wherein said detoxification and concentration process resulted in removal of 99% of furfural in the acid hydrolysed xylose rich lignocellulosic composition.

3. A process for the production of crystalline xylitol from detoxified and concentrated lignocellulosic biomass obtained by the process as claimed in claim 1, in the presence of a whole-cell biocatalyst, wherein the process comprises the steps of: a) concentrating the dotoxified and concentrated xylose rich biomass derived liquid hydrolysate broth further by using a rotary vacuum evaporator to achieve a xylose concentration of 66, 100 and 150 g/L; b) monitoring the xylitol production from concentrated biomass hydrolysate (at varying xylose concentration) obtained at step a) by Pichia caribicca MTCC 5703 strain in a bioreactor with a supervisory control and a data acquisition (SCADA) system; c) controlling the fermentation parameters by maintaining the biomass obtained at step b) at a temperature in the range of 28-30 C., agitation at 150-200 rpm, and the pH of the system at 6.0-6.5; d) allowing the cells to settle down of the biomass obtained at step c) after fermentation; e) siphoning out the xylitol rich broth obtained at step d) from the fermenter and decolorizing by activated carbon treatment (5% w/v); f) filtering and concentrating the filtrate with a xylitol content of 8-12% by weight obtained at step e) in a rotary evaporator at 80 C. under vacuum (0.5 atm) to achieve a xylitol content of 80-90% by weight; g) cooling the filtrate obtained at step f) gradually from 60 C. to 25 C. over a period of about 1-2 hours while controlling the temperature and then keeping at a temperature in the range of 20 C. to 0 C. for 5 days to complete the crystallization of xylitol; h) separating and drying the crystals to obtain crystalline xylitol.

4. The process as claimed in claim 3, wherein said produced xylitol from lignocellulosic biomass hydrolysate is with high yields and selectivity, wherein the maximum yield is 0.87 g/g with a product selectivity of >96%.

5. The process as claimed in claim 3, wherein said process results in 85% xylitol recovery from the broth in the form of crystals with a degree of purity of >96.9%.

6. The process as claimed in claim 3, wherein the Xylitol crystals demonstrated no toxic effect on HepG2 cell lines when assayed for cytotoxicity studies.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 a) represents the front view of a designed or cusotmized glass chamber with dimensions (in cm) for detoxification of hydrolysate andfurfural removal and concentrating the feed wherein: 1Removable glass wall; 2Borosilicate glass tray; 3Tin sheet coated with black paint; 4PVC casing; 5Polyethylene foam; 6Plywood; b) represents the side view of the glass chamber with dimensions (in cm) wherein the numbers indicate: Removable glass wall; 2Borosilicate glass tray; 3PVC casing; 4Tin sheet coated with black paint; 5Polyethylene foam; 6Plywood; c) represents the top view of the glass chamber with dimensions (in cm) wherein the nubers indicate: 1Borosilicate tray; 2Black paint coated tin sheet; 3Condensate collectors; 4Thermocol base; 5Plywood base.

[0036] FIG. 2 represents a model glass chamber for detoxification of hydrolysate.

[0037] FIG. 3 represents xylitol production with cell recycle. F.sub.1 to F.sub.5 indicates the 5 fermentation cycles with cell recycle

[0038] FIG. 4 represents the time scale graph of xylitol production at 0.5 vvm (volume of air/volume of media) and xylose concentration of 150 g/L

[0039] FIG. 5 represents xylitol crystals recovered from the fermentation broth

[0040] FIG. 6 represents a-c SEM images for recovered xylitol crystals

[0041] FIG. 7 represents .sup.13C NMR spectra of (a) commercial xylitol crystals from Himedia with a degree of purity of 98% compared to (b) xylitol produced through fermentation from lignocellulosic hydrolysate.

[0042] FIG. 8 represents .sup.1H NMR spectra of (a) commercial xylitol crystals compared to (b) xylitol crystals produced through fermentation.

[0043] FIG. 9 represents XRD patterns of (a) commercially available xylitol and (b-c) xylitol produced through fermentation and recovered at different crystallization temperatures.

