METHOD FOR FORMING A STORAGE STABLE HYDROLYSATE FROM A LIGNOCELLULOSIC MATERIAL

Abstract

The present disclosure generally relates to a method and a system for forming a storage stable hydrolysate from a lignocellulosic material and to a hydrolysate formed by such a method. It also relates to the use of the hydrolysate to reduce and/or control microbial contamination during storage and/or fermentation. Additionally, the present disclosure relates to a method and a system for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process.

Claims

1. A method for forming a storage stable hydrolysate from a lignocellulosic material comprising: a) pretreating said lignocellulosic material to form a pretreated lignocellulosic composition comprising a solid component and a liquid component; said solid component comprising at least lignin and cellulose, b) removing at least 80%, preferably at least 90% of said liquid component from said pretreated lignocellulosic composition to form a separated lignocellulosic component, c) diluting said lignocellulosic component with a dilution liquid to form an aqueous slurry, d) subjecting said aqueous slurry to hydrolysis in the presence of at least one saccharification enzyme to form a hydrolysate, e) adding an antimicrobial compound to said hydrolysate in an amount sufficient to form a storage stable hydrolysate, wherein said antimicrobial compound comprises at least one sulfur oxyanion.

2. The method of claim 1, wherein said aqueous slurry formed in said step c) has a suspended solids content of from 10% to 35% by weight.

3. The method of claim 1, wherein said hydrolysis step d) is performed in the presence of oxygen.

4. The method of claim 1, wherein the concentration of said antimicrobial compound in said hydrolysate is between 1 and 100 mM, preferably between 5 and 25 mM.

5. The method of claim 1, wherein said antimicrobial compound is selected from dithionite and sulfite.

6. The method of claim 1, further comprising the step of subjecting said hydrolysate formed in said step d) or said step e) to separation to remove at least a portion of residual solid components formed during said hydrolysis.

7. The method of claim 1, further comprising the step of controlling the amount of antimicrobial compound in said storage stable hydrolysate by measuring the sulfur ion content in said storage stable hydrolysate, comparing said sulfur ion content with a reference sulfur value, and optionally, adding an additional amount of said antimicrobial compound if said sulfur ion content is lower than said reference sulfur value.

8. The method of claim 1, further comprising washing the separated lignocellulosic component of step b) to comprise a ratio of solid-to-liquid of 40:60-70:30.

9. A hydrolysate formed by the method of claim 1.

10. The hydrolysate of claim 9, being storable up to three weeks, e.g. at least five weeks, such as at least eight weeks at room temperature without getting infected.

11. Use of the hydrolysate of claim 9 to reduce and/or control microbial contamination during storage and/or fermentation of a target chemical.

12. A method for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process comprising: providing a storage stable hydrolysate according to claim 9, subjecting said storage stable hydrolysate to fermentation.

13. The method of claim 12, wherein said fermentation is a fed-batch or a continuous fermentation.

14. The method of claim 12, wherein said method further comprises the step of storing said hydrolysate at least three weeks, preferably at least five weeks, more preferably at least eight weeks at room temperature prior to fermentation.

15. A system for forming a storage stable hydrolysate, wherein said system comprises: a pretreatment unit (2) for pretreating said lignocellulosic material to form a pretreated lignocellulosic composition comprising a liquid component and a solid component, said solid component comprising at least lignin and cellulose, means (7) for removing at least 80%, preferably at least 90% of said liquid component from said pretreated lignocellulosic composition to form a separated lignocellulosic component, means (10) for diluting said lignocellulosic component with a dilution liquid to form an aqueous slurry, a hydrolysis unit (12) arranged to receive said aqueous slurry and to hydrolyze said aqueous slurry in the presence of at least one saccharification enzyme to form a hydrolysate, means (18) for adding an antimicrobial compound to said hydrolysate to form a storage stable hydrolysate, wherein said antimicrobial compound comprises at least one sulfur oxyanion.

16. A system according to claim 15, wherein said hydrolysis unit (12) is configured to receive an air stream through an inlet (21) of said hydrolysis unit (12) and to discharge said air stream by means of an outlet (22) of said hydrolysis unit (12).

17. A system according to claim 15, further comprising means (25) for controlling the amount of antimicrobial compound in said storage stable hydrolysate, wherein said means (25) comprises means for measuring the sulfur ion content of said storage stable hydrolysate, and optionally means for adding additional antimicrobial compound in case the sulfur ion content is lower than a reference value.

