KR20130123246A

KR20130123246A - Method for culturing microalge thraustochytrid using palm empty fruit bunch hydrolysate and method for preparing biooil through the same

Info

Publication number: KR20130123246A
Application number: KR1020120046550A
Authority: KR
Inventors: 서정우; 김철호; 유안나; 홍원경
Original assignee: 한국생명공학연구원
Priority date: 2012-05-02
Filing date: 2012-05-02
Publication date: 2013-11-12

Abstract

The present invention relates to a method for producing a bio-oil containing high concentrations of DHA through cultivation of Thraustochytrid microalgae using a saccharified liquid of the fiber-based palm oil industry by-products, more specifically, Trausuto The present invention relates to a method for producing a biooil, which comprises culturing a saccharified solution of a filtrate palm oil industry byproduct in a cultured microalgae of a kitride (Thraustochytrid) microalgae in a medium, and then recovering the generated biooil. .
Bio-oil production method according to the present invention can produce bio-oil from abundant non-food fiber-based biomass through the culture of Thraustochytrid microalgae, biofuel such as food resource insecurity and raw material price increase It can overcome the limitation of development and secure the commercial competitiveness of microbial fermentation oil.

Description

Method for Culturing Microalge Thraustochytrid Using Palm Empty Fruit Bunch Hydrolysate and Method for Preparing Biooil Through the Same}

The present invention relates to a method for producing bio-oil from fibrous biomass using Thraustochytrid microalgae, and more specifically, to the Traustochytrid microalgae is a fiber-based palm oil industry by-product. The present invention relates to a method for preparing microbial oil, wherein the microbial oil is recovered by culturing in a medium containing saccharified solution of the microorganism.

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 oil-soluble plants and microalgae, which are the main sources of biooil raw materials for the manufacture of biodiesel, have a very important advantage of utilizing abundant sunlight and recycling carbon dioxide, but in various environments such as time, space, season and climate. There are disadvantages that are affected by factors, and in some cases, doubts about the effectiveness of biodiesel from photosynthetic oils may arise due to food shortages and new environmental problems caused by mass cultivation of raw crops. The situation is being raised.

Recently, the fermentation culture method of organic nutrition microorganism has attracted attention as a mass production method of bio oil, Chlorella as a representative oil production microorganism protothecoides , Yarrowia lipolytica , Rhodosporidium toruloides , Rhodotorula glutinis . Their fermentation process studies are actively underway. The most important factor to secure commercial competitiveness of microbial fermentation oil as biodiesel raw material is to utilize industrial waste, waste resources, surplus biomass as nutrient source, and ultimately to utilize rich non-edible fibrous biomass ( 1). Examples of non-edible cellulose-based biomass resources include wood-based, agricultural by-products, and municipal waste (Korean Patent Publication 10-2008-097651, US Patent Publication 2009-648483).

On the other hand, the Thraustochytrid-based heterotrophic microalgae are oil-retaining microorganisms capable of producing biooils containing polyunsaturated fatty acids such as DHA (docosahexaenoic acid) up to 70% of the dry cell mass. Korean Patent Publication No. 10-2011-0122424). DHA is an essential fatty acid for the brain, eye tissue and nervous system, and is known to play an important role in the development of vision and motor neuron ability, especially in infants. In addition, the amount of dementia has been reported to be significantly reduced in the brain, and various anti-aging functions such as suppressing macular degeneration of presbyopia are newly revealed. Despite these useful physiological functions, the human body is unable to synthesize sufficient amounts of DHA on its own and is recognized as an essential nutrient that must be supplied from the outside world. It is recommended. Therefore, DHA has been commercialized as a variety of products, such as health functional food, and the commercial value of DHA is very high because it is likely to be used as a raw material for pharmaceuticals. Thus, fermented oil of Thraustochytrid microalgae containing high concentrations of DHA connects the biodiesel industry with high value-added industries utilizing DHA, so that the market competitiveness of biodiesel is different from general microbial fermented or photosynthetic oils. It is expected to be able to provide.

The present inventors earnestly endeavored to develop a method for producing biooil through cultivation of microalgae using abundant fibrous biomass as a nutrient, and as a result, Thraustochytrid microalgae KRS101 (isolated from soil of mangrove region) When KCTC11686BP) was cultured using the saccharified liquid of the fiber-based palm oil industry by-product as a nutrient source, it was confirmed that the bio-oil containing DHA was produced, thus completing the present invention.