[0044] FIG. 10 represents the process flow diagram for xylitol production.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention provides a simultaneous process for the detoxification and concentration of xylose rich biomass derived liquid hydrolysate using a custom-designed glass chamber utilizing sunlight as the energy source. The xylose rich liquid hydrolysate obtained from acid and steam explosion of lignocellulosic biomass is detoxified and concentrated in a customized glass chamber using sunlight as the energy source. The detoxified lignocellulosic biomass is substantially free of furfural.

[0046] The lignocellulosic biomass is obtained from any agricultural waste, preferably from corn cob or sugarcane bagasse.

[0047] Source of corn cob and sugarcane bagasse: [0048] 1. Corncob: Farmland in Arki town, Distric Solan, HP (Cordinates of the field: 31 929.6539 N, 76 5820.9953 E). [0049] 2. Sugarcane bagasse: Local sugar mill in Doiwala, Uttarakhand (Co-ordinates: 30 1011N 78 723E.

[0050] The process for the detoxification and concentration of xylose rich biomass derived liquid hydrolysate comprises the steps of: [0051] i. hydrolysing the biomass for the preatreatment by using dilute sulphuric acid, wherein the ratio of biomass: sulphuric acid is 1:8 at a temperature in the range of 138 C.-140 C. with a holding time period in the range of 85-90 minutes at 100-120 rpm; [0052] ii. passing the discharged biomass slurry obtained at step i) through a squeezer for solid-liquid separation to obtain xylose rich liquid solution [0053] iii. keeping the xylose rich compositon obtained at step ii) in the solar concentrator and exposing to sunlight at a temperature in the range of 25 C.-45 C. for a period in the range of 47-48 hours to obtain the substantially furfural free xylose rich acid hydrolysed detoxified hydrolysate.

[0054] The detoxification process resulted in removal of 95% of furfural in the acid hydrolysed xylose rich lignocellulosic composition or broth.

[0055] The detoxification process resulted in removal of 99% of furfural in the acid hydrolysed xylose rich lignocellulosic composition or broth.

[0056] Several experimnets have been conducted by using detoxified and non-detoxified lignocellulosic hydrolysate for different time period. Experimental results with detoxified and non-detoxified lignocellulosic hydrolysate are summarised below in Table-1:

TABLE-US-00001 TABLE 1 Effect of detoxification on xylitol production by the biocatalyst Corncob hydrolysate without detoxification Detoxified corncob hydrolysate Time Xylose Glucose Xylitol Furfural Time Xylose Glucose Xylitol Furfural (h) (g/L) (g/L) (g/L) (g/L) (h) (g/L) (g/L) (g/L) (g/L) 0 31.45 3.79 0 2.4 0 65.07 6.9 0 0 22 29.62 2.75 0 1.48 19 51.06 0 10.61 0 46 28.64 2.54 0 1.19 43 38.41 0 18.42 0 96 28.6 2.23 0 1.19 68 23.62 0 29.73 0 96 15.74 0 34.594 0 114 6.689 0 46.406 0

[0057] A glass chamber termed as solar concentrator at step iii) is designed or customized or tailored to detoxify and concentrate the hydrolysate obtained as described above. The structure is illustrated in FIG. 1 a-c. It comprises of two rectangular glass panels on either side, and two triangular panels at either end joined to form a trapezoidal structure fixed on a wooden base with an overall Length:Width:Height ratio of 1.62:1.38:1. Referring to FIG. 1 a, the solar concentrator comprises a removable glass wall (1); a Borosilicate glass tray (2); a PVC casing (3); Tin sheet coated with black paint (4); a polyethylene foam (5) and Plywood (6). The panel on the front is movable vertically to allow entry of sample tray inside the glass chamber. The junctions between the glass panels and the supporting base structure is sealed with insulating tapes. The walls of the structure allow entry of incident sunlight and prevent exposure to any external air, rain, or moisture. The bottom of the chamber comprises of a tin base coated with black paint to absorb incident sunlight. Two drain pipes are placed on the two walls to collect and drain any evaporated water in pure form.

