CN109022498B

CN109022498B - Method for reducing discharge of acetone-butanol-ethanol fermentation waste liquid

Info

Publication number: CN109022498B
Application number: CN201810887243.XA
Authority: CN
Inventors: 谭天伟; 张长伟; 蔡的; 秦培勇; 司志豪; 陈长京; 周明园; 曾海旺
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2018-08-06
Filing date: 2018-08-06
Publication date: 2022-06-21
Anticipated expiration: 2038-08-06
Also published as: CN109022498A

Abstract

The invention relates to a method for reducing the discharge of acetone-butanol fermentation waste liquid. The method can be reused for the next batch of fermentation without treatment or after simple treatment after the volatile organic products in the fermentation culture are simply filtered, so that the waste fermentation mash (fermentation wastewater) containing acetic acid and butyric acid is recycled for multiple batches, the reabsorption of organic acid byproducts is promoted, and the discharge amount of biological butanol fermentation wastewater is reduced. Simple process, strong operability and environmental protection. By implementing the method, the problems of large wastewater discharge, high environmental pollution, insufficient utilization of organic acid by-products and the like in the ABE fermentation production process can be effectively solved.

Description

Method for reducing discharge of acetone-butanol-ethanol fermentation waste liquid

Technical Field

The invention belongs to the technical field of bioseparation, biofermentation and biorefinery, and particularly relates to a method for reducing the emission of acetone-butanol-ethanol fermentation waste liquid.

Background

(n) butanol is an important platform compound and a potential alternative liquid fuel. By utilizing biotechnology, the realization of fermentation production of bio-based butanol can effectively relieve the dependence of human society on non-renewable petrochemical energy and chemicals, reduce environmental pollution, and increasingly receive high attention from the industrial industry. The production of biobutanol products by acetone-butanol-ethanol (ABE) fermentation has a long history, but suffers from high product inhibition, the butanol concentration in ABE fermentation products is usually only 1-2% (w/w), and the fermentation efficiency of solvent products such as acetone butanol is low. One of the main consequences of the above-mentioned series of problems is that the fermentation wastewater discharge per biobutanol production is greater than the consumption of bioethanol. It is reported that for every 1 ton of ABE solvent produced, 45-50 tons of wastewater are produced, and about 35 tons of wastewater are treated, except that a portion of the wastewater can be recycled. The waste water contains a large amount of fermentation byproducts such as organic acid and the like and hydrolysis byproducts of lignocellulose raw materials, so that the chemical oxygen demand is large, the treatment cost is high, and the environmental hazard is huge.

In recent years, some reports have been made on treatment of wastewater from biological butanol fermentation. These reports have made use of biobutanol wastewater for the preparation and production of other biochemicals. For example, an anaerobic baffled reactor is used to remove COD and by-produce methane from wastewater from acetone butanol fermentation (Bioresource Technology,2011,102, 7407-. The acetone butanol fermentation wastewater is treated by combining the physical and chemical treatment of photosynthetic bacteria and the feed protein is prepared (water treatment technology, 1995,21: 291-. The preparation of microbial oil and bacterial cellulose and the like is realized by adopting oleaginous microorganisms or cellulose-producing microorganisms to treat acetone butanol wastewater (Carbohydrate Polymers,2016,136, 198-202; Renewable Energy,2013,55, 31-34). After a series of subsequent biological processes, although high value-added byproducts are obtained and high value-added utilization of waste water organic matters is realized, one of the common problems of the methods is that the reduction of the fermentation waste water amount cannot be realized. The fermentation wastewater of the post-process still needs to be treated by the traditional wastewater treatment mode.

Therefore, there is a problem in that research and development of a process for effectively recycling byproducts such as acetic acid and butyric acid while reducing the amount of wastewater from biobutanol fermentation are required.

Disclosure of Invention

The invention aims to solve the technical problem of providing a method for reducing the discharge of acetone butanol ethanol fermentation waste liquid aiming at the defects of the prior art. The method can effectively realize the reutilization of byproducts such as acetic acid, butyric acid and the like while reducing the amount of the biological butanol fermentation wastewater.

Therefore, the invention provides a method for reducing the discharge of the waste liquid of the acetone butanol ethanol fermentation, which comprises the following steps:

step B, obtaining fermentation liquor containing volatile organic products through ABE fermentation;

step C, carrying out separation treatment on the fermentation liquor containing the volatile organic products, and obtaining residual fermentation liquor after separating the volatile organic products;

d, mixing the lignocellulose pretreatment residues with the residual fermentation liquor, and carrying out enzymolysis to obtain lignocellulose enzymolysis liquid;

e, recycling the lignocellulose enzymolysis liquid obtained in the step D to the step B, repeating the step B to the step E, and performing circulating fermentation and enzymolysis;

wherein the volatile organic product comprises one or more of acetone, butanol and ethanol.

According to the process of the present invention, the separation treatment comprises separation of volatile organic products and/or solid-liquid separation.

In some embodiments of the invention, the method of separating volatile organic products comprises one or more of liquid-liquid extraction, adsorption, pervaporation, gas stripping, vacuum separation, osmotic extraction, supercritical extraction, and rectification.

According to some embodiments of the invention, in step C, the fermentation broth containing the volatile organic products is subjected to solid-liquid separation and after separation of the volatile organic products, a first remaining fermentation broth is obtained.

In some embodiments of the present invention, the concentration of acetic acid in the first remaining fermentation broth is greater than or equal to 0.1g/L, preferably 0.1-10 g/L, more preferably 0.1-6 g/L, and even more preferably 0.5-4 g/L.

In some embodiments of the present invention, the concentration of butyric acid in the first residual fermentation broth is greater than or equal to 0.1g/L, preferably 0.1-10 g/L, more preferably 0.1-4 g/L, and even more preferably 0.1-3 g/L.

In some embodiments of the invention, the butanol concentration in the first residual fermentation broth is below 3 g/L.

In some embodiments of the invention, the ethanol concentration in the first residual fermentation broth is below 2g/L.

In some embodiments of the invention, the acetone concentration in the first residual fermentation broth is below 2g/L.

In some embodiments of the invention, the total residual sugar concentration in the first remaining fermentation broth is less than 20 g/L.

According to some embodiments of the invention, in step C, after the fermentation broth containing the volatile organic product is subjected to solid-liquid separation and the volatile organic product is separated, the obtained first residual fermentation broth is subjected to acid reduction treatment to obtain a second residual fermentation broth.

In some embodiments of the present invention, the concentration of acetic acid in the second residual fermentation liquid is 0 to 6g/L, more preferably 0 to 4g/L, and still more preferably 0 to 2g/L.

In some embodiments of the present invention, the concentration of butyric acid in the second residual fermentation broth is 0 to 6g/L, more preferably 0 to 4g/L, and still more preferably 0 to 2g/L.

In the invention, the acid reduction treatment mode comprises biological treatment and/or physical treatment.

In some embodiments of the invention, the physical process treatment comprises activated carbon adsorption and/or resin adsorption.

In some embodiments of the invention, the biological process treatment comprises microbial oil fermentation and/or bacterial cellulose fermentation.

In some embodiments of the invention, the biological process is a microbial oil fermentation comprising:

step M1, mixing the lignocellulose pretreatment residues with the first residual fermentation broth, and carrying out enzymolysis to obtain a first lignocellulose enzymolysis liquid;

step M2, inoculating microbial lipid yeast into the first lignocellulose hydrolysate obtained in the step M1, and performing microbial lipid fermentation to obtain microbial lipid fermentation liquor;

and step M3, using the microbial oil fermentation supernatant obtained by solid-liquid separation of the microbial oil fermentation broth as a second residual fermentation broth.

In some embodiments of the present invention, the microbial lipid fermenting bacteria include one or more of microbial lipid producing bacteria capable of metabolically utilizing low molecular chain organic acid as a substrate, preferably rhodotorula glutinis and/or rhodosporidium toruloides.

Accordingly, it is preferred that the microbial lipid fermentation include, but are not limited to, Rhodotorula glutinis fermentation and/or Rhodosporidium toruloides fermentation.

According to some embodiments of the invention, in step D, the lignocellulose pretreatment residue is mixed with the remaining fermentation broth and pH buffering salt and then subjected to enzymatic hydrolysis to obtain a lignocellulose enzymatic hydrolysate.

In the invention, the lignocellulose enzymolysis solution comprises lignocellulose enzymolysis supernatant and/or lignocellulose enzymolysis stock solution containing insoluble solid phase.

