KR101681670B1 - Method for Culturing Microalge Thraustochytrid Using Palm Empty Fruit Bunch Hydrolysate and Method for Preparing Biooil Through the Same - Google Patents

Abstract

The present invention relates to a method for producing a high-concentration DHA-containing bio-oil by culturing a micro-algae of Thraustochytrid using a glycated liquid of a fibrous palm oil industry by-product, and more specifically, The present invention relates to a method for producing a bio-oil, which comprises culturing a micro-algae of a Thraustochytrid-type microalgae in a fibrous palm oil industry by-product in a culture medium to produce a bio-oil, and then recovering the bio-oil .
The method for producing bio-oil according to the present invention can produce bio-oil from abundant non-food fiber biomass through cultivation of micro-algae of Thraustochytrid, It is possible to overcome the limit of development and to secure the commercial competitiveness of microbial fermentation oil.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a method for culturing micro-algae using a saccharification solution and a method for producing bio-oil using the micro-

The present invention relates to a method for producing a bio-oil from a fibrous biomass using micro-algae based on Thraustochytrid, and more particularly, to a method for producing bio-oil from a fibrous palm oil industrial by- In a medium containing a saccharification solution to produce microbial oil, and then recovering the microbial oil.

Biodiesel, which is produced from oil of sustainable plants such as rapeseed, soybean, and palm, is a representative bio-energy that has already been commercialized, and its production is rapidly increasing worldwide. However, due to the high cost of raw materials in terms of production costs, biodiesel is relatively ineffective compared to diesel raw materials derived from crude oil. Therefore, despite the various advantages of the environment and agriculture economy, biodiesel is not competitive in the market without the government tax relief benefits. In the future, rising oil prices due to energy depletion are expected to provide market competitiveness to biodiesel. However, the recent surge in raw material prices due to the increase in biodiesel production is a new factor that further deteriorates the competitiveness of biodiesel Is emerging.

In addition, photosynthetic oils of sustainable plants and microalgae, which are the main sources of bio-oil raw materials for the production of biodiesel, have a very important advantage of using abundant sunlight and recycling carbon dioxide. However, in various environments such as time, space, , And some of them have doubts about the effectiveness of biodiesel because it can cause new environmental problems due to lack of food and mass cultivation of raw material crops. It is a situation that is being raised.

Recently, the fermentation culture method of organic nutrition microorganisms has attracted attention as a mass production method of bio oil, and typical representative production microorganisms include Chlorella protothecoides , Yarrowia lipolytica , Rhodosporidium toruloides , Rhodotorula glutinis . Studies on these fermentation processes are actively underway. The most important factor to secure commercial competitiveness of microbial fermentation oils as raw materials for biodiesel is to utilize industrial waste, waste resources and surplus biomass as nutrients, ultimately to utilize abundant non-edible fibrous biomass 1). Non-edible fiber sub-total biomass resources include woody, agricultural by-products, and municipal wastes (Korean Patent Laid-Open No. 10-2008-097651, US Patent Publication No. 2009-648483).

On the other hand, Thraustochytrid-based heterotrophic microalgae are biofuels containing polyunsaturated fatty acids such as DHA (docosahexaenoic acid), which are sustainable microorganisms capable of producing up to 70% of dry cell mass Korean Patent Publication No. 10-2011-0122424). DHA is an essential fatty acid in the brain, eye tissues and nervous system, and is known to play an important role in developing the visual and motor skills of babies. In addition, it has been reported that the amount of dementia is significantly reduced in the brain of demented patients, and various anti-aging functions such as inhibition of presbyopia and macular degeneration are newly discovered. Despite these useful physiological functions, the human body can not synthesize sufficient amount of DHA itself. Therefore, it is recognized as an essential nutrient to be supplied from the outside, and the authorized bodies of the World Health Organization and other countries in the world constantly consume more than 1 g of DHA per day . Therefore, DHA is commercialized as a diverse product such as health functional food, and DHA has a high commercial value because it is highly available as a raw material for pharmaceuticals. Therefore, the fermentation oil of Thraustochytrid microalgae containing DHA at a high concentration, by linking the high-value-added industry and the biodiesel industry using DHA, is different from general microbial fermentation oil or photosynthetic oil, It is expected to be able to provide.