[0058] Present invention provides a process for the production of crystalline xylitol from lignocellulosic biomass using a whole-cell biocatalyst. The novel mesophilic yeast utilizes the glucose in liquid hydrolysate for cell biomass accumulation and/or NADPH regeneration, which is utilized as a cofactor for xylose reduction into xylitol with high conversion efficiency.

[0059] In an embodiment, the present invention provides a process for the production of crystalline xylitol from lignocellulosic biomass using a whole-cell biocatalyst, wherein the process comprises of fermenting the detoxified and concentrated biomass hydrolysate to xylitol by using a generally regarded as safe (GRAS) organism Pichia caribicca MTCC 5703.

[0060] The biocatalyst Pichia caribicca MTCC 5703 is evaluated for its efficacy when recycled. The biocatalyst synthesized xylitol with no substantial effect on its yield over 500 hours, also refer FIG. 5.

[0061] The process comprises the steps of: [0062] a) concentrating the dotoxified and concentrated xylose rich broth further by using a rotary vacuum evaporator to achieve a xylose concentration of 66, 100 and 150 g/L; [0063] b) investigating the xylitol production from concentrated biomass hydrolysate (at varying xylose concentration) obtained at step a) by Pichia caribicca MTCC 5703 strain in a bioreactor with a supervisory control and a data acquisition (SCADA) system; [0064] c) controlling the fermentation parameters by maintaining the biomass obtained at step b) at a temperature in the range of 28-30 C., agitation at 150-200 rpm, and the pH of the system at 6.0-6.5; [0065] d) allowing the cells to settle down of the biomass obtained at step c) after fermentation; [0066] e) siphoning out the xylitol rich broth obtained at step d) from the fermenter and decolorizing by activated carbon treatment (5% w/v); [0067] f) filtering and concentrating the filtrate with a xylitol content of 8-12% by weight obtained at step e) in a rotary evaporator at 80 C. under vacuum (0.5 atm) to achieve a xylitol content of 80-90% by weight; [0068] g) cooling the filtrate obtained at step f) gradually from 60 C. to 25 C. over a period of about 1-2 hours while controlling the temperature and then keeping at a temperature in the range of 20 C. to 0 C. for 5 days to complete the crystallization of xylitol; [0069] h) separating and drying the crystals to obtain crystalline xylitol.

[0070] Present invention provides a biocatalytic process that produces xylitol from lignocellulosic biomass hydrolysate with high yields and selectivity, wherein the maximum yield is 0.87 g/g with a product selectivity of >96%. Single-step crystallization results in 85% xylitol recovery from the broth in the form of crystals with a degree of purity of >96.9%. The crystals demonstrated no toxic effect on HepG2 cell lines when assayed for cytotoxicity studies.

[0071] As the initial xylose concentration in the broth increased, the xylitol yield increased linearly up to 100 g/L, after which a slight decrease in the return is observed. This indicates that initial xylose concentration in the broth has an influence on the xylitol yield.

[0072] FIG. 5 depicts .sup.13C NMR for Xylitol crystals. 50 mg of the crystals (FIG. 5) are suspended in 600 L of deuterated water and subjected to .sup.13C NMR for structural confirmation.

[0073] Quantitative .sup.1H NMR (Bruker Avance III, Switzerland) is done to estimate the degree of purity with potassium phthalate as the standard reference material (SRM), refer FIGS. 7 and 8.

[0074] For further characterization by SEM and XRD, refer FIGS. 6 and 9 respectively.

