ALKALINE HYDROLYSIS OF WASTE CELLULOSE

Abstract

The present invention relates to a process which makes it possible to obtain a plurality of organic compounds that can be used as chemical intermediates through the use of waste cellulosic biomass as a raw material. Through this process fermentable saccharides can be extracted, separated and recovered from said waste cellulosic biomass.

Claims

1. A process for the production of C5-C6 sugars from waste cellulosic biomass containing nitrogen, comprising the steps of: (a) placing said biomass in contact with a basic aqueous solution of pH > 12 at a temperature between 60 and 120° C., obtaining a mixture containing at least 5% by dry weight of said cellulosic biomass in relation to total weight of the solution; (b) separating said mixture into a solid fraction comprising cellulose and a liquid fraction; (c) subjecting said solid fraction to one or more washes with water; (d) subjecting the solid fraction resulting from step c) to a hydrolysis treatment resulting in a hydrolysate comprising C5-C6 sugars, in which the waste cellulosic biomass is a post-consumer biomass and/or a post-industrial biomass.

2. The process according to claim 1, in which the waste cellulosic biomass is derived from a hygiene product.

3. The process according to claim 1, in which the waste cellulosic biomass comes from wastewater treatment plants.

4. The process according to claim 1, in which the waste cellulosic biomass has an impurity content of less than or equal to 50% by weight, relative to the dry weight of the biomass.

5. The process according to claim 1, in which the waste cellulosic biomass contains impurities comprising a super-absorbent polymer.

6. The process according to claim 5, in which said cellulosic biomass has a super-absorbent content of less than or equal to 35% by weight relative to the dry weight of the biomass.

7. The process according to claim 1, in which the total nitrogen content is from 0.35% to 3.5% by weigh, relative to the dry weight of the cellulosic biomass.

8. The process according to claim 1, in which the waste cellulosic biomass contains impurities comprising phosphorus.

9. The process according to claim 1, in which the solid fraction obtained at the end of step c) has an impurity content of less than or equal to 30% by weight, relative to the dry weight of the solid fraction.

10. The process according to claim 1, 9, in which the solid fraction obtained at the end of step c) has a total nitrogen content of less than 0.35% by weight, relative to the dry weight of the solid fraction.

11. The process according to claim 1, in which the solid fraction obtained at the end of step c) has a phosphorus content of less than 500 mg/Kg, relative to the dry weight of the solid fraction.

12. The process according to claim 1 comprising a subsequent step e) of separating said C5-C6 sugars from said hydrolysate.

13. The process according to claim 12 comprising a subsequent step of purifying and/or concentrating the C5-C6 sugars obtained from step e) through one or more operations selected from adsorption, dialysis, reverse osmosis, crystallisation, chromatography, evaporation, distillation.

14. The process according to claim 1 comprising a subsequent step of growing a microbial strain capable of producing chemical intermediates and/or polyhydroxyalkanoates in the presence of a carbon source comprising the C5-C6 sugars hydrolysed in step d).

15. The process according to claim 14 comprising a step of growing a microbial strain capable of producing 1,4-butanediol in the presence of a carbon source comprising the C5-C6 sugars hydrolysed in step d).

16. The process according to claim 1, in which said waste cellulosic biomass undergoes mechanical comminution treatment prior to step a).

17. The process according to claim 1, in which in step a) the biomass is placed in contact with a basic aqueous solution for a time of between 30 minutes and 24 hours.

18. A composition of C5-C6 sugars obtained from waste cellulosic biomass having an impurity content of less than 45% by weight in relation to the dry weight of the composition and a total nitrogen content of from 0.1% by weight to 0.5% by weight.

19. The process according to claim 2, in which the waste cellulosic biomass has an impurity content of less than or equal to 50% by weight, relative to the dry weight of the biomass.

20. The process according to claim 3, in which the waste cellulosic biomass has an impurity content of less than or equal to 50% by weight, relative to the dry weight of the biomass.

Description

EXAMPLES

Example 1

Step a)

[0135] Cellulosic biomass from adult absorbent products used for this example had a moisture content of 10.45%, impurity content of 27, 4% by weight and total nitrogen 0.56% by weight, relative to the dry weight of the biomass. The impurities content was determined by subtracting the polysaccharide content from the dry weight of the biomass according to Technical Report NREL/TP-510-42618, 2012 as reported above.