The present inventors have made intensive efforts to develop a method for producing bio-oils by cultivating microalgae using a fibrous biomass rich in nutrients. As a result, they have found that Thraustochytrid-type microalga KRS101 KCTC11686BP) as a nutrient source of a byproduct of fibrous palm oil industry as a nutrient source, it was confirmed that DHA-containing bio oil was produced. Thus, the present invention was completed.

It is an object of the present invention to provide a method for culturing microalgae using a saccharified liquid of a fibrous palm oil industrial by-product as a nutrient source.

Another object of the present invention is to provide a method for producing a bio-oil using a saccharified liquid of a by-product of the oily palm oil industry as a nutrient source.

In order to accomplish the above object, the present invention provides a method for producing a palm oil, comprising the steps of: (a) immersing the fibrous palm oil industry by-product in an alkali solution, treating the mixture at 100 to 130 ° C for 30 minutes to 3 hours, washing with water, ; (b) adding distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-product and stirring to prepare a fibrous palm oil industrial by-product saccharified liquid; And (c) culturing the fibrous palm oil industrial by-product glycated fluid by inoculating microalgae. The present invention also provides a method for culturing microalgae using the fibrous palm oil industrial by-product.

(A) immersing the fibrous palm oil industrial by-product in an alkali solution, treating the flask at 100 to 130 ° C for 30 minutes to 3 hours, washing it with water, and drying and pretreating it; And (b) inoculating distilled water, saccharifying enzyme and microalgae into the pretreated fibrous palm oil industrial by-product, and simultaneously performing saccharification and cultivation, wherein the microalgae are cultured using the fibrous palm oil industrial by- to provide.

The present invention also relates to a method for producing a palm oil composition, comprising the steps of: (a) immersing a fibrous palm oil industrial by-product in an alkali solution, treating at 100 to 130 ° C for 30 minutes to 3 hours, washing with water, (b) adding distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-product and stirring to prepare a fibrous palm oil industrial by-product saccharified liquid; (c) inoculating and cultivating a microalgae of a threustochytrid system in a glycated solution of a fibrous palm oil industry by-product; And (d) obtaining a bio-oil from the cultured micro-algae of Thraustochytrid-type microalgae, wherein the by-product of the fiber-based palm oil industry is used as a carbon source.

(A) immersing the fibrous palm oil industrial by-product in an alkali solution, treating the flask at 100 to 130 ° C for 30 minutes to 3 hours, washing it with water, and drying and pretreating it; (b) inoculating distilled water, a saccharifying enzyme and a Thraustochytrid-type microalgae into the pretreated fibrous palm oil industry by-product, and performing saccharification and culture simultaneously; And (c) obtaining a bio-oil from the cultured micro-algae of Thraustochytrid-type microalgae, wherein the byproduct of the fiber-based palm oil industry is used as a carbon source.

The method for producing bio-oil according to the present invention can produce bio-oil from abundant non-food fibrous industrial byproduct resources through cultivation of micro-algae of Thraustochytrid, It is possible to overcome the limit of fuel development and to secure the commercial competitiveness of microbial fermentation oil.

Fig. 1 shows the results of analysis of the composition of the palm oil industrial by-product saccharified liquid.
Fig. 2 shows the results of the reaction surface analysis for the culture conditions of the microalgae KRS101 using the glycated fluid of the fibrous palm oil industry by-product.
Fig. 3 shows the result of culturing the microalgae KRS101 strain using the palm oil industrial by-product saccharified liquid.
Fig. 4 shows the result of simultaneous saccharification culture of microalgae KRS101 strain using palm oil industrial by-products.
FIG. 5 shows the results of investigation of the effect of the inoculation amount on the simultaneous saccharification culture of the microalgae KRS101 strain using the by-product of the palm oil industry.

In one aspect, the present invention relates to a process for producing a fiber-based palm oil by-product (PEFB), which comprises (a) immersing a fibrous palm oil industrial by-product (PEFB) in an alkali solution, treating at 100 to 130 ° C for 30 minutes to 3 hours, washing with water, ; (b) adding distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-product and stirring to prepare a fibrous palm oil industrial by-product saccharified liquid; And (c) a step of inoculating and cultivating a microalgae in a glycated solution of a fibrous palm oil industry by-product, and culturing the microalgae using a fibrous palm oil industrial by-product.