In some embodiments of the invention, the concentration of fermentable sugars in the lignocellulosic hydrolysate is 20 to 100g/L, preferably 20 to 80g/L, and more preferably 40 to 60 g/L.

In some embodiments of the invention, the lignocellulosic hydrolysate is reused in step B after adjusting the pH to 5.5-7.5, preferably to 6-7.

The invention provides a method for reducing the discharge of acetone-butanol fermentation waste liquid. The method can be reused for the next batch of fermentation without treatment or after simple treatment after the volatile organic products in the fermentation culture are simply filtered, so that the waste fermentation mash (fermentation wastewater) containing acetic acid and butyric acid is recycled for multiple batches, the reabsorption of organic acid byproducts is promoted, and the discharge amount of biological butanol fermentation wastewater is reduced. Simple process, strong operability and environmental protection. By implementing the method, the problems of large wastewater discharge amount, high environmental pollution, insufficient utilization of organic acid by-products and the like in the ABE fermentation production process can be effectively solved.

Drawings

The invention will be described below with reference to the accompanying drawings.

FIG. 1 is a schematic diagram of batch free fermentation-pervaporation separation and residual fermentation broth recycling processes for producing acetone butanol ethanol by lignocellulose fermentation.

FIG. 2 is a schematic diagram of batch free fermentation, gas stripping separation and Rhodotorula glutinis fermentation broth recycling process for producing acetone butanol ethanol by lignocellulose fermentation.

FIG. 3 is a simplified diagram of the recycling process of the residual fermentation broth after the fermentation of lignocellulose for producing acetone butanol ethanol, batch free fermentation, gas stripping separation and activated carbon adsorption.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings.

As mentioned above, the main treatment method for biological butanol fermentation wastewater is to use biological butanol wastewater for the preparation and production of other biochemicals. Although the method obtains the by-product with high added value after a series of subsequent biological processes and realizes the high added value utilization of the organic matters in the wastewater, the common problem of the method is that the reduction of the amount of the fermentation wastewater cannot be realized, and the fermentation wastewater of the post-process still needs to be treated by using the traditional wastewater treatment mode. In view of this, the present inventors have conducted extensive studies on biobutanol fermentation and wastewater treatment thereof.

The inventor researches and discovers that during the ABE fermentation process, the lignocellulose enzymolysis liquid is used as a fermentation raw material, after the fermentation is finished, solid-phase substances such as thalli and the like and target products of volatile organic products (such as ethanol, acetone and butanol) in a fermentation culture are simply filtered, and after the treatment such as no treatment or simple dilution, concentration and the like, the residual fermentation liquid containing organic acid byproducts (butyric acid and acetic acid) is used as an enzymolysis buffer solution for lignocellulose enzymolysis to prepare the fermentation raw material for the next batch of fermentation production, so that the recycling of multiple batches of biological butanol waste water can be realized, the reabsorption of the organic acid byproducts is promoted, and the emission of the biological butanol fermentation waste water is reduced. The present invention has been made based on the above findings.

Therefore, the method for reducing the emission of the waste liquid from the acetone butanol ethanol fermentation in the first aspect of the invention comprises the following steps:

and E, recycling the lignocellulose enzymolysis liquid obtained in the step D in the step B, repeating the step B to the step E, and performing circulating fermentation and enzymolysis.

The volatile organic product in the invention comprises one or more of acetone, butanol and ethanol, and is preferably a solution containing acetone, butanol and ethanol.

It will be understood by the skilled person that the fermentation broth containing volatile organic products as described above is understood to be a fermentation culture containing volatile organic products which, in addition to containing mainly volatile organic products, also contain organic acid by-products of the fermentation process and additionally a small amount of solid matter.

In the present invention, the organic acid by-product mainly includes acetic acid and butyric acid.

It should be understood by those skilled in the art that the method for reducing the discharge of the acetone butanol ethanol fermentation waste liquid in the present invention can be understood as a method or a process specially developed and designed by the present inventors for greatly reducing the discharge of fermentation liquid waste water in the ABE fermentation process, simultaneously realizing the effective utilization of organic acid by-products such as acetic acid and butyric acid in the fermentation liquid waste water, and improving the added value thereof, and recycling the remaining fermentation liquid from which the volatile organic products are removed.

According to the process of the present invention, the separation treatment comprises separation of volatile organic products and/or solid-liquid separation. Preferably, the separation treatment comprises separation of volatile organic products and solid-liquid separation.

In the present invention, the order of the operation of "solid-liquid separation" and the operation of "separation of volatile organic products" in the separation treatment is not particularly limited, and for example, the volatile organic products may be separated by first subjecting a fermentation broth containing acetone butanol ethanol to solid-liquid separation, or the volatile organic products may be separated by first subjecting a fermentation culture containing acetone butanol ethanol to solid-liquid separation, or the volatile organic products may be separated by first subjecting a fermentation broth containing acetone butanol ethanol and organic acid by-products to solid-liquid separation.

In the present invention, the timing of separating the volatile organic product from the fermentation broth containing acetone butanol ethanol is not particularly limited, and for example, the separation of the volatile organic product such as acetone butanol ethanol may be performed after the completion of the fermentation, or the separation of the volatile organic product such as acetone butanol ethanol may be performed by an in-situ separation means during the fermentation.

The separation method of the present invention for separating the volatile organic product from the fermentation broth containing acetone butanol ethanol is not particularly limited, and for example, the volatile organic product can be separated from the fermentation broth containing acetone butanol ethanol by a separation method that is conventional in the art; in some embodiments of the invention, the method of separating volatile organic products comprises one or more of liquid-liquid extraction, adsorption, pervaporation, gas stripping, vacuum separation, osmotic extraction, supercritical extraction, and rectification.

The method of the solid-liquid separation in the present invention is not particularly limited, and for example, the solid-liquid separation can be carried out by a separation method which is conventional in the art; in some embodiments of the invention, the method of solid-liquid separation comprises, but is not limited to, one or more of centrifugation, suction filtration, and microfiltration.

According to some embodiments of the invention, in step C, the fermentation broth containing the volatile organic products is subjected to solid-liquid separation and after separation of the volatile organic products, a first remaining fermentation broth is obtained. The first remaining fermentation broth contains organic acid by-products (i.e., acetic acid and butyric acid).

It will be appreciated by those skilled in the art that, in the present invention, the first remaining fermentation broth containing organic acid by-products (i.e., acetic acid and butyric acid) obtained by subjecting the fermentation broth containing acetone butanol ethanol and organic acid by-products to solid-liquid separation in step C and separating out volatile organic products may be used for lignocellulose (pretreatment residue) enzymolysis, and the obtained enzymolysis liquid may be used for the next batch of ABE fermentation.

In the enzymolysis process, the first residual fermentation liquor can play a role of an enzymolysis buffer solution due to the fact that the first residual fermentation liquor contains acetic acid and butyric acid, and meanwhile, the first residual fermentation liquor can play a role of an enzymolysis medium due to the fact that a large amount of water is contained in the first residual fermentation liquor; and the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation. The inventor researches and discovers that in order to better realize the function of the enzymolysis buffer solution of the first residual fermentation liquor, the concentrations of acetic acid and butyric acid in the first residual fermentation liquor are respectively and independently more than or equal to 0.1 g/L.

In some embodiments of the invention, the concentration of acetic acid in the first remaining fermentation broth is greater than or equal to 0.1g/L, preferably 0.1-10 g/L, more preferably 0.1-6 g/L, and even more preferably 0.5-4 g/L; the concentration of butyric acid in the first residual fermentation liquid is more than or equal to 0.1g/L, preferably 0.1-10 g/L, more preferably 0.1-4 g/L, and even more preferably 0.1-3 g/L.

In some embodiments of the invention, the butanol concentration in the first residual fermentation broth is below 3 g/L; the ethanol concentration in the first residual fermentation liquid is below 2 g/L; the concentration of acetone in the first residual fermentation liquid is below 2g/L.

According to some embodiments of the present invention, in step C, after the fermentation liquid containing the volatile organic product is subjected to solid-liquid separation and the volatile organic product is separated, the obtained first residual fermentation liquid is subjected to acid reduction treatment to obtain a second residual fermentation liquid. The second remaining fermentation broth may or may not contain organic acid byproducts (i.e., acetic acid and butyric acid). In general, the content of organic acid by-products (i.e., acetic acid and butyric acid) in the second residual fermentation broth is less than or equal to the content of organic acid by-products (i.e., acetic acid and butyric acid) in the corresponding first residual fermentation broth.