An object of the present invention is to provide a method for culturing microalgae using the saccharified liquid of the fiber-based palm oil industry by-product as a nutrient source.

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

In order to achieve the above object, the present invention (a) after fibrous palm oil industry by-product immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pretreatment ; (b) adding a distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-products and stirring to prepare a fibrous palm oil industry by-product saccharification liquid; And (c) inoculating the microalgae into the fibrous palm oil industry by-product saccharification liquid and culturing the microalgae using the fibrous palm oil industry by-product.

The present invention also comprises the steps of: (a) fibrous palm oil by-products are deposited in an alkaline solution, and then treated at 100-130 ° C. for 30 minutes to 3 hours, followed by washing with water and drying to pretreat; And (b) inoculating the pretreated fibrous palm oil industry by-products with distilled water, saccharase and microalgae, and simultaneously performing the saccharification and cultivation process using the fibrous palm oil industry by-products. to provide.

The present invention also comprises: (a) fibrous palm oil by-products are deposited in an alkaline solution, and then treated at 100-130 ° C. for 30 minutes to 3 hours, followed by washing with water and drying to pretreat; (b) adding a distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-products and stirring to prepare a fibrous palm oil industry by-product saccharification liquid; (c) inoculating the cultured palm oil industry by-product saccharification solution by inoculating Thraustochytrid microalgae; And (d) provides a method for producing a bio-oil, characterized in that using the fibrous-based palm oil industry by-products comprising the step of obtaining a bio-oil from the cultured Traustochytrid microalgae.

The present invention also comprises the steps of: (a) fibrous palm oil by-products are deposited in an alkaline solution, and then treated at 100-130 ° C. for 30 minutes to 3 hours, followed by washing with water and drying to pretreat; (b) inoculating the pretreated fibrous palm oil industry by-products with distilled water, saccharase and Thraustochytrid microalgae, and simultaneously saccharifying and culturing; And (c) provides a method for producing a bio-oil, characterized in that using the fibrous-based palm oil industry by-products comprising the step of obtaining a bio-oil from the cultured Traustochytrid microalgae.

Bio-oil production method according to the present invention can produce bio-oil from abundant non-food-based industrial by-product resources through the cultivation of Thraustochytrid-based microalgae, such as unstable supply of food resources and rising raw material prices It can overcome the limitations of fuel development and secure the commercial competitiveness of microbial fermentation oil.

Figure 1 shows the results of analyzing the composition of the palm oil industry by-product saccharification liquid.
Figure 2 shows the response surface analysis method for the culture conditions of microalgae KRS101 using the fibrous palm oil industry by-product saccharification solution.
Figure 3 shows the results of culturing the microalgal KRS101 strain using the palm oil industry by-product saccharification solution.
Figure 4 shows the results of co-glycosylation of microalgae KRS101 strain using the palm oil industry by-products.
5 is a result of examining the effect of inoculation amount on the co-glycosylation of microalgae KRS101 strain using the palm oil industry by-products.

In view of consistency, the present invention (a) fibrous palm oil industry by-products (PEFB) after immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pretreatment ; (b) adding a distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-products and stirring to prepare a fibrous palm oil industry by-product saccharification liquid; And (c) inoculating microalgae into the fibrous palm oil industry by-product saccharification liquid and culturing the microalgae using the fibrous palm oil industry by-products.

The present invention, after physicochemical pretreatment of fiber-type Palm Fruit Bunch Fiber containing excessive amounts of hemicellulose and lignin, is used for the culture of microalgae. In one embodiment of the present invention, PEFB saccharification solution for culturing Thraustochytrid microalgae KRS101 (KCTC11686BP) isolated from the soil of the mangrove region was prepared as follows.

First, crushed palm oil by-products having a size of about 1 to 2 mm for pretreatment were immersed in 1M NaOH solution for 30 minutes, subjected to autoclave (121 ° C., 15 psi, 1 hour), and then thoroughly washed with water and dried with NaOH. 50 g of pre-treated EFB was added to 500 ml of a mixed solution containing distilled water and enzyme (PEFB added amount of 10% (w / v)) and saccharified for 3 days with stirring at 45 rpm at 150 rpm. In this case, 40 FPU (Filter Paper Assay Unit) was used for 1 g of dry weight of EFB pretreated by the above-described method. As a result of analyzing the components of the PEFB saccharification solution, glucose and xylose were present at 68 g / L and 22 g / L, respectively, and HMF and furfural were not detected.

