JP2007195406A - Method for producing and fermenting ethanol - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing and fermenting ethanol by which the ethanol can efficiently be produced using biomass and a contribution can be made even to a waste liquor treatment causing problems. <P>SOLUTION: The method for producing and fermenting ethanol is carried out as follows. Metroxylon sagu succeeded in artificial cultivation as a plant resource produced in the tropics kept in a wood state is saccharified to remove non-degraded fibers, etc., and POSS (palm oil separator sludge) produced during oil pressing of oil palm widely cultivated in Southeast Asia or a latex mother liquor, etc., obtained from a natural rubber sap are added as an organic nutrient component. An alcohol-producing bacterium Zymomonas mobilis or an alcoholic yeast Saccharomyces cerevisiae is cultivated therein to produce alcohol. Thereby, effects as a method for producing energy from the biomass can be obtained and the contribution can be made to environmental countermeasures in the tropics as a method for treating the waste liquor. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  Considering the current situation in which global warming and depletion of petroleum resources proceed in parallel, it is a common challenge for humankind to suppress the consumption of oil and replace energy sources with renewable resources that are photosynthetic products. In addition, as uncertainty about the future of petroleum, which is a limited resource, has increased, movements to add ethanol to gasoline for automobiles are expanding around the world, and not only in Brazil and the United States, but also in countries in China and Southeast Asia The competition for developing raw materials and new production methods for fuel ethanol production is intensifying.

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

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 development of more rational waste liquid treatment technology is desired from the viewpoint of environmental countermeasures. Oil palm extraction waste liquid is treated by anaerobic methane fermentation and reduced to plantation, and natural rubber latex mother liquor is treated by lagoon to make it harmless. However, it is not always sufficient.
Japanese Patent Publication No. 6-46941

  Now, since organic nutrients such as amino acids and vitamins are generally required for the growth of microorganisms, when using starch raw materials in alcoholic fermentation, CSL and malt extract can be used for both yeasts and bacteria such as Zymomonas. Natural organic nutrient sources such as Natural rubber latex mother liquor is an attractive organic nutrient source containing abundant microbial growth promoting factors (Patent Document 1). This is a solution obtained by concentrating the mother liquor and spray-drying it into powder, which is rich in nutritional value but expensive in terms of cost and cannot be a raw material for ethanol fermentation.

  Oil palm extraction waste liquid is considered to be an inexpensive organic nutrient source that can supply microbial growth factors rich in proteins and vitamins contained in the oil palm pulp, but has not yet been used as a fermentation raw material. It has only been reported that acetone / butanol fermentation is possible from the viewpoint of effective use of waste liquid.

  Then, this invention aims at providing the ethanol production fermentation method which can produce ethanol efficiently using biomass, and can also contribute to the waste liquid process used as a subject.

  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 liquefying enzyme and a saccharifying enzyme to the prepared pulverized product 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.

  If this configuration is adopted, as will be apparent from the examples described later, sago palm can be utilized in a state that is almost close to the raw wood, and ethanol can be produced efficiently.

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

  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.

  With these configurations, waste liquids that are difficult to process can be used effectively.

  ADVANTAGE OF THE INVENTION According to this invention, while being able to produce alcohol efficiently from the sago palm as biomass, not only can a waste liquid be processed but it can be utilized effectively.

  If sago palm is crushed, the fiber is separated, starch is separated, and this is refined and dried to make starch powder. Corn starch, cassava starch, potatoes and sweet potatoes are also a raw material for fermentation as starch resources. become. However, it is believed that the use of sago palm as a raw material for fermentation as raw materials is impossible with conventional common sense, and no one has yet considered it. If starch is not separated from sago palm and sago timber can be directly used as a raw material for ethanol fermentation, the yield from sago palm will be improved and the process will be simplified. It must be a fuel production method from a periodical photosynthesis product.

  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 alcohol yeast Saccharomyces cerevisiae were cultured in the range of 2-50% of medium addition concentration using crystalline glucose medium and sago starch saccharified solution as carbon source and POSS as organic nutrient source. As a result, in all cases, the growth of the bacteria was not observed, or even if the growth was slight (see Examination Example 1).

Then, activation of the active ingredient was tried by hydrolyzing the organic substance contained in POSS in advance. Fermentation tests were performed on POSS hydrolyzed with 6N—H ₂ SO _{4 at} 110 ° C. for 6 hours, and enzymatically decomposed with Flavorzyme 1000 L (Novo) and Alcalase 2.4 LFG (Novo) protease. No activity was expressed. Alcohol fermentation could not be carried out at all even with a sago starch saccharified solution and a natural rubber latex mother liquor as an organic nutrient source (see Study Example 2).