The present invention relates to a method of physically and chemically pretreating a fiber-type emulsion palm fruit bunch fiber containing an excess of hemicellulose and lignin and then using a saccharified saccharified liquid for culturing microalgae As a method, in one embodiment of the present invention, a PEFB saccharification solution for culturing a Thraustochytrid-based microalga KRS101 (KCTC11686BP) isolated from soil in the Mangungbu area was prepared as follows.

First, for the pretreatment, crushed palm oil byproducts of 1 ~ 2mm size were immersed in 1M NaOH solution for 30 minutes, and autoclave (121 ℃, 15 psi, 1 hour) was performed. 50 g of the pretreated EFB (PEFB added amount 10% (w / v)) was added to 500 ml of a mixture solution containing distilled water and enzyme, and saccharification was carried out for 3 days with stirring at 45 rpm at 150 rpm. At this time, a 40 FPU (Filter Paper Assay Unit) was used for 1 g of dry weight of EFB pretreated with the above method. The contents of glucose and xylose were 68 g / L and 22 g / L, respectively, and HMF and furfural were not detected.

The microalgae KRS101 was cultivated in the glycated fluid of the fibrous palm oil industry by-product prepared by the above method. As a result, the concentration of PEFB glycosylated glucose was 53.8 g / L, the concentration of yeast extract was 6.03 g / L, the concentration of seawater salt was 20.1 g / The highest oil content (15.35 g / L) and DHA content (5.28 g / L) were found at pH 5.7 of the initial culture. After 36 hours of culture, glucose was consumed and then xylose was consumed. The maximum oil production was 12.53 g / L (% oil g / g glucose) and the content of DHA was 5.39 g / L, 43% of total fatty acids.

In another aspect, the present invention provides a method for producing a palm oil, comprising: (a) immersing the fibrous palm oil industry by-product in an alkali solution, treating the mixture at 100 to 130 ° C for 30 minutes to 3 hours, washing with water, And (b) inoculating distilled water, saccharifying enzyme and microalgae into the pretreated fibrous palm oil industry by-product, and simultaneously performing saccharification and cultivation, and culturing the microalgae using the fibrous palm oil industrial byproduct .

In one embodiment of the present invention, the saccharification enzyme and the microalgae KRS101 were added to the pretreated byproducts of the fibrous palm oil industry, and saccharification and cultivation were performed in the same manner. In the simultaneous saccharification culture in which 5% PEFB was added, Glucose was completely consumed and the use of xylose started to be started (Fig. 3A). The total amount of carbon source produced by 5% PEFB was 33.7 g / L and 10.8 g / L for glucose and xylose, respectively. The maximum yield of the oil produced was 1.7 g / L (0.57 g / L day) and the content of DHA in the total fatty acids was more than 45% (FIG. 3C).

As shown in FIG. 3B, in the simultaneous saccharification culture experiment in which 10% PEFB was added, 57.3 g / L of glucose and 26.2 g / L of xylose were produced and only 31.1 g / L of glucose was consumed until the fifth day of culture. However, the production of oil was twice as high as that of 5% PEFB (3.4 g / L, 0.68 g / L day), compared with 10% PEFB input.

According to another aspect of the present invention, there is provided a method for producing a palm oil, comprising: (a) immersing a fibrous palm oil industrial by-product in an alkali solution, treating the mixture at 100 to 130 ° C for 30 minutes to 3 hours, washing with water, (b) adding distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-product and stirring to prepare a fibrous palm oil industrial by-product saccharified liquid; (c) inoculating and cultivating a microalgae of a threustochytrid system in a glycated solution of a fibrous palm oil industry by-product; And (d) a step of obtaining a bio-oil from the cultured Thraustochytrid-based microalgae, wherein the by-product of the fiber-based palm oil industry is used as a carbon source .

According to another aspect of the present invention, there is provided a method for producing a palm oil, comprising: (a) immersing the fibrous palm oil industry by-product in an alkali solution, treating the mixture at 121 DEG C and 15 psi for 30 minutes to 3 hours, (b) inoculating distilled water, a saccharifying enzyme and a Thraustochytrid-type microalgae into the pretreated fibrous palm oil industry by-product, and performing saccharification and culture simultaneously; And

(c) obtaining a bio-oil from the cultured Thraustochytrid-type microalgae, wherein the by-product of the fibrous palm oil industry is used as a carbon source.

