WO2007083746A1

WO2007083746A1 - Fermentation method for producing ethanol

Info

Publication number: WO2007083746A1
Application number: PCT/JP2007/050799
Authority: WO
Inventors: Ayaaki Ishizaki; Keishi Shimazaki
Original assignee: New Century Fermentation Research Co., Ltd.
Priority date: 2006-01-23
Filing date: 2007-01-19
Publication date: 2007-07-26
Also published as: JP2007195406A

Abstract

A method comprising: saccharifying sago palm timber, which has been successfully artificially grown as tropical plant resources, as such; removing undecomposed fibers, etc.; adding, for example, waste liquor obtained in the course of pressing oil palm, which has been also broadly grown in Southeast Asia, or latex liquor obtained from natural rubber sap as an organic nutritional component thereto; and culturing an alcohol-producing bacterium such as Zymomonas mobilis or an alcohol-producing yeast such as Saccharomyces cerevisiae therein to thereby allow the organism to produce alcohol. According to this method, it is possible to establish an energy production effect from biomass. Moreover, it contributes to the environmental program in the tropical region as a method of processing the waste liquor.

Description

Specification

Ethanol production fermentation method

Technical field

[0001] Considering the current situation in which global warming and oil resource depletion proceed in parallel, it is a common challenge for mankind to reduce oil consumption and replace energy sources with renewable resources that are photosynthetic products. It has become. In addition, as uncertainty about the future of petroleum, which is a limited resource, has increased, the movement to add ethanol to gasoline for automobiles is expanding all over the world, and not only in Brazil and the United States, but also in other countries in China and Southeast Asia, There is a growing competition for the development of raw materials for fuel ethanol production and the development of new production methods.

[0002] In particular, the tropical regions of Southeast Asia are rich in photosynthetic capacity, have high potential for biomass production, and have many undeveloped plant resources. Among them, sago palm is a promising photosynthetic resource for which cultivation techniques are currently being established. If this cultivation is realized, it will become a large source of photosynthetic energy comparable to oil palm, and an ethanol production source on par with US corn. Seem.

[0003] Oil palm is a typical tropical industrial crop along with natural rubber. However, development of waste liquid countermeasure technology related to these has been delayed, and the development of more rational waste liquid treatment technology is desired from the viewpoint of environmental measures. Oil palm extraction wastewater is treated by anaerobic methane fermentation and reduced to plantation, and natural rubber latex mother liquor is treated by lagoon to make it harmless, but this is not always sufficient.

Patent Document 1: Japanese Patent Publication No. 6-46941

Disclosure of the invention

Problems to be solved by the invention

[0004] Generally, organic nutrients such as amino acids and vitamins are necessary for the growth of microorganisms. Therefore, when starch raw materials are used in alcoholic fermentation, even if they are yeast, they are bacteria such as Zymo monas. It is also necessary to use natural organic nutrient sources such as CSL and malt extract. Natural rubber latex mother liquor is attractive because it contains abundant microbial growth-promoting factors. (Reference 1: Japanese Patent No. 6-46941). This is a concentrate obtained by concentrating the mother liquor and then spray-dried and powdered. It is nutritious but expensive in terms of cost, and cannot be a raw material for ethanol fermentation.

[0005] Oil palm oil effluent is considered to be an inexpensive organic nutrient source capable of supplying microbial growth factors rich in proteins and vitamins contained in the oil palm pulp, but has never been used as a fermentation raw material. The viewpoint power of effective use of waste liquid Acetone 'It is reported that butanol fermentation is possible, and it is only.

[0006] Accordingly, an object of the present invention is to provide an ethanol production fermentation method that can efficiently produce ethanol using biomass, and can contribute to waste liquid treatment, which is a problem.

Means for solving the problem

[0007] The ethanol production fermentation method according to the first invention includes a first step of preparing a pulverized product of sago palm, and saccharification by adding at least one of a liquid enzyme and a sugar enzyme to the prepared pulverized material. A second step of obtaining a sugar solution and a third step of culturing at least one of alcohol-producing bacteria and alcohol yeast using the sugar solution to produce ethanol.

[0008] By adopting this configuration, as will be apparent from the examples described later, it is possible to efficiently produce ethanol by utilizing sago palm in a state almost similar to a raw wood. As illustrated in Example 5, since sago palm logs have not yet been identified and contain various microbial growth factors, yeasts and bacteria that still have alcohol-fermenting ability will proliferate, and the starch contained in the logs will be reduced. Can be converted to alcohol. However, since there are not enough nutrients, implementing the method described below will enable more efficient alcoholic fermentation and promote effective use of resources.