18. A system (1) for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process comprising: a system for forming a storage stable hydrolysate according to claim 15 at least one fermentation vessel (23) arranged downstream of and in fluid connection with said system for forming a storage stable hydrolysate.

19. A method for decreasing microbial contamination of a hydrolysate formed from a lignocellulosic material, comprising using dithionite or sulfite.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0144] The various aspects of the present disclosure, including its particular features and advantages, will be readily understood from the following detailed description and the accompanying drawings, in which:

[0145] FIG. 1 schematically illustrates a separate hydrolysis and fermentation (SHF) process according to the present disclosure.

[0146] FIG. 2 illustrates growth of Lactobacillus and production of lactic acid in a spruce hydrolysate detoxified with various concentrations of sodium sulfite.

[0147] FIG. 3 illustrates the growth of Lactobacillus and production of lactic acid in a pure and sugar rich hydrolysate containing various concentrations of sodium sulfite.

DETAILED DESCRIPTION

[0148] The present invention will now be described more fully hereinafter with reference to the accompanying drawings. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided for thoroughness and completeness, and fully convey the scope of the present invention to the skilled person.

[0149] FIG. 1 illustrates a system for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process. The system 1 comprises a pretreatment unit 2, e.g. comprising a reactor, a vessel or a container, wherein the lignocellulosic material is pretreated. The present disclosure is not limited to a particular type of pretreatment method, but any pretreatment method may be utilized. Typically, the temperature in the pretreatment unit 2 is from 150 to 230° C., the pH is from 1 to 3 and the pretreatment time may be from 3 minutes to 60 minutes. The pretreatment unit 2 comprises an inlet 3 and an outlet 4. A feed stream of lignocellulosic material 5 enters the pretreatment unit 2 through the inlet 3, and the pretreated lignocellulosic composition 6 is discharged through the outlet 4. The pretreated lignocellulosic composition 6 comprises a liquid component and a solid component (comprising lignin and cellulose and/or hemicellulose).

[0150] The system comprises means 7 to remove at least 80%, preferably at least 90% of the liquid component from the pretreated lignocellulosic composition 6 to form a separated lignocellulosic component 8. As illustrated in FIG. 1, this may be achieved by means of a separation unit 7 arranged downstream of and in fluid communication with the pretreatment unit 2. Alternatively, it may be arranged inside the pretreatment unit 2. The separation unit 7 is configured to separate the majority of the liquid component from the solid component. As the separation unit 7 removes the liquid component, it also functions as a dewatering unit. The separation unit 7 may comprise a screen, a filter, a decanter screw, a centrifuge or similar equipment to separate and remove the liquid component from the pretreated lignocellulosic composition.

[0151] The liquid component is removed from the separation device, as illustrated by the liquid stream 9. The liquid component comprises water and byproducts formed during the pretreatment process. The liquid stream 9 may be utilized outside the process, e.g. for ethanol fermentation. The separated lignocellulosic component (comprising the solid component of the pretreated lignocellulosic composition) 8 is discharged from the separation unit 7 and is thereafter diluted.

[0152] Means 10 for diluting the lignocellulosic component 8 with a dilution liquid, preferably water, to form an aqueous slurry 11 may be performed in a separate dilution vessel or tank as illustrated in FIG. 1. Dilution can e.g. be accomplished by means of a dilution screw, or a mixer/pump/standpipe with dilution liquid addition. A stream of dilution liquid, e.g. water is added to the lignocellulosic component 8 downstream of the separation unit 7 such that an aqueous slurry 11 is formed. It is also conceivable that the stream of water is added to the separation unit 7 such that the separation unit 7 acts both to separate the “dirty” liquid component and to replace it with fresh water.

[0153] The system 1 further comprises a hydrolysis unit 12 for hydrolyzing the aqueous slurry, which has both been pretreated and cleaned; i.e. separated and diluted with water.

[0154] The hydrolysis unit 12 comprises an inlet 13 for receiving the aqueous slurry 11, and an outlet 14 for discharge of the hydrolysate 15 after the hydrolysis has been completed.

[0155] A second separation unit 16 may be arranged to receive the hydrolysate 15 in order to remove residual solids formed during hydrolysis. As illustrated in FIG. 1, residual solid components 17 are removed from the hydrolysate 15.

[0156] The system also comprises means 18 for adding antimicrobial compound to the hydrolysate 15 before separation in the second separation unit 16 or to the separated hydrolysate stream 19. It is also conceivable to add the antimicrobial compound directly to the hydrolysis unit 12 upon completion of the hydrolysis reaction. Antimicrobial compound may, if needed, also be added to the fermentation vessel 23. The dotted arrows in FIG. 1 illustrates points in the process where the antimicrobial compound can be added.