  However, surprisingly, by adding appropriate amounts of oil palm oil extraction waste liquid and natural rubber latex mother liquor to the sugar liquid obtained by pulverizing the raw sago palm as it is, and saccharifying the starch without separating starch, Zymomonas mobilis In Saccharomyces cerevisiae, very good fermentability was obtained, and the fermentation performance was exactly the same as that of standard ethanol fermentation using CSL as glucose. Unlike patent document 1, the natural rubber latex mother liquor used in the present invention is not concentrated spray-dried, but is the mother liquor itself, and can be used as a raw material for ethanol fermentation at low cost.

  Thus, the inventors have completed a biomass ethanol production process that surpasses the US corn starch-CSL combination as described below.

  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.

(Examination example 1)
After weighing 100 g of Sago palm starch produced by Sarawak Chemical Co., Malaysia, suspended in 500 milliliters of water and adjusting to pH 6.5, 100 μl of Termamyl-120L (Novo) was added and kept at 90 ° C. for 1 hour. Gelatinization was performed.

  Thereafter, the pH was adjusted to 4.5, 100 μL of Detrozyme (Novo) was added, and the mixture was heated to 70 ° C. and subjected to a saccharification reaction for 24 hours.

  This sugar solution is diluted with water to a sugar concentration of 130 g / liter “5”, whereas the oil palm extraction waste fluid (Palm Oil Separator Sludge = POSS) of Malaysia Palm Oil Research Institute (Malaysia Palm Oil Board = MPOB) “1” Were mixed and adjusted to pH 5.8, 50 ml were dispensed into a large test tube, and autoclaved at 120 ° C. for 10 minutes.

  To this, YM broth (Difco Laboratories, Detroit) was adjusted to a specified concentration, and 10 ml was dispensed into a test tube and sterilized by heating at 115 ° C. for 10 minutes. Zymomonas mobilis NRRL B-14023 was seed-cultured at 1 ml. Inoculated and left to stand at 30 ° C. for 48 hours.

  Slight growth of bacteria was observed in 24 hours of culture, but almost no ethanol was produced. Bacterial growth was observed at 48 hours of culture, but the ethanol concentration of the culture end solution was low and remained at only 5 g / liter.

(Examination example 2)
The same sago starch saccharified solution as that used in Study Example 1 was used. Natural sugar latex mother liquor “1” of National Timber and Forest Products of Indonesia is mixed with this sugar liquor “1”, adjusted to pH 5.8, and then dispensed 50 ml into a large test tube. Minute autoclave sterilization.

  To this, YM broth (Difco Laboratories, Detroit) was adjusted to a prescribed concentration, and 10 ml was dispensed into a test tube, which was then sterilized by heating at 115 ° C. for 10 minutes. Zymomonas mobilis NRRL B-14023 1 ml Was inoculated and cultured at 30 ° C. for 48 hours, but no bacterial growth was observed, and ethanol was not detected in the culture solution.

Example 1
Sago palm was obtained from National Timber and Forest Products, Indonesia. This is a sago palm log having a thickness of about 1-2 cm ^{2 and a} thickness of 3-4 mm, and hot-air drying in an oven to a moisture content of about 50%.

  250 g of this was weighed and pulverized with a mortar stick, then further pulverized with a mortar, 900 ml of water was added, pH was adjusted to 6.5 with 0.1 N NaOH, and Termamyl-120L (Novo) 100 μl was added. Warm to 90 ° C. and hold for 1 hour.

  Subsequently, while stirring with a magnetic stirrer, the pH was adjusted to 4.5 with 0.1 N HCl, dextrozyme (Novo) 100 μL was added, the mixture was heated to 70 ° C., and saccharification reaction was performed for 24 hours.

  In 8 hours after the start of the reaction, the hydrolysis rate reached 95%, and thereafter the degradation rate hardly changed. The saccharified liquid was 1050 milliliters, and the sugar concentration was 160 g / liter.

  The sugar solution was centrifuged at 3,000 g for 2 minutes to separate the solid content. This sugar solution is diluted with water to a sugar concentration of 130 g / liter “5”, whereas the oil palm extraction waste fluid (Palm Oil Separator Sludge = POSS) of Malaysia Palm Oil Research Institute (Malaysia Palm Oil Board = MPOB) “1” Were mixed and adjusted to pH 5.8, 50 ml were dispensed into a large test tube, and autoclaved at 120 ° C. for 10 minutes.