[0009] In the ethanol production fermentation method according to the second invention, in addition to the first invention, prior to the third step, oil palm oil extraction waste liquid is added to the sugar liquid.

In the ethanol production fermentation method according to the third invention, in addition to the first invention, the natural rubber latex mother liquor is added to the sugar solution prior to the third step.

[0011] With these configurations, it is possible to effectively use waste liquids that are difficult to process.

The invention's effect [0012] According to the present invention, the sago palm power as biomass can efficiently produce alcohol and can be used effectively as well as treating waste liquid.

Brief Description of Drawings

[0013] FIG. 1 is a graph showing the course of ethanol fermentation according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Sago palm pulverizes the raw wood, separates the fiber, separates the starch, refines it, and dries it into starch flour, which can be used as corn starch, cassava starch, potato or sweet potato starch. It becomes a fermentation raw material as a starch resource. However, it is believed that using sago palm as a raw material for fermentation as raw material is impossible with conventional common sense, and no one has yet considered it. If it is possible to use sago palm as a raw material for ethanol fermentation directly without separating starch from sago palm, the yield from sago palm will be improved and the process will be simplified. It must be a fuel production method from groundbreaking photosynthetic products.

[0015] Based on these findings, the present inventors examined whether oil palm oil extraction waste liquid (Palm Oil Separator Sludge = POSS) can be an organic nutrient source for ethanol fermentation. Ethanol bacteria Zymomonas mobilis and alcoholic yeast Saccharomyces cerevisiae were cultured in the range of 2-50% with POSS as an organic nutrient source using crystal glucose medium sago starch sugar solution as a carbon source. As a result, in all cases, the growth of the bacteria was not observed, or the growth was slight (see Examination Example 1).

[0016] Therefore, an attempt was made to activate the active ingredient by pre-hydrolyzing the organic substances contained in POSS. POSS hydrolyzed with 6N—H 2 SO 4 at 110 ° C for 6 hours, Flavo

twenty four

Fermentation tests were also conducted on urzyme 1000L (Novo) and Alcalase 2.4LFG (Novo) proteases, but no microbial growth-promoting activity was expressed. Alcohol fermentation was completely impossible even when the natural sugar latex mother liquor was used as the organic nutrient source for the starch sugar solution of sago palm (see Study Example 2).

[0017] However, surprisingly, the sago palm logs were ground as they were, and the starch was not separated, but the fe "was obtained. The 7-sugar cane was special even in ¾accharomyces cerevisiae even against Zymomonas mobilis. Alcohol fermentation is possible without supplementing nutrients. By adding appropriate amounts of lupal oil extraction waste liquor and natural rubber latex mother liquor, the fermentability was markedly improved, and a fermentation performance exactly equivalent to standard ethanol fermentation using CSL for glucose was obtained. Unlike the literature 1, the natural rubber latex mother liquor used in the present invention is a mother liquor itself that is not concentrated and spray-dried, and is inexpensive and can be used as a raw material for ethanol fermentation.

[0018] As described below, the present inventors have completed a process for producing nitrogen ethanol that surpasses the combination of US corn starch CSL.

Hereinafter, the present invention will be described in more detail with reference to examination examples and examples. The following examples are merely illustrative, and it goes without saying that the present invention is not limited to these examples.

[0020] (Examination example 1)

Malaysia Sarawak Chemical sago starch lOOg was weighed, suspended in 500 milliliter water and adjusted to pH 6.5, then Termamyl—120L (Novo) 100 μliter was prepared at 90 ° C for 1 hour. The starch was gelatinized.

[0021] After that, set ρΗ to 4.5, add Dextrozyme (Novo) 100 μlitter and heat to 70 ° C.

As a result of the sugar-sugar reaction for 24 hours, 480 milliliters of sugar solution of glucose 165 gZ litter was obtained.

[0022] This sugar solution diluted with water to a sugar concentration of 130g / liter is "5" and oil palm extraction waste fluid (Palm Oil Separator Sludge = POSS) of Malaysia Palm Oil Laboratory (Malaysia Palm Oil Board = MPOB). ) Mix “1”, adjust to pH 5.8, dispense 50 milliliters into a large test tube, and sterilize by autoclaving at 120 ° C for 10 minutes.