[0157] The hydrolysis unit 12 may be configured to receive an air stream 20 through an inlet 21 and to discharge the air stream 20 through an outlet 22 of the hydrolysis unit 12.

[0158] In other words, the air stream 20 is mixed with the feedstock within the hydrolysis unit 12. This may be achieved by simultaneously stirring the feedstock in the hydrolysis unit 12. The air stream 20 allows for the enzymes present in the system to become more efficient, yielding a quicker and more efficient hydrolysis.

[0159] The air inlet 21 may be the same as the inlet 13 for receiving the aqueous slurry 11 and the air outlet 22 may be the same as the outlet 14 for discharging the hydrolysate stream 15. Alternatively, the air inlet 21 is arranged in a bottom portion of the hydrolysis unit 12 and the air outlet 22 is arranged in a top portion of the hydrolysis unit 12, as illustrated in FIG. 1.

[0160] The system 1 may be connected to a fermentation vessel 23 arranged in fluid communication with and downstream of the hydrolysis unit 12.

[0161] A product recovery unit 24, such as distillation or ion exchange chromatography may be connected to the system downstream of the fermentation vessel.

[0162] The system may also comprise means 25 for controlling the amount of antimicrobial compound. Such means 25 comprises means for measuring the sulfur ion content of the hydrolysate (15 or 19) after a sufficient amount of antimicrobial has been added. The measuring means may be a sulfur probe or a sulfur sensor arranged or inserted into the process to measure the sulfur content after the antimicrobial compound has been added to the hydrolysate. FIG. 1 illustrates suitable positions in the process, when the sulfur ion content is preferably monitored (see illustrative dotted arrows from antimicrobial control means 25). In embodiments, the sulfur ion content is measured inline; i.e. the measurements are performed directly in the process line. The sulfur ion content may be measured inline or online. If the sulfur ion content is measured online, a sample is diverted from the process line, e.g. in a bypass loop from the main process line (not shown). Means for measuring the sulfur ion content may also (or alternatively) be arranged within the fermentation vessel 23 for securing an efficient and infection free fermentation.

[0163] If the fermentation vessel 23 also comprises means to control the microbial fermentation by means for measuring a residual sugar indicator parameter, RSI, the content of sulfur may be measured simultaneously with such parameter.

[0164] After a storage stable hydrolysate has been formed, i.e. after the addition of antimicrobial to the hydrolysate stream 15, or optionally hydrolysate stream 19, the hydrolysate may be removed from the process and stored during a period of time, before being used for subsequent fermentation in the fermentation vessel 23.

[0165] Although FIG. 1 illustrates a system 1 for reducing and/or controlling microbial contamination in a separate hydrolysis and fermentation (SHF) process, the same components are used in a system for forming a storage stable hydrolysate (apart from the fermentation vessel 23 and product recovery unit 24).

[0166] Terms, definitions and embodiments of the first aspect of the present disclosure apply mutatis mutandis to the other aspects of the present disclosure, and vice versa.

Example 1: Bacterial Contamination in a Non-Separated Lignocellulosic Hydrolysate

[0167] Non-detoxified spruce hydrolysate (with added 2 g/l yeast extract; 0.5% sodium phosphate; 0.5 g/l diammonium phosphate; 2 g/l peptone and diluted with water to 30 g/l glucose) was filter sterilized and used to represent feed media. An inoculum was prepared using MRS media and the same contaminated sample as used in previous experiments, and was incubated at 37° C. overnight (16 hours). Sodium sulfite was added at concentrations of 0 mM, 5 mM, 10 mM, 20 mM and 40 mM, respectively, to 25 ml serum bottles with 22.5 ml of the feed media. After inoculation, the bottles were placed on a multiple magnetic stirring plate at room temperature, with samples taken periodically and analyzed using HPLC.

[0168] The hydrolysate used may represent a feed stream where no separation of the pretreatment liquid and solid component has taken place. In other words, hydrolysis and fermentation inhibitors are still present in the feed stream.

[0169] As illustrated in FIG. 2, the addition of an amount of 5 to 40 mM of sodium sulfite has no antimicrobial effect on the hydrolysate. Instead, the sulfite added acts as a detoxifying agent on the fermentation inhibitors still present in the hydrolysate. The result is an increased bacterial growth when sodium sulfite is added.