[0136] 6.7 kg of such biomass were added to a cylindrical reactor equipped with a mechanical stirrer with alternating paddles, a temperature control system, pH meter and drip funnel, in a final concentration of 10% and 59.3 litres of a basic aqueous solution, resulting in a mixture with a pH of 13.3. The resulting mixture was then heated to a temperature of 90° C. by means of a heating oil jacket and maintained under gentle agitation for 4 hours.

Step b)

[0137] The mixture obtained at the end of step a) was separated by a centrifuge filter bag, yielding 10 kg of a solid fraction including cellulose and 56 litres of a liquid fraction.

Step c)

[0138] The solid fraction comprising cellulose from step b) was washed successively with 330 litres of water at a temperature of 20° C. until a pH of approximately 8 was reached.

[0139] At the end of step c) the solid fraction had an impurity content of 5% by weight and a total nitrogen content of 0.28% by weight, relative to the dry weight of the solid fraction.

Step d)

[0140] The solid fraction from step c) underwent enzyme hydrolysis treatment.

[0141] 3.6 kg of dry solid fraction was added to 23.3 litres of 50 mM phosphate buffer at pH 5 in a cylindrical reactor equipped with a mechanical stirrer with alternating paddles, a temperature control system and a pH control system, and 569 ml of Viscamyl™ Flow (an enzyme complex containing enzymes with cellulolytic and hemicellulolytic action) and 24 ml of nonanoic acid was added. The reaction was maintained at 50° C. under gentle agitation for 48 h.

Step e)

[0142] On completion of the hydrolysis reaction, the hydrolysate was centrifuged, filtered through sieves with a mesh size of up to 25 micrometers and subjected to tangential ultrafiltration using regenerated cellulose membranes with 10 kDa pores, resulting in a liquid fraction (sugar solution) with a glucose concentration in solution of 55 g/L, determined by ion chromatography. At the end of step e) the liquid fraction had a C5-C6 sugar content equal to 76.66% by weight with respect to the dry weight of the liquid fraction. The content of impurities, obtained by subtracting the content of glucose, xylose, oligosaccharides and additives of step d) from the dry weight of the liquid fraction, was equal to 15.93% by weight, with respect to the dry weight of the liquid fraction.

[0143] The sugar content was analysed using a Metrohm Professional IC Vario 940 ion chromatograph, equipped with an amperometric detector and fitted with a Metrosep Carb 2 250 mm x 4.0 mm x 5 .Math.m column and Metrosep Carb 2 Guard/4.0 pre-column, using the following operating conditions: [0144] Flow: 0.7 mL/minute [0145] Oven temperature: 30° C. [0146] Detector temperature: 35° C. [0147] Eluent: 40 mM NaOH + 40 mM NaOAc.

Example 2

[0148] The liquid fraction obtained at the end of step e) was concentrated using a rotary evaporator under vacuum at 50° C., resulting in a syrup with a glucose concentration in solution of 484.4 g/L, determined by liquid chromatography.

[0149] The syrup obtained was used as a carbon source in a fermentation process for the production of 1,4-BDO.

[0150] A strain of Escherichia coli with a metabolic pathway for the synthesis of 1,4-BDO was inoculated into a 250 ml Erlenmeyer flask containing 50 ml of first culture medium (10 g/l of Tryptone enzymatic digest from casein Sigma, 5 g/l of Yeast extract Sigma, 0.5 g/l of NaCl, 10 g/l of glucose). The flask was then shaken at 275 rpm overnight, at a temperature of 35° C., yielding a preinoculum.

[0151] Subsequently an aliquot of the preinoculum was transferred to a 1000 ml Erlenmeyer flask containing 200 ml of a second culture medium (12.78 g/L M9 Minimal Salt Teknova; 10 g/L first-generation glucose; 1 ml/L 1 M MgSO.sub.4; 1 ml/L 0.1 M CaCl.sub.2; 1ml/L Trace Elements Teknova T1001; 0.5 ml/L Streptomycin sulphate salt 100 mg/ml).

[0152] The Erlenmeyer flask was incubated at 35° C., shaking the contents at 275 rpm for approximately 8 hours. After this incubation period the optical density reached an OD value (optical density measured at 600 nm) of approximately 3 to 4 OD and the culture was used to inoculate a seed fermenter.

[0153] After reaching proper cell biomass, an aliquot of the seed fermentation was used to inoculate at OD 4 a production fermenter containing 1 litre of medium (1.73 g/L KH.sub.2PO.sub.4; 0.83 g/L (NH.sub.4).sub.2SO.sub.4; 0.30 g/L Na.sub.2SO.sub.4; 0.038 g/L Ca Citrate*4H.sub.20; 0.20 g/L Citric Acid C.sub.6H.sub.8O.sub.7; 1 M MgSO.sub.4 (2 ml/L); Trace Elements Teknova T1001 2 ml 1/L; Antifoam 204 Sigma 0.1ml/L) and 20 g/L of first-generation glucose to promote the growth of the microorganism before the addition of sugar solution from Example 1.