[0075] The SEM micrograms (in FIG. 6, a-c) shows a myriad of small microcrystalline molecules with an average crystal size of 400-600 m. The recorded micrographs are highly charged and heavily agglomerated form with tight packing, which corroborated to the crystal structure elucidated in XRD analysis. The .sup.13C-NMR spectrums (FIG. 7b) demonstrated 3 characteristics peaks in .sup.13C-NMR: =74 (m,1C), 72.3 (m, 2C), 64.4 (m, 2C) for the recovered crystals. A single peak near to 64.4 represented two-terminal CH.sub.2 groups in the molecule. A small peak at 72.3 ppm with an intense signal observed at 74 ppm represented the C.sub.3 and C.sub.2 carbon atom respectively within the molecule, which confirmed its structure as xylitol (FIG. 7b). .sup.1H NMR analysis (FIGS. 8 a and b) revealed that the purity of the obtained crystals was 96.9% and resembled the commercial crystals (purity of 98%). The crystals had a moisture content of less than 1% with xylose (1.5% w/w) and glycerol (2% w/w), accounting for the impurities.

[0076] Powdered X-ray diffractions showed a high impact on the crystallinity of the xylitol produced via biocatalytic route when compared to the commercially available xylitol. The XRD pattern of xylitol processed through bio-catalytic route (FIG. 9, b-c) shows peaks at 2 of 15.6, 18.7, 20.3, 23.6, and 27.1 which attribute to (331), (511), (440), (533) and (642) planes of xylitol crystalstype cubic system belonging Fd3m space group (JCPDS Card No. 39-1380; lattice parameter a=24.681 ). With respect to commercial xylitol, the X-ray diffraction pattern found identical. The only significant change found, is in the intensity of the peaks, where the intensity of some peaks undergoing reduction while others being increased compared with the commercial xylitol crystals. Comparing the X-ray diffraction patterns (commercial and bio-catalyzed xylitol), one can assume, the commercial crystals are exposed with (020) and (220) planes whereas the as prepared xylitol was preferentially presented with (022) plane. Table-2 below summarizes the data related to composition analysis of xylitol.

TABLE-US-00002 TABLE 2 Compositional analysis of xylitol Component Recovered crystals Commercial crystals Xylitol (wt %) 96.9 98.0 Other polyols (wt %) Not detected <1.5 Xylose (wt %) <1.0 <0.5 Glycerol (wt %) <1.0 Not detected Moisture content (wt %) <1.0 <0.5

[0077] The schematic diagram for the process is represented in FIG. 10 depicting the different steps in the process of xylitol production from agricultural residue.

[0078] Along with detoxification, the process concentrates the xylose in the liquid hydrolysate by 2 fold which results in energy saving by 0.72 kW/per L of the hydrolysate (equivalent to Rs 4.68). [0079] Economic saving: Rs 7.5 (material)+Rs 4.68 (Energy)=Rs 12.18/L of hydrolysate [0080] (Assuming 3% w/v, AC loading with a bulk price of Rs 250/kg of adsorbent) [0081] 1 kW of electricity equivalent to Rs 6.5.

[0082] In terms of economic significance, the current invention saves both in terms of material and energy expenditure.

EXAMPLES

[0083] The following examples are given by way of illustration and therefore should not be construed to limit the scope of the invention.

[0084] The quantitative analysis of the components xylose, glucose, xylitol, acetic acid, glycerol, furfural, and HMF have been carried out in HPLC with the following conditions:

[0085] Column: Aminex HPX-87H (3007.8 mm) manufactured by Bio-Rad Laboratories Column oven temperature: 55 C.

[0086] Eluent: 5 mM H.sub.2SO.sub.4 with a pump flow rate of 0.55 mL/min in isocratic mode Detection: RI detector.

Example-1: Detoxification and Concentration of Lignocellulosic Hydrolysate

[0087] The transparent glass chamber was placed under direct sunlight. A glass tray filled with 2 L of neutralized acid-treated lignocellulosic hydrolysate (pH 4.5) with a furfural and xylose content of 2.4 g/L and 33 g/L respectively was placed inside the transparent glass chamber. The evaporation of liquid hydrolysate begins after nearly 1 h of exposure to sunlight (FIG. 2). The volume of the liquid hydrolysate is reduced to almost half of the initial volume after 48 h of the treatment and analyzed for furfural and xylose content. The analysis of the concentrated hydrolysate has shown that the efficiency of furfural removal by the method was >99% and increased the xylose concentration by two-fold.