In some embodiments of the invention, the concentration of acetic acid in the second residual fermentation broth is 0-6 g/L, more preferably 0-4 g/L, and still more preferably 0-2 g/L; the concentration of butyric acid in the second residual fermentation liquid is 0-6 g/L, more preferably 0-4 g/L, and still more preferably 0-2 g/L.

In some embodiments of the invention, the physical method treatment comprises activated carbon adsorption and/or resin adsorption.

It will be appreciated by those skilled in the art that, in the present invention, the first remaining fermentation broth containing organic acid by-products (i.e. acetic acid and butyric acid) obtained in step C by subjecting the fermentation broth containing acetone butanol ethanol and organic acid by-products to solid-liquid separation and separating out volatile organic products can be used directly for lignocellulose (pretreatment residue) enzymatic hydrolysis without any further treatment, and the obtained enzymatic hydrolysate can be used for the next batch of ABE fermentation; or subjecting the first residual fermentation liquid containing the organic acid byproducts (namely acetic acid and butyric acid) obtained by performing solid-liquid separation on the fermentation liquid containing acetone butanol ethanol and the organic acid byproducts and separating volatile organic products in the step C to further acid reduction treatment, using the obtained byproduct (namely acetic acid and butyric acid) containing or not containing the organic acid for enzymolysis of lignocellulose (pretreatment residue), and using the obtained enzymolysis liquid for next batch of ABE fermentation.

In the enzymolysis process, the second residual fermentation liquor contains a small amount of acetic acid and butyric acid, so that the second residual fermentation liquor can play a certain role of an enzymolysis buffer solution and does not inhibit fermentation, and meanwhile, the second residual fermentation liquor contains a large amount of water and can play a role of an enzymolysis medium; and the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation.

The inventor has unexpectedly discovered that the second residual fermentation broth which does not contain acetic acid and butyric acid still can play a role of a certain enzymolysis buffer solution and cannot inhibit fermentation due to the fact that the second residual fermentation broth contains a small amount of other organic acids such as lactic acid, and meanwhile, the second residual fermentation broth can play a role of an enzymolysis medium due to the fact that the second residual fermentation broth contains a large amount of water; and the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation.

According to some embodiments of the invention, the biological process treatment is microbial oil fermentation, comprising:

In some specific embodiments of the invention, the biological process is a microbial oil fermentation comprising:

step N1, mixing the lignocellulose pretreatment residues with the first residual fermentation broth, and carrying out enzymolysis to obtain a first lignocellulose enzymolysis liquid;

step N2, inoculating a Rhodotorula glutinis strain into the first lignocellulose enzymolysis liquid obtained in the step N1, and performing Rhodotorula glutinis fermentation to obtain a Rhodotorula glutinis fermentation liquid;

and step N3, using the supernatant of the fermentation of the rhodotorula glutinis obtained by solid-liquid separation of the fermentation liquor of the rhodotorula glutinis as the second residual fermentation liquor.

Similar to the above, in the enzymolysis process of step N1, the first residual fermentation broth can function as an enzymolysis buffer solution because it contains acetic acid and butyric acid, and at the same time, the first residual fermentation broth can function as an enzymolysis medium because it contains a large amount of water; the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for fermentation of Rhodotorula glutinis.

The inventor researches and discovers that the rhodotorula glutinis can metabolize organic acid in fermentation liquor, so that the organic acid is eaten and converted into grease. The fermentation is facilitated when the concentration of the organic acid in the fermentation liquor is low, the organic acid is converted as a carbon source, and the fermentation inhibition is caused when the concentration of the acid is high. When multiple batches of waste liquid are recycled, the concentration of the organic acid in the fermentation liquid is gradually increased along with the increase of the batches due to accumulation caused by slow concentration and metabolism, so that the fermentation effect of the subsequent batches is gradually deteriorated due to the gradual accumulation of the organic acid when the fermentation waste water is recycled in multiple batches, and the concentration of the organic acid in the fermentation waste liquid can be reduced after the fermentation waste water is treated by the rhodotorula glutinis, so that the fermentation effect of the fermentation waste water recycled in multiple batches is better, and the method is mainly embodied in the aspects of product concentration, conversion rate of a substrate to a product, solvent production rate and the like.

It will be readily appreciated that the solid-liquid separation of the fermentation broth of Rhodotorula glutinis in step N3 above is for the removal of Rhodotorula glutinis. It is understood by those skilled in the art that the microbial oil in the rhodotorula glutinis is a high value-added product and is mainly used for the oil recovery of the subsequent process. In step N3, if the Rhodotorula glutinis is removed, the Rhodotorula glutinis mixed with Clostridium butyricum in the fermentation system in the subsequent fermentation batch, such a fermentation mode belongs to mixed fermentation, is difficult to control, and the synergistic or inhibitory effect of the two floras is unknown.

The method of solid-liquid separation in step N3 in the present invention is not particularly limited, and for example, solid-liquid separation can be carried out by a separation method which is conventional in the art; in some examples, for example, the solid-liquid separation methods include, but are not limited to, centrifugation, suction filtration, and microfiltration.

The rhodotorula glutinis fermentation supernatant obtained by solid-liquid separation of the rhodotorula glutinis fermentation culture in the step N3 contains or does not contain organic acid by-products (acetic acid and butyric acid), and can be used as the second remaining fermentation broth for the enzymolysis process in the step D.

Similar to the above, in the enzymolysis process in step D, the second residual fermentation broth (rhodotorula glutinis fermentation supernatant) can play a role of a certain enzymolysis buffer solution and does not inhibit fermentation because of containing a small amount of acetic acid and butyric acid, and meanwhile, the second residual fermentation broth can play a role of an enzymolysis medium because of containing a large amount of water; and the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation.

For the second residual fermentation liquid (rhodotorula glutinis fermentation supernatant liquid) without acetic acid and butyric acid, the second residual fermentation liquid can still play a role of a certain enzymolysis buffer solution and cannot inhibit fermentation due to the fact that the second residual fermentation liquid contains a small amount of other organic acids such as lactic acid, and meanwhile, the second residual fermentation liquid can play a role of an enzymolysis medium due to the fact that the second residual fermentation liquid contains a large amount of water; and the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation.

In some embodiments of the present invention, the process for recycling the fermentation broth (second residual fermentation broth) of microbial oil (i.e. fermentation broth) for fermentation of lignocellulose to produce acetone butanol ethanol is shown in fig. 2, and as can be seen from fig. 2, the method for reducing the discharge of waste acetone butanol ethanol fermentation liquid in the present invention comprises the following steps:

(1) inoculating a fermentation substrate containing fermentable sugars with an acetobutylcarbinol-producing ethanol fermentation strain (e.g., c. acetobutylicum ATCC824) to perform ABE fermentation to obtain a fermentation broth containing acetobutylcarbinol [ and organic acid by-products (acetic acid and butyric acid) ];

(2) carrying out solid-liquid separation on fermentation liquor containing acetone butanol ethanol [ and organic acid byproducts (acetic acid and butyric acid) ], and separating out a volatile organic product (acetone butanol ethanol product) through pervaporation to obtain first residual fermentation liquor containing the organic acid byproducts (acetic acid and butyric acid);

(3) mixing lignocellulose pretreatment residues with a first residual fermentation broth containing organic acid byproducts (acetic acid and butyric acid), and adding cellulase for enzymolysis to obtain a first enzymolysis liquid;

(4) adjusting the pH of the first enzymolysis liquid to 5-6 by using a pH regulator to obtain the first enzymolysis liquid with the pH of 5-6;

(5) inoculating microbial oil fermentation strain (such as Rhodotorula glutinis) into the first enzymolysis solution with pH of 5-6, and fermenting to obtain microbial oil fermentation liquid;

(6) mixing lignocellulose pretreatment residue and microbial oil fermentation supernatant (second residual fermentation liquid) obtained by solid-liquid separation of microbial oil fermentation liquid, adding cellulase for enzymolysis to obtain microbial oil enzymatic hydrolysate (second enzymolysis liquid);

(7) adjusting the pH of the microbial oil lipase hydrolysate (second hydrolysate) to 5.5-7.5, preferably 6-7, with a pH adjusting agent to obtain a microbial oil lipase hydrolysate (second hydrolysate) having a pH of 5.5-7.5, preferably 6-7 as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation.

From the above, the present invention can realize the multi-batch recycling of the fermentation wastewater, wherein the ABE fermentation and the microbial oil fermentation can be performed alternately, the first residual fermentation liquid containing organic acid by-products (acetic acid and butyric acid) and the microbial oil fermentation supernatant (second residual fermentation liquid) which are subjected to solid-liquid separation and volatile organic product removal are used for the lignocellulose enzymolysis during the fermentation process, and the prepared second enzymolysis liquid is used as the fermentation substrate containing fermentable sugar for the ABE fermentation of the next batch, so that the recycling of the fermentation wastewater can be realized, and the recycling batch can reach more than 6 batches.