Example 2: Controlling Bacterial Contamination in a Sugar-Rich Hydrolysate

[0170] The aim of this experiment was to test the potential of sodium sulfite to control bacterial contamination in the storage tanks which contain sugar-rich hydrolysate streams before they are used in fermentations. The hydrolysate is representative of a hydrolysate according to the present disclosure.

[0171] To test the theory, synthetic media was made containing about 90 g/l glucose, 0.5% sodium phosphate/phosphoric acid buffer (pH 5.5), 0.5 g/l diammonium phosphate, 2 g/l yeast extract and 2 g/l peptone. This solution (45 ml) was added to 50 ml serum bottles, along with a magnetic stirrer bar, and autoclaved, where after sodium sulfite at concentrations of 5, 10, 20 and 40 mM was added, while some bottles had no sulfite added, which served as control (0 mM). In an Erlenmeyer flask, 150 ml MRS media was inoculated using a sample previously collected from a contaminated fermentation at SEKAB, previously showed to involve lactic acid bacteria. This culture was incubated at 37° C. for 36 hours and used to inoculate all the serum bottles (10% v/v). The serum bottles were then sealed with an airtight septum and a metal ring clamp and two needles were inserted through the septum to allow for sampling. The serum bottles were placed on a magnetic stirrer plate at room temperature (since storage tanks are typically kept at room temperature), with stirring at 100 rpm. Samples were taken periodically and analyzed for glucose and lactic acid using HPLC. Experiments were performed in triplicate. The experiments were continued for 11 days.

[0172] The only growth that took place in the experiment was in the control bottles, both based on the visual evaluation, which indicated no growth or turbidity in any other bottles, as well as the lactic acid data obtained with HPLC analysis. This seems to support the theory that sodium sulfite inhibits the growth of lactic acid bacteria under these conditions. As illustrated in FIG. 3, sodium sulfite at all concentrations tested (5, 10, 20 and 40 mM) inhibited the growth of lactic acid bacteria (the concentration of lactic acid was 0 g/l).

Example 3: Controlling Bacterial Contamination in a Sugar-Rich Hydrolysate Comprising 10% Non-Detoxified Spruce Hydrolysate

[0173] Experiments in this example were performed according to the description in example 2 with the difference that the sugar-rich hydrolysate contained 10% (v/v) non-detoxified spruce hydrolysate. This was done to simulate sugar-rich hydrolysate with some inhibitory impurities present that may arise from insufficient separation and/or washing.

[0174] In the “Experiment A” samples, serum bottles were inoculated with Lactobacillus sp. at time 0 hours and incubated at room temperature for 21 days. Samples were taken periodically and analyzed for glucose and lactic acid (not shown here) using HPLC.

[0175] In the “Experiment B” samples, serum bottles were inoculated twice—firstly on the first day and again two weeks later, to simulate a re-infection and test the longevity of the inhibitory effect of sodium sulfite.

[0176] In Experiment “C”, the third group of serum bottles were inoculated after two weeks of storage at room temperature and inoculated with Lactobacillus (after 14 days).

[0177] Table 1 illustrates the glucose consumption at different time points for experiments A-C.

TABLE-US-00001 TABLE 1 Glucose concentration at different time points Glucose (g/L) Sample 0 h 168 h 336 h 508 h Experiment A 0 mM 84.1 79.3 77.5 74.1 5 mM 83.8 71.9 66.3 65.9 10 mM 84.8 72.9 64.6 63.8 20 mM 83.4 75.2 70.1 67.2 40 mM 82.0 80.1 77.0 82.5 Experiment B 0 mM 83.3 69.1 69.0 71.2 5 mM 83.8 72.6 66.8 69.0 10 mM 81.7 71.5 67.9 73.4 20 mM 83.1 69.0 72.4 72.1 40 mM 85.2 86.6 86.2 86.3 Experiment C 0 mM 82.1 81.1 83.8 78.8 5 mM 85.4 84.8 83.4 77.1 10 mM 84.8 81.7 81.9 75.5 20 mM 85.9 82.3 83.5 79.5 40 mM 80.9 81.0 85.6 82.6

[0178] Growth was observed in all three experiments for all samples except in those containing 40 mM sodium sulfite, indicated by the glucose consumption in these samples (see table 1). Similar to example 1, a detoxification effect was observed in the samples containing 5-20 mM sodium sulfite, which grew faster than the sample with no sulfite added (0 mM). Thus, in contrast to example 1, where addition of sodium sulfite resulted in a detoxification effect and facilitated growth of Lactobacillus sp., sodium sulfite levels of 40 mM instead introduced an antimicrobial effect in this example. While 10% (v/v) of lignocellulosic hydrolysate containing impurities may be considered a moderate to low level of impurities, this examples clearly shows the importance of creating sugar-rich hydrolysates with a minimum of inhibitory compounds. Presence of such compounds results in a reaction between the sodium sulfite and the inhibitory compounds, causing a detoxification of the hydrolysate and facilitating bacterial growth rather than introducing an antimicrobial effect. To displace the effect of detoxification caused by sodium sulfite addition (even at moderate levels of impurities) requires higher amounts of sodium sulfite, i.e. 40 mM in this experiment compared to 5 mM in examples 2 and 4.