[0154] The sugar from Example 1, purified and concentrated, was progressively fed into the fermenter in a fed-batch process so as to keep the glucose concentration in the culture medium constant in the range 30-60 g/L, and then gradually reduced until a glucose concentration at the end of fermentation (about 30 - 40 hours after inoculation) of about 0 g/L was obtained.

[0155] The bioreactor was maintained under stirring >700 rpm and air flow at 0, 6755 Pa*m3/s, and optimized pH and temperature conditions.

[0156] Samples of the reaction medium were taken at different times to assess the production of 1,4-BDO by analysis using ion chromatography.

[0157] The 1,4-BDO content was determined using a Metrohm Professional IC Vario 940 ion chromatograph, equipped with amperometric detector and fitted with a Metrosep Carb 2 250 mm x 4.0 mm x 5 .Math.m column and a Metrosep Carb 2 Guard/4.0 pre-column, using the following operating conditions: [0158] Flow: 0.7 mL/minute [0159] Oven temperature: 30° C. [0160] Detector temperature: 35° C. [0161] Eluent: 50 mM NaOH + 5 mM NaOAc.

[0162] Based on the data collected, Titre and Productivity were determined (Table 1), where: [0163] “Titre” (g/1): weighted concentration of 1,4-BDO in the reaction medium at the end of the fermentation time; [0164] “Productivity” (g/1/h): weighted average synthesis rate of 1.4-BDO, calculated as

Titre/Hours of Fermentation

[0165] The results obtained are shown in Table 1.

Example 3

[0166] The same fermentation process described in Example 2 was performed using a concentrated syrup prepared by mixing 25% by weight of glucose from Example 1 as a carbon source, and 75% by weight of first-generation glucose.

[0167] Fermentation was complete approximately 40 hours after inoculation.

[0168] The results obtained are shown in Table 1.

Comparative Example 4

[0169] 665.7 g of cellulosic biomass from adult absorbent products (with a moisture content of 9.59%, impurity content of 27, 4% by weight and nitrogen content of 0.56% by weight, relative to the dry weight of the biomass) was subjected directly to an enzyme hydrolysis without any additional treatment.

[0170] Cellulosic biomass from adult absorbent products was introduced into a cylindrical reactor equipped with a mechanical stirrer with alternating paddles, a temperature control system and a pH control system, in the presence of 11.87 L of 50 mM phosphate buffer at pH 5, 95.1 ml of Viscamyl™ Flow (enzyme complex containing enzymes with cellulolytic and hemicellulolytic action) and 12 ml of nonanoic acid. The reaction was maintained at 50° C. with gentle agitation for 140 hours.

[0171] On completion of the hydrolysis reaction the hydrolysate was centrifuged, filtered through sieves with a mesh size of up to 25 micrometers and underwent tangential filtration through regenerated cellulose membranes with 10 kDa pores, resulting in a liquid fraction with a glucose concentration in solution of 29.5 g/L, determined by ion chromatography.

[0172] The liquid fraction obtained was concentrated using a rotary evaporator under vacuum at 50° C., resulting in a syrup with a concentration of glucose in solution of 467.9 g/L, determined by ion chromatography.

[0173] The same fermentation process described in Example 2 was performed using as the carbon source a mixture prepared by mixing 25% by weight of glucose produced in Comparative Example 4 and concentrated, and 75% by weight of first-generation glucose.

[0174] Fermentation was stopped about 27 hours after inoculation due to a drastic reduction in the microorganism vital parameters.

[0175] The results obtained are shown in Table 1.

TABLE-US-00001 Example % sugar from cellulosic biomass fed during fermentation Titre (g/L) Productivity (g/L/h) 2 100 86.26 2.17 3 25 119.81 2.67 4 comparative 25 22.94 0.856

[0176] The results obtained clearly show that the process according to the invention can be used to obtain C5-C6 sugars from a waste cellulosic biomass suitable for use by a microbial strain capable of producing 1,4-butanediol. Such sugars, used alone (Example 2) or in a mixture with first-generation sugars (Example 3), do not interfere with the cell viability and are efficiently converted by it into 1,4-butanediol, as demonstrated by the titre and productivity values shown in Table 1.