In some embodiments of the present invention, in step D, the lignocellulose pretreatment residue is mixed with a residual fermentation broth (a first residual fermentation broth or a second residual fermentation broth) containing organic acid byproducts (acetic acid and butyric acid) and pH buffer salt, and then subjected to enzymolysis to obtain a lignocellulose enzymolysis solution.

It should be understood by those skilled in the art that, in the step D, the remaining fermentation broth containing organic acid byproducts (acetic acid and butyric acid) can play a role of an enzymolysis buffer solution with a certain stable pH and does not inhibit fermentation, and at the same time, the second remaining fermentation broth can play a role of an enzymolysis medium due to a large amount of water; the enzymatic hydrolysate thus obtained can be used as a fermentation substrate containing fermentable sugars for the next batch of ABE fermentation, while phosphate buffered saline can also provide a stable pH.

In some embodiments of the invention, the pH buffering salts include, but are not limited to, one or more of phosphoric acid/sodium dihydrogen phosphate (potassium, ammonium), citric acid/sodium citrate (potassium, ammonium), acetic acid/sodium acetate (potassium, ammonium), butyric acid/potassium butyrate (potassium, ammonium) at a pH of 4 to 6 and acid group concentrations below 0.5M.

In some embodiments of the present invention, the batch free fermentation-pervaporation separation and recycling process for acetone butanol ethanol production by lignocellulose fermentation in the present invention is shown in fig. 1, and as can be seen from fig. 1, the method for reducing the discharge of acetone butanol ethanol fermentation waste liquid in the present invention comprises the following steps:

(3) mixing lignocellulose pretreatment residues (pretreated biomass) with a first residual fermentation broth containing organic acid byproducts and a phosphoric acid/potassium dihydrogen phosphate buffer solution, and adding cellulase for enzymolysis to obtain an enzymolysis solution;

(4) adjusting pH of the enzymolysis solution to 5.5-7.5, preferably 6-7 with pH regulator to obtain enzymolysis solution with pH of 5.5-7.5, preferably 6-7 as fermentation substrate containing fermentable sugar for next batch of ABE fermentation.

In the invention, the fermentation raw material for ABE fermentation in the step B contains fermentable sugar, which can be added externally or obtained by enzymolysis of lignocellulose pretreatment residues. This is understood to mean that during the fermentation of the ABE of the first batch, fermentable sugars may be added externally or may be obtained from the lignocellulosic pretreatment residues by enzymatic hydrolysis; in the ligninolytic process for providing fermentable sugars for the first lot of ABE fermentation, lignocellulolytic hydrolysis may be performed using an enzymatic hydrolysis buffer that is conventional in the art, and for example, a 0.02M phosphate/potassium dihydrogen phosphate (pH 4.7) buffer may be mixed with the lignocellulolytic pretreated residue and subjected to enzymatic hydrolysis to obtain a lignocellulolytic solution.

In some embodiments of the invention, the temperature of the enzymatic hydrolysis of the lignocellulose is controlled at 30-60 ℃.

In some embodiments of the present invention, the pH of the enzymolysis buffer used for enzymolysis is 4-7, preferably 4-6, and more preferably 4.5-5.5.

The lignocellulose pretreatment residues are prepared by crushing lignocellulose raw materials and then pretreating the crushed lignocellulose raw materials.

In some embodiments of the invention, the lignocellulosic feedstock comprises, but is not limited to, one or more of sugar cane straw, wheat straw, and corn stover.

In some embodiments of the invention, the lignocellulosic feedstock is comminuted to above 40-80 mesh. This is understood to mean that the lignocellulosic feedstock is comminuted to a particle size below that corresponding to a 40-80 mesh size.

The method for pretreating the lignocellulosic raw material in the present invention is not particularly limited, and for example, pretreatment may be performed by one or more of high-temperature high-pressure cooking, acid treatment and alkali treatment.

In some embodiments of the invention, the dried lignocellulosic feedstock is ground to a particle size of 40 mesh to 80 mesh or less. Fully mixing the crushed lignocellulose raw material with a prepared acid solution or alkali solution, and pretreating at the temperature of 100-150 ℃, preferably 120-140 ℃ for 1-4h, preferably 1-2 h. After that, it was cooled to room temperature and centrifuged at 10000rpm to obtain solid lignocellulose pretreatment residue. And washing the lignocellulose pretreatment residues with deionized water until the surface is neutral. And the resulting neutral lignocellulosic pretreatment residue was oven dried at 105 ℃ to oven dry.

The acid solution used for the pretreatment in the above examples is not particularly limited, and for example, 5% (w/v) dilute sulfuric acid may be used.

The alkali solution used for the pretreatment in the above examples is not particularly limited, and for example, a 10% (w/v) aqueous solution of ethylenediamine may be used.

In the above-mentioned examples, the method for detecting the pH value of the pretreated residue to the surface is not particularly limited, and the pH value of the pretreated residue to the surface can be detected by a conventional method, for example, the pH value of the surface of the wet pretreated residue can be directly detected by a pH test paper.

The inventor researches and discovers that acidic or alkaline lignocellulose pretreatment residues after pretreatment can influence the construction of a buffer system in the subsequent enzymolysis process. For example, if the alkali is strong, the influence is great, and if the alkali is not strong, the alkali concentration requirement of potassium hydroxide and the like can be met. The result of the residue not being washed or washed to neutrality is that phenols and organic acids which are toxic to bacteria are dissolved in the pretreated lye, and if the residue is not washed, the part of inhibitors can affect the subsequent fermentation process, and the residue is preferably washed to neutrality.

For another example, after pretreatment with acid liquor, a large amount of acid radical ions exist in the enzymatic hydrolysate, and when the method is adopted for subsequent fermentation, too high concentration of anionic acid radicals exist in the enzymatic hydrolysate to generate too high osmotic pressure, so that the subsequent metabolism of the fermentation strain is remarkably inhibited.

The present inventors have discovered that, because the ABE fermentation process can be broken down into two stages, fermentation in subsequent ABE fermentations may produce only acid, not alcohol, without adjusting the initial pH prior to ABE fermentation. The inventor further researches and discovers that the yield of the acetone butanol and the ethanol in the subsequent ABE fermentation is higher by adopting a pH value regulator to regulate the pH value of the enzymolysis liquid to 5.5-7.5, preferably 6-7 before the ABE fermentation.

In some embodiments of the invention, the lignocellulosic hydrolysate is recycled to step B after adjusting the pH to 5.5-7.5, preferably to 6-7.

According to the invention, the pH regulator comprises one or more of metal oxide, metal hydroxide, ammonia gas and ammonia water.

In some preferred embodiments of the invention, the metal cations comprising the metal oxide or metal hydroxide comprise K⁺、Na⁺、Ca²⁺、Mg²⁺、Co²⁺、Cu²⁺、Zn²⁺、Fe²⁺And Mn²⁺One or more of (a).

In some particularly preferred embodiments of the invention, the pH adjusting agent comprises one or more of potassium hydroxide and sodium hydroxide and aqueous ammonia.

The method of ABE fermentation in the present invention is not particularly limited. According to some embodiments of the invention, in step B, the acetobutanol-producing ethanol fermentation strain is inoculated into the lignocellulose enzymolysis of the exogenous fermentable sugar or the fermentable sugar-containing lignocellulose for ABE fermentation to obtain the acetone butanol-containing ethanol fermentation liquid.

In the invention, the acetone butanol ethanol fermentation strain comprises one or more of clostridium acetobutylicum, clostridium beijerinckii, clostridium pasteurianum, clostridium saccharobutyricum, clostridium glycoacetate, clostridium polybutylicum and butanol-producing genetic engineering bacteria.

The genetically engineered bacteria are one or more genetically engineered bacteria which are transformed by genetic engineering, have the capacity of producing butanol and have acetic acid and butyric acid metabolic pathways.

In the invention, the temperature of ABE fermentation is controlled to be 30-45 ℃.

In the invention, the fermentation mode of the ABE fermentation is suitable for batch fermentation, fed-batch fermentation and continuous fermentation; immobilized fermentation, thallus recovery fermentation and free fermentation; simultaneous saccharification and fermentation; semi-synchronous saccharification and fermentation, and the like.