Hereinafter, the present invention will be described in more detail with reference to examples. It is to be understood by those skilled in the art that these embodiments are only for illustrating the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

Establishment of culture condition of microalgae KRS101 strain by reaction surface analysis

Culture conditions of KRS101 (KCTC11686BP) strain of subspecies Aurantiochytrium subsp. Genus Thraustochytrid were investigated using palm oil fumigation (PEFB) as a nutrient source. The microalgae KRS101 strain was inoculated into medium containing 60 g / L of carbon source glucose, 10 g / L of yeast extract, 6 g / L of nitrogen source, and 6 g / L of artificial sea salt, and cultured for 3 days at 28 ° C and 125 rpm for inoculation , And the cell growth was analyzed by measuring the optical density (OD) at 600 nm while culturing at 28 ° C at 125 rpm.

The PEFB saccharified liquid for culturing microalgae strain KRS101 was prepared as follows. First, for the pretreatment, crushed palm oil byproducts of 1 ~ 2mm size were immersed in 1M NaOH solution for 30 minutes, and autoclave (121 ℃, 15 psi, 1 hour) was performed.

 50 g of the pretreated EFB (PEFB added amount 10% (w / v)) was added to 500 ml of a mixture solution containing distilled water and enzyme, and saccharification was carried out for 3 days with stirring at 45 rpm at 150 rpm. At this time, a 40 FPU (Filter Paper Assay Unit) was used for 1 g of dry weight of EFB pretreated with the above method.

As a result of analysis of components of the saccharified liquid of PEFB, glucose and xylose were present at 68 g / L and 22 g / L, respectively, and HMF and furfural were not detected (FIG. 1).

The content of oil in the microalgae KRS101 strain was analyzed using the modified Bligh-Dyer method (Burja et al., 2007). The reaction was carried out at 28 ° C and 200 rpm for 1 hour with 6.25 mL of chloroform, 12.5 mL of methanol and 5 mL of 50 mM K ₂ HPO ₄ buffer (pH 7.4), and 6.25 mL of chloroform and 6.25 mL of K2HPO4 buffer were added to the dried cell mass of 125 mg, And allowed to stand for 30 minutes to separate into a water layer and an oil-containing organic solvent layer. The chloroform layer was carefully transferred to an aluminum plate which had been previously weighed, and then dried at 80 ° C. for 30 minutes, and then the weight of the oil was measured. The total oil content was calculated as follows.

Total oil content (%, oil g / dry cell weight 100 g) = (WL-WD) xVCx100 / VPxWS

WL: Weight of aluminum plate

WD: Weight of aluminum plate + lipid

VC: Total volume of Chloroform

VP: Volume of Chloroform transferred to aluminum plate

WS: weight of the used cells (dry weight)

On the other hand, the content of DHA contained in the oil was measured by gas chromatography. An appropriate amount of dried cells was suspended in 3 mL of a methanol-sulfuric acid solution (96: 4 (v / v)%) and reacted at 90 ° C for 1 hour to produce a fatty acid ester. The resultant was extracted with 0.3 mL of nucleic acid and analyzed by gas chromatography Respectively.

The optimum culture conditions of the microalgae KRS101 (FIG. 2) were examined through the reaction surface analysis method, the concentration of PEFB glycosylated glucose was 53.8 g / L, the concentration of yeast extract was 6.03 g / L, (15.35 g / L) and DHA content (5.28 g / L) at the initial culture pH of 5.7 (Table 1).