  To this, YM broth (Difco Laboratories, Detroit) was adjusted to a specified concentration, and 10 ml was dispensed into a test tube, which was then sterilized by heating at 115 ° C. for 10 minutes. Zymomonas mobilis NRRL B-14023 1 ml And incubating at 15 ° C. for 15 hours.

  The ethanol at the end of the culture was analyzed by gas chromatography to obtain an ethanol concentration of 63 g / liter.

(Example 2)
The same sago palm tree saccharified solution as that used in Example 1 was used.

  Natural sugar latex mother liquor “1” of National Timber and Forest Products of Indonesia is mixed with this sugar liquor “1”, adjusted to pH 5.8, and then dispensed 50 ml into a large test tube. Minute autoclave sterilization.

  To this, YM broth (Difco Laboratories, Detroit) was adjusted to a prescribed concentration, and 10 ml was dispensed into a test tube, which was then sterilized by heating at 115 ° C. for 10 minutes. Zymomonas mobilis NRRL B-14023 1 ml And incubating at 15 ° C. for 15 hours.

  The ethanol at the end of the culture was analyzed by gas chromatography to obtain an ethanol concentration of 41 g / liter.

(Example 3)
The same sago palm tree saccharified solution as that used in Example 1 was used.

  This sugar solution is diluted with water to a sugar concentration of 130 g / liter “5”, whereas the oil palm extraction waste fluid (Palm Oil Separator Sludge = POSS) of Malaysia Palm Oil Research Institute (Malaysia Palm Oil Board = MPOB) “1” Were mixed and adjusted to pH 5.8, 50 ml were dispensed into a large test tube, and autoclaved at 120 ° C. for 10 minutes.

  To this, YM broth (Difco Laboratories, Detroit) was adjusted to a prescribed concentration, 10 ml was dispensed into a test tube, and heat-sterilized at 115 ° C. for 10 minutes, and then inoculated with alcoholic yeast Saccharomyces cerevisiae. The fermentation was performed at 5 ° C. for 24 hours.

  The ethanol at the end of the culture was analyzed by gas chromatography to obtain an ethanol concentration of 58 g / liter.

Example 4
The same sago palm chips obtained from National Timber and Forest Products of Indonesia as used in Example 1 were used. This was weighed 200 grams, ground with a mortar and further ground with a mortar. The mixture was suspended in 1.3 liters, adjusted to pH 6.5 with 0.1 N NaOH, Termamyl-120L (Novo) 100 μL was added, heated to 90 ° C. and kept for 1 hour.

Next, while stirring with a glass rod, the pH is adjusted to 4.5 with 0.1 N HCl, Dextrozyme (Novo) 100 μL is added, heated to 70 ° C. and subjected to a saccharification reaction for 24 hours to a sugar concentration of 140 g / A 1.35 liter sugar solution of liter was obtained.
Milliliter This sugar solution was centrifuged at 3,000 g for 2 minutes to separate the solid content, and then diluted to a sugar concentration of 120 g / liter.

  To this was added 400 milliliters of oil palm oil expelled waste liquid (POSS) from National Timber and Forest Products, which had previously been freed of solids (SS) by centrifugation at 9,000 g for 3 minutes, adjusted to pH 5.8, and then adjusted to 120 ° C. 10 After autoclaving, the mixture was charged into a 2-liter glass jar.

  YM broth (Difco Laboratories, Detroit) was adjusted to a prescribed concentration, and then inoculated with Zymomonas mobilis NRRL B-14023 50 milliliter which was put into 100 milliliter Erlenmyer Flask and seeded in a medium sterilized by heating at 115 ° C. for 10 minutes. And ethanol fermentation at 150 rpm, pH 5.5 and 30 ° C.

  FIG. 1 shows the course of alcoholic fermentation (POSS 20%), and the horizontal axis is time “CT”. In FIG. 1, “RS” indicates the residual sugar concentration, “DCW” indicates the dry cell weight, and “EtOH” indicates the ethanol concentration. After 14 hours of culture, ethanol was 47 g / liter (6 vol%) and the residual sugar concentration was 0.

The graph which shows the ethanol fermentation progress by this invention

Claims (5)

A first step of preparing a sago palm crushed material,
A second step of adding at least one of a liquefying enzyme and a saccharifying enzyme to the prepared pulverized product to saccharify and 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. 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. 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. The ethanol-producing fermentation method according to any one of claims 1 to 3, wherein the alcohol-producing bacterium is Zymomonas mobilis. The ethanol-producing fermentation method according to any one of claims 1 to 4, wherein the alcoholic yeast is Saccharomyces cerevisiae.

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