In the invention, the acetone butanol ethanol producing fermentation strain is obtained by seed culture of corresponding strains.

In some embodiments of the present invention, the fermentation for producing acetone and butanol may be a single-strain fermentation, or a co-fermentation of a butanol-producing strain mixed with one or more other strains.

This is understood to mean that the fermentation to produce acetone butanol ethanol can be carried out using the following inoculation:

(1) inoculating a seed culture solution obtained by seed culture of a single strain corresponding to the acetone butanol ethanol fermentation strain, and then carrying out fermentation production of the acetone butanol ethanol;

(2) inoculating a mixed seed solution formed by mixing seed solutions independently cultured by more than two strains corresponding to the acetone butanol ethanol producing fermentation strain to produce acetone butanol ethanol;

(3) inoculating a mixed seed solution obtained by mixed culture of more than two strains corresponding to the acetone butanol ethanol fermentation strain to produce acetone butanol ethanol;

(4) the method comprises the following steps of (1) adopting a seed solution which is formed by mixing a strain corresponding to an acetone butanol ethanol production fermentation strain and a seed solution which is separately cultured by a strain corresponding to other one or more strains to inoculate the mixed seed solution, and then carrying out fermentation production of the acetone butanol ethanol;

(5) the fermentation production of the acetone butanol ethanol is carried out after the mixed seed liquid obtained by the mixed culture of the strain corresponding to the fermentation strain for producing the acetone butanol ethanol and the strain corresponding to one or more other strains is inoculated.

In some preferred embodiments of the present invention, the acetone-butanol-ethanol production is performed by fermentation after inoculation of a mixed seed solution formed by mixing a seed solution cultured separately from a strain corresponding to a strain of acetobutanol-ethanol-producing fermentation strain and another strain corresponding to one or more other strains.

In some embodiments of the invention, the media formulation for clostridium beijerinckii seed culture is as follows: 40g/L glucose, 1g/L potassium monohydrogen phosphate, 1g/L potassium dihydrogen phosphate, 2.2g/L ammonium acetate, 1mg/L para aminobenzoic acid, 0.01mg/L biotin, 0.2g/L magnesium sulfate, 0.01g/L manganese sulfate and 0.01g/L ferrous sulfate. An anaerobic environment was established with sterile nitrogen.

In other embodiments of the invention, the media formulation for the clostridium acetobutylicum seed culture is as follows: 40g/L of glucose, 0.2g/L of magnesium sulfate, 1g/L of potassium monohydrogen phosphate, 1g/L of monopotassium phosphate, 2.2g/L of ammonium acetate, 1mg/L of p-aminobenzoic acid, 0.01mg/L of biotin, 0.2g/L of magnesium sulfate, 0.01g/L of manganese sulfate and 0.01g/L of ferrous sulfate.

In still other embodiments of the present invention, the media formulation of the clostridium acetobutylicum seed solution is as follows: 35g/L of glucose, 3g/L of yeast extract powder, 2g/L of beef peptone, 0.2g/L of magnesium sulfate, 1g/L of monopotassium phosphate and 2g/L of ammonium acetate.

In the invention, the fermentation temperature of the rhodotorula glutinis is controlled to be 30-45 ℃.

In the invention, the fermentation mode of the rhodotorula glutinis is suitable for batch fermentation, fed-batch fermentation and continuous fermentation; immobilized fermentation, thallus recovery fermentation and free fermentation; simultaneous saccharification and fermentation; semi-synchronous saccharification and fermentation, and the like.

In the invention, the rhodotorula glutinis strain is obtained by seed culture of corresponding strains.

In some preferred embodiments of the present invention, Rhodotorula glutinis fermentation is performed after inoculating a seed solution obtained by seed culture of a strain corresponding to Rhodotorula glutinis.

In some embodiments of the present invention, the media formulation for seed culture of Rhodotorula glutinis is comprises: 2g/L of (NH)₄)₂SO₄、2g/LNa₂SO₄1.5g/L MgSO 1₄7g/L KH₂PO₄1.5g/L yeast extract powder and 40g/L glucose.

The term "lignocellulose" as used herein refers to an organic fiber obtained by chemically treating natural wood or a woody material, and has a flocculent appearance and a white or off-white color. Lignocellulose is mainly composed of cellulose (30-50% of dry matter), hemicellulose (20-40% of dry matter) and lignin (15-25% of dry matter), and also contains small amount of structural protein, lipid and ash. Wherein, the cellulose is a homogeneous polymer formed by the linear connection of glucose units through beta-1, 4 glycosidic bonds, is a macromolecular polysaccharide, is insoluble in water and common organic solvents, and is a main component of plant cell walls. Cellulose fibers interact with each other through hydrogen bonds and can form crystalline regions and amorphous regions. The "hemicellulose" is a heteropolysaccharide composed of different types of monosaccharides (including five-carbon and six-carbon sugars), with a proportion of xylan of about 50%. The lignin is an amorphous aromatic high polymer with a molecular structure rich in an oxo-phenylpropanol structure or a derivative structural unit thereof. The lignin is dispersed among the cellulose fibers, but the lignin and the cellulose fibers usually have no direct chemical bond connection, and the lignin mainly plays a role in resisting pressure; the hemicellulose penetrates between the lignin and the cellulose fiber and plays a role in connecting the lignin and the cellulose fiber, and then a very firm cellulose-hemicellulose-lignin network structure is formed. This structure of lignocellulose is the result of natural selection of plants during long-term evolution, and lignocellulosic biomass is therefore highly resistant to biological or non-biological attack in the environment.

The terms "lignocellulosic pretreatment residue", "solid lignocellulosic pretreatment residue" and "lignocellulosic residue" as used herein are used interchangeably.

The term "acetone-butanol-ethanol fermentation" (abbreviated as ABE fermentation) as used herein refers to a fermentation for the production or production of acetone, butanol and ethanol; the fermentation culture may also be considered an acetone-butanol fermentation primarily for the production or production of acetone and butanol, since it contains less ethanol.

The term "water" as used herein means one or more of deionized water, distilled water and ultrapure water, unless otherwise specified or indicated.

Examples

In order that the invention may be more readily understood, the invention will now be described in further detail with reference to the accompanying drawings and examples, which are given by way of illustration only and are not limiting to the scope of the invention. The starting materials or components used in the present invention can be obtained commercially or by conventional methods unless otherwise specified.

Example 1: and performing ABE fermentation on the sugarcane straw residue enzymatic hydrolysate pretreated by using ethylenediamine, and directly recycling the residual fermentation liquor for performing the next batch of enzymatic fermentation after reduced pressure distillation.

(1) And (3) washing the juiced sugarcane straws with deionized water until no free sugar remains on the surface, and drying at 105 ℃ to be absolutely dry. And then crushing the mixture to obtain powder with the particle size of less than or equal to 40 meshes. The sugarcane straw residues and 10 percent (w/v) ethylene diamine aqueous solution which is prepared in advance are fully mixed and pretreated for 1h at the temperature of 120 ℃. After that, the reactor was cooled to room temperature and centrifuged at 10000rpm to obtain solid sugar cane straw pre-treatment residue. Washing the residue with deionized water until the surface is neutral. The resulting neutral residue was oven dried at 105 ℃ to dryness. The solid yield was found to be 49.8%, and the cellulose, hemicellulose and lignin contents in the solid residue were 59.4%, 18.6% and 14.2%, respectively.

(2) Mixing the dried sugarcane straw pretreatment residues with a 0.01M phosphoric acid/potassium dihydrogen phosphate buffer solution prepared in advance (pH is 4.8). 20FPU/g of cellulase enzyme (from KDN group) was added to the solid-liquid mixture. After that, enzymatic hydrolysis was carried out at 55 ℃ and 200rpm for 72 hours. After the enzymolysis is finished, the enzymolysis liquid is filtered by a 400-mesh nylon filter cloth to obtain supernatant. The glucose and xylose concentrations in the supernatant were 29.1g/L and 10.6g/L, respectively, as determined by high performance liquid chromatography.

(3) Adjusting the pH of the enzymolysis supernatant to 7 by using concentrated ammonia water. Thereafter, Clostridium acetobutylicum ATCC824 (OD) was inoculated at an inoculation amount of 5% (v/v)₆₀₀>0.8). The formula of the culture medium of the seed liquid is as follows: 40g/L glucose, 1g/L potassium monohydrogen phosphate, 1g/L potassium dihydrogen phosphate, 2.2g/L ammonium acetate, 1mg/L para aminobenzoic acid, 0.01mg/L biotin, 0.2g/L magnesium sulfate, 0.01g/L manganese sulfate and 0.01g/L ferrous sulfate. An anaerobic environment was established with sterile nitrogen.