Table-3 below indicates data related to Xylose composition:

TABLE-US-00003 Composition of acid Xylose Glucose Acetic acid HMF Furfural hydrolysate (g/L) (g/L) (g/L) (g/L) (g/L) Before treatment 33.547 3.467 1.267 0.38 2.446 After detoxification 66.71 6.90 2.584 0.604 0

Example-2: Xylitol Fermentation of Detoxified and Non-Detoxified Liquid Hydrolystae by Biocatalyst

[0088] The detoxified and non detoxified liquid hydrolysate (as in Table 3) were evaluated as the feed for xylitol production by the bioctalyst. The liquid hydrolysates supplemented with salts (composition mentioned in table 4) was used for fermentation studies. Grown Pichia caribicca cells MTCC 2703 were innoculated in 2 L baffled flasks with 1 L of fermentation media to achieve a sugar to cell ratio of 1:10. Cells were cultivated in a shaker incubator at 29 C. and analyzed for xylitol producing ability from the two different feeds. The bioctalyst cultivated on untreated liquid hydrolysate demonstrated little or no xylose consumption without any xylitol production. However, cells cultivated on detoxified hydrolysate produced xylitol with significant yields and demonstrated 95% xylose consumption (Table 1). This indicated that detoxification using the custom designed glass chamber had a positive impact on xylose conversion and xylitol yields.

Table-4: Composition of salts used in fermentation media

TABLE-US-00004 TABLE 4 Composition of salts used in fermentation media Salts g/L Ammonium sulfate 1 Di-sodium phosphate 0.15 Monopotassium phosphate 0.15 Yeast extract 1 Magnesium sulphate 0.06

Example-3: Xylitol Production from Liquid Hydrolysate with Cell Recycling

[0089] The biocatalyst was evaluated for its recyclability and stability studies. The detoxified corncob hydrolysate (supplemented with salts as mentioned in Table-4) was fermented by the biocatalyst in a 7 L bioreactor. The fermentation parameters were controlled, such that the temperature was 28 C., the agitation of the fermentation and pH maintained at 150 rpm and pH 6.0, respectively. After the first fermentation cycle, the biocatalyst was recovered by centrifugation and evaluated for 4 successive fermentation cycles with the addition of fresh hydrolysate in the fermenter. The biocatalyst showed excellent stability up to a fermentation period of >500 hours with no loss in activity (FIG. 3). The maximum xylitol titer attained was 50.33 g/L with a yield of 0.79-0.81 g/g. The xylitol titer in the broth after successive recycling increased by 10.2%.

Example-4: Fermentation of Lignocellulosic Hydrolysate with Varying Initial Xylose Concentration

[0090] The detoxified corncob hydrolysate (as in Table 3) supplemented with salts (as mentioned in Table 4) and concentrated to attain xylose concentrations of 100, and 150 g/L was fermented using the biocatalyst in the bioreactor. The aeration inside the bioreactor was maintained at 3.5 L/min (0.5 vvm), temperature-controlled at 28 C. and pH maintained at 6.0. The sugar to cell ratio inside the fermenter was 10:1. The xylitol yield ranges from 0.78-0.87 g/g when the initial xylose concentration varies from 66 to 100 g/L, and attains a value of 0.83 g/g when the initial xylose concentration is nearly 150 g/L (Table 5, FIG. 4). Glucose, present in the hydrolysate during fermentation (6.90-12.8 g/L) was rapidly consumed within the first 10 hours. It was used up by the cell for cell biomass accumulation since a significant increase in dry cell weight was observed. A part of it was also be utilized for maintenance energy in ATP generation and cofactor (NADPH) regeneration, which was revealed by the transcript abundance of Glucose 6 phosphate dehydrogenase genes in the transcriptome data analysis. Ethanol or any other metabolite (from glucose) during fermentation is not detected. The biocatalyst produced a maximum of 113.9 g/L xylitol (from 150 g/L xylose in lignocellulosic hydrolysate) with a yield of 0.83 g/g using an initial cell concentration of 12-15 g/L. 90% of the xylose was consumed as the substrate during fermentation. The xylitol selectivity was estimated as 96.22%. Glycerol was obtained as a by-product during fermentation with a yield of 0.037 g/g.