[0023] After adjusting YM broth (Difco Laboratories, Detroit) to the specified concentration, 10 ml of aliquot was dispensed into test tubes and heat-sterilized at 115 ° C for 10 minutes. Was inoculated with 1 milliliter and fermented at 30 ° C for 48 hours.

[0024] Slight growth of bacteria was observed in 24 hours of culture. Production of ethanol was almost unrecognized. Bacterial growth was observed after 48 hours of culture, but the ethanol concentration in the culture end solution was low and remained only at 5 gZ litter. [Study Example 2]

The same sago starch sugar liquor as that used in Study Example 1 was used. The sugar solution “1” is mixed with the natural rubber latex mother liquor “1” of National Timber and Forest Products of Indonesia, adjusted to pH 5.8, and then dispensed into a large test tube with 50 milliliters.

Autoclaved at 20 ° C for 10 minutes.

[0026] After adjusting YM broth (Difco Laboratories, Detroit) to the specified concentration, dispense 10 milliliters into a test tube and heat-sterilized at 115 ° C for 10 minutes. Zymomon as mobilis NRRL B— 14023 1 milliliter was inoculated and cultured at 30 ° C for 48 hours, but no bacterial growth was observed, and ethanol was not detected in the culture.

[0027] (Example 1)

Sago was obtained from National Timber and Forest Products in Indonesia. This is a sago palm log with a thickness of about 12 cm ^{2 and a} thickness of 3-4 mm, and dried in hot air in an oven to a moisture content of about 50%.

[0028] Weighed 250 grams, pulverized with a mortar stick, further ground with a mortar, added 900 milliliters of water, adjusted to pH 6.5 with 0. IN-NaOH, and Termamyl- 120L (Novo) 100 liters And heated to 90 ° C and kept for 1 hour.

[0029] Next, while stirring with a magnetic stirrer, adjust the pH to 4.5 with 0.IN—HC1.

Dextrozyme (Novo) 100 μlitter added, warmed to 70 ° C and saccharified for 24 hours o

[0030] The hydrolysis rate reached 95% 8 hours after the start of the reaction, and the degradation rate remained almost unchanged thereafter. The sugar solution was 1050 milliliters and the sugar concentration was 160 g / liter.

[0031] This sugar solution was centrifuged at 3, OOOg for 2 minutes to separate the solid content. Sugar concentration of this sugar solution with water

Mix "5" diluted in 130gZ litter with "1" from Palm Oil Separator Sludge (POSS) of Malaysia Palm Oil Laboratory (Malaysia Palm Oil Board = MPOB) to pH 5.8 After adjustment, 50 milliliters were dispensed into a large test tube and autoclaved at 120 ° C for 10 minutes.

[0032] After adjusting YM broth (Difco Laboratories, Detroit) to the specified concentration, dispense 10 milliliters into a test tube and heat-sterilized at 115 ° C for 10 minutes. onas mobilis NRRL B—14023 1 milliliter was inoculated and incubated at 30 ° C. for 15 hours.

[0033] Ethanol in the culture end solution was analyzed by gas chromatography to obtain an ethanol concentration 63 gZ litter.

[0034] (Example 2)

The same sago palm sugar syrup solution as used in Example 1 was used.

[0035] Natural sugar latex mother liquor "1" from Indonesia's National Timber and Forest Products was mixed with this sugar liquor "1", adjusted to pH 5.8, and then dispensed 50 milliliters into a large test tube. Autoclaved at 120 ° C for 10 minutes.

[0036] After adjusting YM broth (Difco Laboratories, Detroit) to a specified concentration, 10 ml of aliquot was dispensed into a test tube and sterilized by heating at 115 ° C for 10 minutes. Zymomon as mobilis NRRL B— 14023 1 milliliter was inoculated and fermented at 30 ° C for 15 hours.

[0037] Ethanol in the culture ending liquid was analyzed by gas chromatography to obtain an ethanol concentration 41 gZ litter.

[0038] (Example 3)

The same sago palm sugar syrup solution as used in Example 1 was used.

[0039] This sugar solution was diluted with water to a sugar concentration of 130 g / liter, and “5” was used for oil palm oil waste (Palm Oil Separator Sludge = POSS) from Malaysia Palm Oil Laboratory (Malaysia Palm Oil Board = MPOB). ) Mix “1”, adjust to pH 5.8, dispense 50 milliliters into a large test tube, and sterilize by autoclaving at 120 ° C for 10 minutes.