After inoculation, the fermentor was continuously purged with sterile high purity nitrogen (> 99.999%) for 2h to create an anaerobic fermentation environment. Thereafter, the fermenter was continued at 37 ℃ and 50rpm for a further 72h batch fermentation. And after the fermentation is finished, taking the fermentation supernatant for centrifugal sample preparation. And detecting the concentrations of glucose and xylose in the fermentation supernatant by using a high performance liquid chromatography, and detecting organic acid byproducts such as ABE, acetic acid and butyric acid in the fermentation supernatant by using a gas chromatography. No glucose and xylose residues are detected in the fermentation supernatant. The concentrations of acetone, butanol and ethanol in the fermentation liquor are respectively 2.5g/L, 8.9g/L and 1.5 g/L. The concentrations of acetic acid and butyric acid in the fermentation liquor are respectively 2.4g/L and 1.3 g/L.

(4) The supernatant of the first fermentation was distilled at 50 ℃ under reduced pressure at 20 kpa. The concentration of ethanol in the fermentation liquor after distillation is 1.1g/L, the concentration of acetone is 0g/L, the concentration of butanol is 0.4g/L, and in addition, the concentrations of acetic acid and butyric acid are 4.4g/L and 2.4g/L respectively. Through high performance liquid chromatography detection, the concentrations of glucose and xylose in the distilled fermentation liquor are respectively 0g/L and 0.2g/L.

In this embodiment, the distilled fermentation broth is equivalent to buffer salt, the sugar is obtained after adding the dregs for enzymolysis, the sugar solution is used as the fermentation medium of the next batch, the distillation aims to evaporate ABE and a part of water in the fermentation broth of the previous batch, and the pH of the waste liquid of the two acids is adjusted to be used as the enzymolysis buffer solution.

(5) Mixing the dried sugarcane straw pretreatment residues with the distilled kettle bottom fermentation waste liquid (the pH is adjusted to 4.8 by 10% ammonia water). 20FPU/g of the cellulase of the pretreatment residue KDN03 (available from KDN group) was added to the solid-liquid mixture. After that, enzymatic hydrolysis was carried out at 55 ℃ and 200rpm for 72 hours. After the enzymolysis is finished, the enzymolysis liquid is filtered by 400-mesh nylon filter cloth to obtain supernatant. The glucose and xylose concentrations in the supernatant were determined to be 30.2g/L and 9.8g/L, respectively, by high performance liquid chromatography. This liquid was used for the next batch of ABE fermentation medium.

(6) After the fermentation medium was sterilized, Clostridium acetobutylicum ATCC824 was inoculated and fermentation continued for 72h using the same procedure as described above for the fermentation process. And after the fermentation is finished, taking the fermentation supernatant for centrifugal sample preparation. And detecting the concentrations of glucose and xylose in the fermentation supernatant by using a high performance liquid chromatography, and detecting organic acid byproducts such as ABE, acetic acid and butyric acid in the fermentation supernatant by using a gas chromatography. No glucose and xylose residue remained in the fermentation supernatant through detection. The concentrations of acetone, butanol and ethanol in the fermentation liquor are respectively 3.9g/L, 8.3g/L and 1.4 g/L. The concentrations of acetic acid and butyric acid in the fermentation liquor are respectively 4.5g/L and 1.9 g/L.

According to the experimental data, when the distilled first batch of fermentation liquor is used for preparing the culture medium, and the second batch of fermentation is completed by using the culture medium, the concentration of acetic acid is basically unchanged, and the concentration of butyric acid is slightly reduced, so that part of organic acid in the first batch of fermentation liquor is recycled, and is converted into products such as acetone butanol and the like in the second batch of fermentation.

Example 2: after the batch of the cellulose acetone butanol is subjected to free fermentation-pervaporation separation, the retentate can be recycled for 5 batches under the conditions of no exogenous nutrition and no detoxification, and the specific process is shown in figure 1.

(1) In this example, a lignocellulosic pretreatment residue obtained by alkaline treatment was subjected to enzymatic saccharification to obtain a fibrous sugar solution. Taking enzymolysis supernatant, adjusting pH, and performing batch ABE fermentation.

(2) After the fermentation was completed, the cells and solid residues were removed by centrifugation. Separating the fermentation supernatant in batches by using a pervaporation membrane.

(3) And adjusting the pH of the obtained residual solution, adding lignocellulose pretreatment residues and cellulase, carrying out enzymolysis saccharification of a second batch, and obtaining a corresponding second batch of enzymolysis solution. And continuing to perform the ABE fermentation and pervaporation separation of the second batch by using the second batch of enzymolysis liquid. The process was cycled 5 times. The glucose and xylose concentrations before and after each batch of fermentation were determined by high performance liquid chromatography. And detecting the ABE concentration and the acetic acid and butyric acid concentration of each batch after the fermentation is finished by using gas chromatography.

The specific operating parameters for the alkaline treatment of lignocellulose are as follows: absolutely dry corn stalks are crushed to the grain size below 40 meshes. The corn stalk dregs and the prepared 1 percent (w/v) sodium hydroxide aqueous solution are fully mixed and pretreated for 1.5 hours at the temperature of 120 ℃. After that, the reactor was cooled to room temperature and centrifuged at 10000rpm to obtain solid straw pretreatment residue. Washing the residue with deionized water until the surface is neutral. The resulting neutral residue was oven dried at 105 ℃ to dryness.

The specific operating parameters of the enzymolysis process are as follows: the corn stalk dregs after alkaline treatment are mixed with 0.02M phosphoric acid/potassium dihydrogen phosphate (pH is 4.7) buffer solution with the solid adding amount of 6% (w/v) or the seeping liquid after ABE product separation by pervaporation in the previous batch of which the pH is adjusted to 4.7 by concentrated ammonia water, and 15FPU/g of cellulase KDN03 (purchased from KDN group) of the pretreatment residues is added into the solid-liquid mixture. After that, enzymatic hydrolysis was carried out at 55 ℃ and 200rpm for 72 hours. After the enzymolysis is finished, the enzymolysis liquid is filtered by a 400-mesh nylon filter cloth to obtain supernatant.

The specific operating parameters of the fermentation process are as follows: after adjusting pH to 7 with concentrated ammonia water, inoculating Clostridium acetobutylicum seed solution (Clostridium acetobutylicum ATCC824, OD) with an inoculum size of 5% (v/v) into the enzymolysis supernatant obtained in the enzymolysis process₆₀₀>0.8). Seed liquid medium is shown in example 1. After inoculation, sterile high-purity nitrogen is continuously introduced into the fermentation tank (>99.999%) for 2h to create an anaerobic fermentation environment. Thereafter, the fermenter was continued at 37 ℃ and 50rpm for a further 72h batch fermentation. After the fermentation is finished, taking the fermentation supernatant for centrifugation.

The operating parameters of the pervaporation process were: the pervaporation membrane is a PDMS/PVDF membrane self-made by the national energy biorefinery center, and by utilizing a first-stage pervaporation device and method provided by the inventor in patent CN 102757984A, fermentation supernatant is placed at the side of a feed liquid, the fermentation supernatant is continuously introduced into the pervaporation membrane module under the action of a peristaltic pump, the flow rate is 1 time of the liquid volume in a raw material storage tank per minute, and the fermentation liquid is poured back to the feed liquid storage tank after being fully contacted with the membrane module. The temperature of the system was controlled at 37 ℃. On the transmission side of the pervaporation membrane, a vacuum environment of 200Pa or less was established by a vacuum pump. And a cold trap immersed in liquid nitrogen was placed on the permeate side catheter for collection of the separated ABE solvent. After pervaporation was carried out for 25 h. And residual permeation is measured to have no butanol residue. Stopping pervaporation, and collecting separated liquid on the retentate side.

The recycling operation parameters of the residual liquid are as follows: adjusting the pH of the residual liquid to 4.7 by using concentrated ammonia water, namely obtaining residual liquid after separating ABE products by pervaporation in the preorder batch of pH adjustment in the enzymolysis process. The liquid can be reused.

The initial glucose and xylose concentrations of each batch of the enzymatic hydrolysate obtained after 5 continuous cycles of the above process are shown in table 1. The ABE, sugar and acid concentrations of the fermentation broth obtained in each batch after 5 continuous circulations in the above process are shown in Table 2.