TABLE-US-00005 TABLE 5 Xylitol production under varying xylose concentration Initial xylose concentration Xylitol titer Xylitol yield Xylitol selectivity in the liquid hydrolysate (g/L) (g/L) (g/g) (%) 66.71 51.03 0.79 94 102 80.43 0.87 98 145.8 113.9 0.83 96.22 Name of Inlet Outlet Block Step equipment Material name Mass (kg) Material name Mass (kg) R1 Biomass Biomass Biomass 3.5 Bioamass 24.5 hydrolysis digester (feed) hydrolysate Water 28 (BHL) H.sub.2SO.sub.4 0.124 R2 Feed Glass BHL 24.5 Concentrated biomass 12 detoxification chamber (in batches) hydrolysate 1 (in batches) (CBHL1) R3 Evaporation Rotary CBHL1 12 Concentrated biomass 5.702 (in batches) vacuum (in batches) hydrolysate 2 evaporator (CBHL2) R4 Fermentation Bioreactor CBHL2 5.702 Xylitol liquor (XL) 4.97 Biocatalyst 0.010 R5 Adsorption XL 4.97 Treated XL (TXL) 4.90 R6 Evaporation Rotary TXL 4.90 Conc. treated xylitol 0.7 (Evap-2) vacuum liquor (CTXL) evaporator R7 Crystallization CTXL 0.7 Xylitol-solid (XS) 0.506

Example-5: Xylitol recovery from broth by crystallization and characterization

[0091] Xylitol crystals (FIG. 5) with yields varying between 75% to 85% were recovered from the fermentation broth by crystallization. The recoveries were higher at a lower temperature (20 C.) when compared to 0 C. The addition of seed crystals did not improve the crystallization yield.

Example-6

[0092] 3.5 kg of biomass can be converted to 0.502 kg of xylitol crystals with a degree of purity of 96.9%. The detailed material balance is depicted in table below (Table 6) as per the process flow diagram (FIG. 10).

TABLE-US-00006 TABLE 6 Name of Inlet Outlet Block Step equipment Material name Mass (kg) Material name Mass (kg) R1 Biomass Biomass Biomass 3.5 Bioamass 24.5 hydrolysis digester (feed) hydrolysate Water 28 (BHL) H.sub.2SO.sub.4 0.124 R2 Feed Glass BHL 24.5 Concentrated biomass 12 detoxification chamber (in batches) hydrolysate 1 (in batches) (CBHL1) R3 Evaporation Rotary CBHL 1 12 Concentrated biomass 5.702 (in batches) vacuum (in batches) hydrolysate 2 evaporator (CBHL2) R4 Fermentation Bioreactor CBHL2 5.702 Xylitol liquor (XL) 4.97 Biocatalyst 0.010 R5 Adsorption XL 4.97 Treated XL (TXL) 4.90 R6 Evaporation Rotary TXL 4.90 Conc. treated xylitol 0.7 (Evap-2) vacuum liquor (CTXL) evaporator R7 Crystallization CTXL 0.7 Xylitol-solid (XS) 0.506

Advantages of the Invention

[0093] The invention describes a simple method for detoxification and concentrating the lignocellulosic biomass hydrolysate in a single step using only solar light as the energy source.

[0094] The fermentation process not only provides good product yield but also produces xylitol with high selectivity.

[0095] The biocatalyst operates at low temperature (28 C.) and pressure (1.2 atm) and has minimum nutritional requirements, which lowers the media cost.

[0096] The biocatalyst can convert xylose into xylitol for 5 successive fermentation cycles and shows stability of more than 500 h without any reduction in product yields thus resulting into the reduction in manufacturing cost.

[0097] The downstream process results in high xylitol recoveries (85%) with high purity (>96%) resulting intothe reduction in downstream purification cost by at least 5%.