[0177] On the other hand Comparative Example 4 demonstrates that sugars obtained from a waste cellulosic biomass which have not undergone the process according to the invention cannot be used in fermentation, even when mixed with a first-generation sugar. Indeed, such sugars have an impurity content that makes them strongly toxic for thecell viability. Indeed, the presence of the impurities caused a drastic reduction in its vital parameters and fermentation was therefore stopped only 28 hours after inoculation.

[0178] In addition, the presence of impurities interfered with the production of 1,4-butanediol, causing lowering of the fermentation parameters.

Example 5

Step a)

[0179] 0.96 Kg of upcycled cellulose biomass from a sewage treatment plant with a moisture content of 6.69% (impurity content of 10.92% and total nitrogen content of 1.14% relative to the dry weight of the biomass) was diluted in a stirred jacketed reactor at a final concentration of 5.5% wt/wt in 15.35 liters of basic solution with a final pH of 13. The resulting mixture was heated at 90° C. and gently stirred for 4 h. At the end of the process the mixture was cooled down and 1.25 Kg of an aqueous solution of H.sub.2SO.sub.4 7% wt was added up to the neutralization of the solution.

Step b)

[0180] The mixture obtained at the endo of step a) was filtered using a filterbag-centrifuge obtaining 2.6 Kg of a solid fraction including cellulose and 14.9 litres of a liquid fraction.

Step c)

[0181] The solid fraction containing cellulose from step b) was washed with 47.1 L of water at 20° C. At the end of step c) the solid fraction showed an impurity content of 5.45% by weight (as above measured as the sum of water extractives and ethanol extractives) and a total nitrogen content of 0.33% by weight, relative to the dry weight.

Step d)

[0182] The washed solid fraction from step c) underwent an enzymatic hydrolysis treatment. 0.617 Kg of the solid fraction was diluted with 5.57 L of deionized H.sub.2O inside a stirred tank bioreactor equipped with mechanical stirrer, thermal jacket to control temperature and pH control system. pH was set to 5 and automatically corrected using H2SO4 0.3 M and NaOH 0.6 M. In this case the reaction was performed without addition of further salts, advantageously obtaining a final sugar solution with reduced conductivity and thus a reduced impact on the fermentation and downstream process. 5.7 mL of nonanoic acid 97% wt and 90 mL of Genencor Viscamyl™ Flow were added to the reaction mixture. The reaction was maintained to 50° C. and gently stirred for about 90 h until no further increment in the concentration of glucose in the solution can be observed

Step e)

[0183] On completion of the hydrolysis reaction, the hydrolysate was decanted to separate the liquid fraction containing sugars by the not-digested solid fraction. The liquid fraction was filtered in tangential flow microfiltration using 0.1 .Math.m membrane and tangential flow ultrafiltration using 5 KDa polyethersulfone (PES) membrane. Retentate was subjected to diafiltration to maximize sugar recovery.

[0184] The obtained liquid fraction had a glucose concentration of 29.5 g/L and xylose concentration of 3.5 g/L.

[0185] Analysis to quantify sugar concentrations were performed with high pressure liquid chromatography (HPLC) using a HPLC Surveyor Thermo Scientific, equipped with Refractive Index Detector (RID) Shodex and fitted with a Phenomenex Rezex ROA-Organic Acid H+ 300 x 7.8 mm column and a Phenomenex Carbo-H 4 x 3.0 mm ID pre-column, using the following operating conditions: [0186] Flow: 0.6 mL/minute, isocratic [0187] Oven temperature: 65° C. [0188] Eluent: 5 mM Sulfuric Acid

[0189] The operations of steps a) to c) have therefore led to an enrichment in cellulose of the upcycled cellulose biomass, and a consequent greater release of glucose. Additionally, they allowed to slightly increase the yield of hydrolysis with respect to the same hydrolysis reaction performed directly on the same starting biomass.

Example 6

[0190] Final purified sugar solution deriving from example 5 step e was concentrated using rotary evaporator working in vacuum at 50° C. The syrup obtained had a glucose concentration of 526 g/L and xylose concentration of 62 g/L.

[0191] The syrup was mixed with 1st generation glucose reaching a glucose final ratio of 30% wt in the mixture (30% glucose from Example 5 and 70% 1st generation glucose). The mixture obtained was used as a carbon source to feed a fermentation process for 1,4-bioBDO production according to example 2 with minor modifications.

[0192] The chromatograpy analysis showed the production of 1,4-BDO with titer of 117 g/L at the end of the fermentation time (about 35 h) and a productivity of 3.38 g/L/h was observed.