TABLE 1 initial glucose and xylose concentrations for each batch of the enzymatic hydrolysate

TABLE 2 fermentation broths ABE, sugar, acid concentrations for each batch

Sugar concentration (g/L)	Batch 1	Batch 2	Batch 3	Batch 4	Batch 5
						Glucose	0	0.92	2.24	4.28	6.24
Xylose	0	3.66	5.32	7.13	8.47
						Acetone (II)	4.51	1.84	1.43	0.69	0.45
Butanol	11.22	7.60	5.54	5.03	4.87
						Ethanol	1.40	0.82	0.87	0.49	0.42
Total ABE	17.13	10.26	7.85	6.28	5.74
						Acetic acid	5.80	8.42	7.57	8.09	8.92
Butyric acid	2.83	2.21	2.61	3.05	3.74

From the above results, it can be seen that the ABE product concentration decreases slightly after recycling 5 batches of fermentation broth according to the process provided in this example. The concentration of organic acid in the fermentation liquor is increased slightly, but the hydrolysis and fermentation of the cellulose and sugar can be realized by continuously using 5 batches of the circulating fermentation liquor. By using the process method provided by the embodiment, the acetone butanol fermentation wastewater is directly recycled on the premise of not greatly influencing the ABE fermentation effect, and the emission reduction of the fermentation wastewater is about 80%.

Example 3: comparison of inhibition degrees of recycling fermentation of cellulose acetate/butyrate-containing cellulose acetone butanol fermentation wastewater with different dilution or concentration times

Diluting the fermentation liquor obtained in the embodiment 2 after pervaporation separation by adding water with different contents, wherein the liquid accounts for 12.5%, 25%, 50%, 75% and 100% respectively, adding a phosphoric acid/potassium dihydrogen phosphate buffer solution, adjusting the concentration of phosphate radical to be 0.20M, mixing the corn straw residue treated by an alkaline method with the buffer solution by adding solid with 6% (w/v), adjusting the pH of each residual solution with different accounts to 4.7 by using concentrated ammonia water, and carrying out enzymolysis according to the enzymolysis method. Then inoculating and fermenting to obtain different ratios of retentate, comparing the inhibition ratios, determining sugar concentration in the enzymolysis solution by high performance liquid chromatography, and determining fermentation broth ABE and acid concentration by high performance gas chromatography.

The glucose concentration and xylose concentration of the obtained retentate from different dilutions of the above process after respective enzymatic hydrolysis are shown in table 3, and the concentrations of ABE, sugar and acid of the retentate fermentation broth from different dilutions are shown in table 4.

TABLE 3 initial glucose and xylose concentrations of the retentate at different dilutions

Ratio of the retentate (v/v)	0％	12.5％	25％	50％	75％	100％
							Glucose	36.96	37.04	36.84	36.65	37.21	37.19
Xylose	10.63	10.77	11.17	10.49	10.24	10.35

TABLE 4 fermentation broths ABE, sugar, acid concentrations for each batch

Ratio of the retentate (v/v)	0％	12.5％	25％	50％	75％	100％
							Glucose	0.27	0.36	0.47	0.46	0.62	0.74
Xylose	1.46	2.97	2.35	2.19	2.73	2.88
							Acetone (II)	4.51	4.08	3.43	2.57	2.61	2.26
Butanol	11.22	10.77	8.51	7.52	7.20	6.54
							Ethanol	1.40	0.87	0.75	0.72	0.82	0.52
Total ABE	17.13	15.72	12.69	10.81	10.63	9.32
							Acetic acid	2.80	2.86	3.21	5.47	6.66	8.41
Butyric acid	1.33	0.61	0.44	0.47	0.54	0.37

After the seepage liquid is diluted by adding water with different contents, the larger the dilution concentration is, the higher the yield of butanol and ABE is, and when the proportion of the seepage liquid is less than 75%, the reuse of the fermentation wastewater can be effectively realized. By using the process method provided by the embodiment, the acetone butanol fermentation wastewater is diluted and recycled on the premise of not greatly influencing the ABE fermentation effect, so that the emission reduction of the fermentation wastewater is about 75% or more.

Example 4: after the free fermentation-gas stripping separation of the cellulose acetone butanol batch, the retentate liquid is continuously subjected to rhodotorula glutinis fermentation, and the rhodotorula glutinis fermentation liquid is recycled for cellulose glycolysis preparation and acetone butanol fermentation of subsequent batches, wherein the specific process is shown in figure 2.

(1) In this embodiment, in the fermentation liquid obtained in example 2 after pervaporation separation, the corn straw residue treated by the alkaline process is mixed with the pervaporation liquid in a solid addition amount of 6% (w/v), the pH of the retentate is adjusted to 4.8 by using concentrated ammonia water, enzymolysis is performed according to the above enzymolysis method, the enzymolysis liquid is filtered by using a 400-mesh nylon filter cloth to obtain a supernatant, and then the pH of the enzymolysis supernatant is adjusted to 5.5 by using concentrated ammonia water.

(2) Then, the Rhodotorula glutinis seed solution (obtained by seed culture of Rhodotorula glutinis CICC 31229) was inoculated at an inoculation amount of 5% (v/v). The formula of the culture medium of the seed liquid is as follows: 2g/L of (NH)₄)₂SO₄、2g/LNa₂SO₄1.5g/L MgSO 1₄7g/L KH₂PO₄1.5g/L yeast extract and 40g/L glucose. Culturing at 30 deg.C and 150rpm, and adjusting pH to 5.5 with NaOH or ammonia water during Rhodotorula glutinis culture. And then, measuring the residual amounts of organic acid acetic acid and butyric acid and the residual amounts of glucose and xylose by adopting a high performance liquid chromatography, measuring the content of grease and the dry weight of thalli by a differential weight method, centrifuging a rhodotorula glutinis fermentation liquor to obtain a supernatant, mixing the corn straw residue treated by an alkaline method with the supernatant by using the solid addition amount of 6% (w/v), adjusting the pH of the mixed solution to 4.8 by using phosphoric acid, carrying out enzymolysis according to the enzymolysis method, and filtering the enzymolysis solution by using 400-mesh nylon filter cloth to obtain the supernatant.

(3) Then, adjusting the pH value of the enzymolysis supernatant to 7 by using strong ammonia water, inoculating after sterilization for ABE fermentation, and after the fermentation is finished, centrifuging to remove thalli and solid residues. The fermentation supernatant was batch separated using pervaporation membranes, the procedure was as described above. Then, the permeation vaporization liquid is subjected to enzymolysis and rhodotorula glutinis fermentation continuously, and the method is shown as above and can be reused for six batches.

The initial glucose and xylose concentrations of each batch of the enzymatic hydrolysate obtained after 5 continuous cycles of the above process are shown in table 5. The ABE, sugar and acid concentrations of the fermentation broth obtained in each batch after 5 continuous circulations in the above process are shown in Table 6.

TABLE 5 initial glucose and xylose concentrations of the retentate at different dilutions

Sugar concentration (g/L)	For the first time	For the second time	The third time	Fourth time	Fifth time	The sixth time
							Glucose	36.46	35.97	35.54	35.68	34.99	34.78
Xylose (XO)	10.32	10.22	10.17	10.49	10.11	10.05

TABLE 6 Key parameters for batch ABE fermentation-pervaporation-Rhodotorula glutinis fermentation in BFW reuse Process

It can be seen that the above total wastewater was recycled for 6 batches, corresponding to 6 cycles of enzymatic hydrolysis. Wherein the 1 st, 3 th and 5 th batches are butanol fermentation, the 2 nd, 4 th and 6 th batches are rhodotorula glutinis fermentation, and sugar substrates of the butanol fermentation and the rhodotorula glutinis fermentation are obtained by saccharifying lignocellulose hydrolysis residues through enzymolysis.

In the process of recycling the wastewater, the concentration difference between glucose and xylose in each batch is not large, and the influence on enzyme hydrolysis is small in the process of using the wastewater. At the end of the first fermentation cycle, 10.9g/L butanol and 16.7g/L total ABE are present, while the butanol and ABE concentrations in the second and third recycles are respectively as low as 10.21g/L, 9.38g/L, 14.98g/L and 14.01g/L, which is not much different from the first fermentation result. The oil content and the dry cell weight are reduced along with the increase of the recycling times, but the reduction range is not large, and the oil content is from 10.21g/L of the dry cell weight and 3.29g/L of the dry cell weight for the first recycling to 9.56g/L of the dry cell weight for the third recycling, namely 3.08g/L of the oil content. The final residual amounts of the acetic acid and the butyric acid are 0, which indicates that the rhodotorula glutinis can utilize the acetic acid and the butyric acid in the ABE fermentation wastewater, so that the inhibition of the acetic acid and the butyric acid on the fermentation is reduced by the ABE fermentation in the recycling process.