[0040] To this, YM broth (Difco Laboratories, Detroit) was adjusted to the specified concentration, then 10 milliliters were dispensed into a test tube and inoculated with alcoholic yeast Saccharomyces cerevisiae seeded with 115 ° C for 10 minutes. Then, static fermentation was performed at 31.5 ° C for 24 hours.

[0041] Ethanol in the culture-finished solution was analyzed by gas chromatography to obtain an ethanol concentration of 58 gZ litter.

[Example 4]

Same Indonesian National Timber and Forest Pro as used in Example 1 The sago palm chips obtained by ducts company power were used. Weigh 200 g of this, pulverize it with a mortar stick, further pulverize with a mortar and suspend in water. 1. 3 liters of suspension. The litter was calo-free, heated to 90 ^o C and kept for 1 hour.

[0043] Next, while stirring with a glass rod, adjust the pH to 4.5 with 0. IN—HC1, add Dextrozy me (Novo) 100 litter, warm to 70 ° C, and warm for 24 hours. The reaction was carried out to obtain 1.35 litter of sugar solution having a sugar concentration of 140 gZ litter.

Milliliter This sugar solution was centrifuged at 3,000 g for 2 minutes to separate solids, and then diluted to a sugar concentration of 120 g / liter.

[0044] National solids (SS) was previously removed by centrifugation at 9000g for 3 minutes.

Timber and Forest Products oil palm oil waste (POSS) 400 milliliters was added to adjust the pH to 5.8, autoclaved at 120 ° C for 10 minutes, and then charged into a 2 litter glass jar.

[0045] Zymomonas mobilis NRRL B—14023 50 milliliter after YM broth (Difco Laboratories, Detroit) was adjusted to the specified concentration and placed in a 100 milliliter Erlenmyer Flask and seeded in a medium sterilized by heating at 115 ° C for 10 minutes. 150 rpm, pH 5.5, 30. Ethanol fermentation in C.

[0046] FIG. 1 shows the course of alcoholic fermentation (POSS 20%), and the horizontal axis is time “CT”. In Fig. 1, “RS” is the residual sugar concentration, “DCW” is the dry cell weight, and “EtOH” is the ethanol concentration. Ethanol 47gZ litter (6vol%) and residual sugar concentration were 0 after 14 hours of culture.

[Example 5]

Sago palm was harvested for harvest by a farmer in Tebing Tinggi Island, Riau, Indonesia, and used as raw trees. This is the state prior to being subjected to Rasper to separate the starch.

[0048] Weigh 500 grams of blocks (lumps) of sago palm, cut them into dice of about 1 centimeter with a knife, and then adjust the pH to 6.5 with 0.1N NaOH, free of 900 milli-jitter of water. Ter mamyl-120L (Novo) 150 Jitter was calorie-free, boyned to 100 ^o C and held for 1 hour. [0049] Next, while stirring with a magnetic stirrer, adjust the pH to 4.5 with 0.IN—HC1, add Dextrozyme (Novo) 150 litter, warm to 70 ° C and perform saccharification reaction for 24 hours went. The sugar solution thus obtained was adjusted to pH 5.8, and then 50 milliliters was dispensed into a large test tube, and autoclaved at 120 ° C for 10 minutes. After adjusting YM broth (Difco Laboratories, Detroit) to the specified concentration, dispense 10 milliliters into a test tube, heat-sterilized at 115 ° C for 10 minutes, and seed culture with Zymomonas mobilis NRRL B—14023 in 1 milliliter. One was inoculated and left to stand at 30 ° C for 48 hours.

[0050] Ethanol in the culture-finished solution was analyzed by gas chromatography to obtain an ethanol concentration 38 gZ litter.

Claims

The scope of the claims

[1] The first step of preparing sago palm crushed material,

A second step in which at least one of a liquid enzyme and a sugar enzyme is added to the prepared pulverized product and then sugared to obtain a sugar solution;

And a third step of culturing at least one of alcohol-producing bacteria and alcohol yeast using the sugar solution to produce ethanol. An ethanol production fermentation method, comprising:

[2] The ethanol production fermentation method according to claim 1, wherein an oil palm extraction waste liquid is added to the sugar liquid prior to the third step.

[3] The ethanol production fermentation method according to claim 1, wherein a natural rubber latex mother liquor is added to the sugar solution prior to the third step.

[4] The ethanol-producing fermentation method according to claim 1, wherein the alcohol-producing bacterium is Zymomonas mobilis.

5. The ethanol production fermentation method according to claim 1, wherein the alcohol yeast is Saccharomyces cerevisiae.