Example 4: Storage of a Sugar-Rich Hydrolysate

[0179] Storage of sugar-rich hydrolysate with addition of 5-40 mM was performed in accordance with the description of example 2. In this example, the samples were prepared and handled as in example 2. In other words, the “Experiment A” samples in this example are identical to those of example 2, and these were used as a reference for the “Experiment B” and “Experiment C” samples, as well as for validating the results of example 2.

[0180] The “Experiment B” samples were inoculated with a Lactobacillus sp. at time 0 hours, and again after 336 hours. This was done to test the infection resistance of samples that were exposed to more than one culture of Lactobacillus sp; i.e. samples that were re-infected after two weeks.

[0181] The samples in “Experiment C” were stored at ambient room temperature for 2 weeks, after which Lactobacillus was added to the flasks, after 336 hours. Samples were incubated for another 7 days and a total of 21 days as for the other two experiments. Table 2 illustrates the glucose levels at various time points throughout the storage period.

TABLE-US-00002 TABLE 2 Glucose concentration at different time points Glucose (g/L) Sample 0 h 172 h 340 h 510 h Experiment A 0 mM 84.4 75.1 63.9 38.3 5 mM 83.3 84.0 83.5 83.2 10 mM 84.1 85.3 82.8 82.2 20 mM 83.7 85.6 86.8 84.7 40 mM 81.5 83.2 83.2 83.6 Experiment B 0 mM 83.4 73.9 62.6 37.2 5 mM 82.3 82.9 82.6 78.3 10 mM 82.9 84.3 81.5 80.8 20 mM 82.8 74.7 86.0 83.3 40 mM 80.2 82.1 82.0 82.5 Experiment C 0 mM 81.7 82.4 84.6 68.7 5 mM 81.2 81.6 85.5 82.0 10 mM 82.2 81.8 84.0 82.4 20 mM 80.8 80.3 84.5 83.3 40 mM 82.0 81.6 86.2 82.0

[0182] The results of “Experiment A” verified the effect observed in example 2; i.e. that bacterial growth occurs in the absence of sodium sulfite. Flasks with added sodium sulfite showed no glucose consumption during three weeks, indicating that no bacteria were able to grow, and thus no consumption of sugar was evident.

[0183] The results of “Experiment B” are the same as “Experiment A”, but allows for a second effect of sodium sulfite addition to be demonstrated; i.e. that a first addition of Lactobacillus sp. does not decrease the effect of sodium sulfite significantly at any concentration above 5 mM to which a second addition of Lactobacillus sp. was made. No signs of bacterial growth was observed during 21 days for experiments containing 5 mM or more of sodium sulfite.

[0184] The results from “Experiment C” demonstrate that 14 days of storage of a sugar-rich hydrolysate at room temperature containing sodium sulfite concentrations above 5 mM does not decrease the antimicrobial effect of sodium sulfite. As seen in table 2, no growth was observed in such samples compared to the reference with no sodium sulfite addition. Hence, sodium sulfite at all concentrations tested (5, 10, 20 and 40 mM) inhibited the growth of Lactobacillus sp. over three weeks of incubation while samples containing no sulfite allowed for bacterial growth (see table 2). These results indicate that storage for at least three weeks can be achieved with additions of 5-40 mM sodium sulfite when no compounds (or substantially no compounds) that can react with the sodium sulfite are present (compared to e.g. example 3). Presence of such compounds rather results in detoxification effects demonstrated in example 1 and in example 3.

[0185] Even though the present disclosure has been described with reference to specific exemplifying embodiments thereof, many different alterations, modifications and the like will become apparent for those skilled in the art.

[0186] Variations to the disclosed embodiments can be understood and effected by the skilled addressee in practicing the present disclosure, from a study of the drawings, the disclosure, and the appended claims. Furthermore, in the claims, the word “comprising” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude a plurality.