Example 5: after the free fermentation-gas stripping separation of the cellulose acetone butanol batch, the fermentation wastewater is adsorbed by active carbon and then undergoes cellulose glycolysis preparation and acetone butanol fermentation of subsequent batches, and the specific process is shown in fig. 3.

(1) In the embodiment, the activated carbon for different purposes in the current market is optimized to carry out acetic acid and butyric acid adsorption verification experiments, and the activated carbon with the optimal adsorption capacity is screened out and is AR-grade wastewater adsorption activated carbon produced by the Aladdin company.

(2) After the fermentation liquid obtained in example 2 is subjected to pervaporation separation, acetic acid and butyric acid inhibitors in the fermentation wastewater (residual fermentation liquid) can be completely eliminated and the decolorization effect can be completely achieved after the adsorption treatment of activated carbon, so that the inhibition of organic acid recycled from the wastewater is removed.

The adsorbed active carbon is subjected to organic acid desorption for 8 hours at a high temperature of 200 ℃, so that the active carbon is recycled.

(3) Then adding phosphoric acid/potassium dihydrogen phosphate buffer solution into the adsorption solution, adjusting the concentration of phosphate radical to be 50mM, mixing the corn straw residue treated by the alkaline method with the buffer solution by the solid addition amount of 6% (w/v), adjusting the pH of the mixed solution to be 4.7 by using concentrated ammonia water, and carrying out enzymolysis according to the enzymolysis method [ adopting cellulase KDN03 (purchased from KDN group) ]. Filtering the enzymolysis liquid with 400 mesh nylon filter cloth to obtain supernatant, adjusting pH of the enzymolysis supernatant to 7 with strong ammonia water, sterilizing, inoculating, performing ABE fermentation, and centrifuging to remove thallus and solid residue. The fermentation supernatant was batch separated using a pervaporation membrane, the procedure being as described above.

Then continuing activated carbon adsorption on the pervaporation liquid, and then carrying out enzymolysis and ABE fermentation, wherein the method is shown as above and the pervaporation liquid is recycled for four batches. And determining the sugar concentration in the enzymolysis liquid by high performance liquid chromatography, and determining the ABE and acid concentrations of the fermentation liquid by high performance gas chromatography.

The glucose concentration and xylose concentration obtained after respective enzymolysis of the obtained retentate of different recycling batches in the process are shown in table 7, and the ABE, sugar and acid concentration of the retentate fermentation liquor of different recycling batches are shown in table 8.

TABLE 7 initial glucose and xylose concentrations for each batch of the enzymatic hydrolysate

Sugar concentration (g/L)	1 st ofBatch	Batch 2	Batch 3	Batch 4	Batch 5
						Glucose	36.40	37.05	37.22	36.82	36.80
Xylose (XO)	10.82	10.93	11.14	10.92	10.68

TABLE 8 ABE, sugar, acid concentrations in batches of fermentation broths

After the permeable residual liquid of each batch is adsorbed by AR active carbon, the effect of completely removing acetic acid and butyric acid in the permeable residual liquid can be realized, the complete decoloration effect can also be realized, and the inhibiting effect of organic acid on ABE fermentation is effectively removed. Fermentation verification experiments show that the yield of butanol and the yield of ABE after each batch of recycling are similar to the result of the first ABE fermentation. By using the process method provided by the embodiment, the acetone butanol fermentation wastewater is adsorbed and recycled by the AR activated carbon on the premise of not influencing the ABE fermentation effect, so that the complete recycling of the fermentation wastewater can be realized.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. Modifications may be made to the invention as defined within the scope of the claims and modifications may be made without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Claims

1. A method for reducing the discharge of acetone butanol ethanol fermentation waste liquid comprises the following steps:

wherein the volatile organic product comprises one or more of acetone, butanol and ethanol;

in the step C, carrying out solid-liquid separation on the fermentation liquor containing the volatile organic products, and separating out the volatile organic products to obtain a first residual fermentation liquor;

in the step C, performing solid-liquid separation on the fermentation liquor containing the volatile organic product, separating out the volatile organic product, and performing deacidification treatment on the obtained first residual fermentation liquor to obtain a second residual fermentation liquor;

the deacidification treatment mode is biological treatment; the biological treatment is microbial oil fermentation, which comprises:

step M2, inoculating a microbial oil fermentation strain into the first lignocellulose enzymolysis liquid obtained in the step M1, and performing microbial oil fermentation to obtain a microbial oil fermentation liquid;

step M3, using the microbial oil fermentation supernatant obtained by solid-liquid separation of the microbial oil fermentation broth as a second residual fermentation broth;

wherein, the microbial oil fermentation strain is Rhodotorula glutinis.

2. The method according to claim 1, wherein the concentration of acetic acid in the first remaining fermentation broth is not less than 0.1 g/L; and/or the concentration of butyric acid in the first residual fermentation liquid is more than or equal to 0.1 g/L; and/or the concentration of butanol in the first residual fermentation broth is below 3 g/L; and/or, the ethanol concentration in the first residual fermentation broth is below 2 g/L; and/or, the concentration of acetone in the first residual fermentation broth is below 2 g/L; and/or the total residual sugar concentration in the first residual fermentation broth is less than 20 g/L.

3. The method according to claim 2, wherein the concentration of acetic acid in the first residual fermentation broth is 0.1-10 g/L; and/or the concentration of butyric acid in the first residual fermentation liquid is 0.1-10 g/L.

4. The method according to claim 3, wherein the concentration of acetic acid in the first residual fermentation broth is 0.1-6 g/L; and/or the concentration of butyric acid in the first residual fermentation liquid is 0.1-4 g/L.

5. The method according to claim 4, wherein the concentration of acetic acid in the first residual fermentation broth is 0.5-4 g/L; and/or the concentration of butyric acid in the first residual fermentation liquid is 0.1-3 g/L.

6. The method according to claim 1, wherein the concentration of acetic acid in the second residual fermentation broth is 0-6 g/L; and/or the concentration of butyric acid in the second residual fermentation liquid is 0-6 g/L.

7. The method according to claim 6, wherein the concentration of acetic acid in the second residual fermentation broth is 0-4 g/L; and/or the concentration of butyric acid in the second residual fermentation liquid is 0-4 g/L.

8. The method according to claim 7, wherein the concentration of acetic acid in the second residual fermentation broth is 0-2 g/L; and/or the concentration of butyric acid in the second residual fermentation liquid is 0-2 g/L.

9. The method according to any one of claims 1 to 8, wherein in step D, the lignocellulose pre-treated residue is mixed with the remaining fermentation broth and pH buffer salt and subjected to enzymatic hydrolysis to obtain a lignocellulose hydrolysate.

10. The method according to any one of claims 1 to 8, wherein the lignocellulosic hydrolysate comprises a lignocellulosic hydrolysate supernatant and/or a lignocellulosic hydrolysate containing an insoluble solid phase, and the concentration of fermentable sugars in the lignocellulosic hydrolysate is 20 to 100 g/L; and (C) adjusting the pH value of the lignocellulose enzymolysis liquid to 5.5-7.5 and recycling the lignocellulose enzymolysis liquid in the step (B).

11. The method according to claim 10, wherein the concentration of fermentable sugars in the lignocellulosic hydrolysate is 20 to 80 g/L; and (4) adjusting the pH value of the lignocellulose enzymolysis liquid to 6-7 and recycling the lignocellulose enzymolysis liquid in the step B.

12. The method according to claim 11, wherein the concentration of fermentable sugars in the lignocellulosic hydrolysate is 40 to 60 g/L.

13. The method according to claim 9, wherein the lignocellulose enzymolysis solution comprises lignocellulose enzymolysis supernatant and/or lignocellulose enzymolysis stock solution containing insoluble solid phase, and the concentration of fermentable sugar in the lignocellulose enzymolysis solution is 20-100 g/L; and (C) adjusting the pH value of the lignocellulose enzymolysis liquid to 5.5-7.5 and recycling the lignocellulose enzymolysis liquid in the step (B).

14. The method as claimed in claim 13, wherein the concentration of fermentable sugars in the lignocellulose enzymolysis solution is 20-80 g/L; and C, adjusting the pH value of the lignocellulose enzymolysis liquid to 6-7 and recycling the lignocellulose enzymolysis liquid in the step B.

15. The method as claimed in claim 14, wherein the concentration of fermentable sugars in the lignocellulosic hydrolysate is 40 to 60 g/L.