Study on the culture condition of microalgae KRS101 using PEFB saccharified solution by reaction surface analysis Std F1 (glycation solution concentration) F2 (nitrogen source concentration) F3 (salt concentration) F4 (pH) Total lipid (g / L) Total lipid (g / g sample) OD600
DHA (%) One 20 5 20 5.3 0.21 0.0047 19.3 44.0 2 40 5 20 5.3 7.06 0.1084 24.3 46.2 3 20 13 20 5.3 0.19 0.0038 23.5 50.6 4 40 13 20 5.3 0.32 0.0049 30.2 49.7 5 20 5 40 5.3 0.45 0.0067 19.9 45.7 6 40 5 40 5.3 7.33 0.0859 24.4 46.5 7 20 13 40 5.3 1.41 0.0203 23.6 50.0 8 40 13 40 5.3 0.53 0.0068 27.6 52.3 9 20 5 20 5.7 0.24 0.0053 20.1 45.1 10 40 5 20 5.7 6.43 0.1026 24.6 45.5 11 20 13 20 5.7 0.18 0.0036 26.5 50.7 12 40 13 20 5.7 0.35 0.0053 29.2 49.9 13 20 5 40 5.7 0.31 0.0051 20.6 44.2 14 40 5 40 5.7 7.32 0.0970 24.9 46.4 15 20 13 40 5.7 0.54 0.0083 21.7 50.4 16 40 13 40 5.7 0.50 0.0059 28.8 49.5 17 10 9 30 5.5 0.20 0.0043 18.5 48.1 18 50 9 30 5.5 3.65 0.0452 32.6 50.3 19 30 One 30 5.5 7.58 0.1206 17.3 40.4 20 30 17 30 5.5 0.25 0.0035 31.5 50.7 21 30 9 10 5.5 0.17 0.0035 30.3 48.8 22 30 9 50 5.5 0.38 0.0045 24.9 45.1 23 30 9 30 5.1 0.30 0.0046 23 49.0 24 30 9 30 5.9 0.27 0.0042 26.4 46.4 25 30 9 30 5.5 0.25 0.0037 26.4 47.8 26 30 9 30 5.5 0.28 0.0046 27.2 49.8 27 30 9 30 5.5 0.30 0.0046 24.4 48.1 28 30 9 30 5.5 0.29 0.0044 26 49.3 29 30 9 30 5.5 0.27 0.0041 24.6 49.5 30 30 9 30 5.5 0.30 0.0047 25.6 48.1

Culture and oil production of microalgae KRS101 strain using PEFB saccharified solution

The microalgae KRS101 strain was fermented using the PEFB saccharified solution under the culture conditions of Example 1. [ As shown in FIG. 2, after 36 hours of cultivation, glucose was consumed in the culture medium, and then xylose was consumed. The maximum oil production was 12.53 g / L (% oil g / g glucose) and the content of DHA was 5.39 g / L, 43% of total fatty acids.

Oil production by simultaneous saccharification culture of PEFB

The microalgae KRS101 strain was co-saccharified using PEFB under the culture conditions of Example 1. For the saccharification enzyme, 40 FPU (Filter Paper Assay Unit) was used for 1 g dry weight of EFB pretreated by the above method. First, in the simultaneous saccharification culture in which the PEFB was added at 5%, the glucose in the culture liquid began to be completely consumed from the third culture day, and the use of xylose started to be started (FIG. 3A). The total amount of carbon source produced by 5% PEFB was 33.7 g / L and 10.8 g / L for glucose and xylose, respectively. The maximum yield of the oil produced was 1.7 g / L (0.57 g / L day) and the content of DHA in the total fatty acids was over 45% (FIG. 3C).

As shown in FIG. 3B, in the simultaneous saccharification culture experiment in which 10% PEFB was added, 57.3 g / L of glucose and 26.2 g / L of xylose were produced and only 31.1 g / L of glucose was consumed until the fifth day of culture. Were rarely used. However, the production of oil was twice as high as that of 5% PEFB (3.4 g / L, 0.68 g / L day).

Effect of Microalgae Inoculation on PEFB Simultaneous Saccharification Fermentation

In this example, 10% PEFB injection simultaneous saccharification culture experiments were carried out while varying microalgae inoculations at 2%, 5%, and 10%. As shown in FIG. 4A, the PEFB saccharification solution was used in proportion to the inoculum amount. In particular, in the simultaneous saccharification culture using 10% of the inoculum amount, glucose was consumed in the PEFB saccharification solution on the 7th day of culture and xylose was used . As a result, the yield of oil increased about 4 times in the simultaneous saccharification culture using microalgae strain (12.6 g / L, 1.8 g / L day).

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (1)

A method for producing a bio-oil characterized by using a fiber-based palm oil industrial by-product, which comprises the following steps, as a carbon source:
(a) immersing the fibrous palm oil industrial by-product, which has been crushed to a size of 1 to 2 mm, in an NaOH solution, treating at 121 캜 and 15 psi for 30 minutes to 3 hours, washing with water,
(b) adding 5% (w / v) of the above-mentioned pretreated fibrous palm oil industry by-product to distilled water and saccharifying enzyme, inoculating a microalgae of Thraustochytrid, and performing saccharification and culture simultaneously ; And
(c) obtaining a bio-oil from the cultured Thraustochytrid-based microalgae.

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