As a result of culturing the microalgae KRS101 in the fibrous palm oil industry by-product saccharification solution prepared by the above method, the concentration of glucose in PEFB saccharification solution 53.8 g / L, the concentration of yeast extract 6.03 g / L, the concentration of sea salt 20.1 g / L, The initial oil pH 5.7 showed the highest oil content (15.35 g / L) and DHA content (5.28 g / L). After 36 hours of incubation, all of the glucose in the culture was consumed, and then xylose was consumed. In culture, the maximum oil yield was 12.53 g / L (% oil g / g glucose), with a DHA content of 5.39 g / L, 43% of total fatty acids.

In another aspect, the present invention (a) after dipping the fibrous palm oil industry by-products in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pretreat; And (b) inoculating the pretreated fibrous palm oil industry by-products with distilled water, saccharase and microalgae, and simultaneously performing saccharification and culturing. It is about.

In one aspect of the present invention, the glycosylation enzyme and microalgae KRS101 were added together to the pretreated fibrous palm oil industry by-products, and glycosylation and culturing were carried out together. Glucose began to be consumed completely, with the start of the use of xylose (FIG. 3A). The total amount of carbon source produced by 5% PEFB injected through the control experiment without inoculation with microalgae strains was 33.7 g / L and 10.8 g / L, respectively. The maximum amount of oil produced therefrom was 1.7 g / L (0.57 g / L day), and the content of DHA in total fatty acids was 45% or more (FIG. 3C).

Meanwhile, as shown in FIG. 3B, 57.3 g / L of glucose and 26.2 g / L of xylose were produced in the co-glycosylation experiment with 10% PEFB, and only 31.1 g / L of glucose was consumed until the 5th day of culture. Was rarely used, but oil production was twice as high as 10% PEFB (3.4 g / L, 0.68 g / L day) compared to 5% PEFB.

In another aspect, the present invention (a) after the fiber-based palm oil industry by immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pretreat; (b) adding a distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-products and stirring to prepare a fibrous palm oil industry by-product saccharification liquid; (c) inoculating the cultured palm oil industry by-product saccharification solution by inoculating Thraustochytrid microalgae; And (d) obtaining a biooil from the cultured Traustochytrid microalgae. The present invention relates to a method for producing a biooil, characterized in that the fibrous palm oil industry by-product is used as a carbon source. .

In another aspect, the present invention (a) after dipping the fibrous palm oil industry by-products in an alkaline solution, after 30 minutes to 3 hours at 15 ℃, 121 ℃, washed with water and dried to pre-treat; (b) inoculating the pretreated fibrous palm oil industry by-products with distilled water, saccharase and Thraustochytrid microalgae, and simultaneously saccharifying and culturing; And

(C) a method for producing a bio-oil, characterized in that using the fibrous-based palm oil industry by-products comprising the step of obtaining a bio-oil from the cultured Traustochytrid microalgae.

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.

Microalgae through Response Surface Methodology KRS101 Establishing Culture Conditions of Strains

Fiber-based examined the culture conditions of the industrial by-products of palm oil (Palm Empty Fruit Bunch, PEFB) teurawooseu sat lead kit (Thraustochytrid) Using Hydrolyzate as nutrient sources based Aurantiochytrium in heterotrophic microalgae KRS101 strains (KCTC11686BP). The microalgae KRS101 strain was inoculated with a single colony in a medium containing 60g / L of carbon source glucose, Yeast extract 10g / L of nitrogen source, and 6g / L of artificial sea salt, and pre-incubated at 125 rpm at 28 ° C for 3 days to be used as inoculum. The cell growth was analyzed by measuring absorbance (OD, optical density) at 600 nm while incubating at 125 rpm at 28 ° C.

PEFB saccharification solution for culturing microalgal KRS101 strain was prepared as follows. First, crushed palm oil by-products having a size of about 1 to 2 mm for pretreatment were immersed in 1M NaOH solution for 30 minutes, subjected to autoclave (121 ° C., 15 psi, 1 hour), and then thoroughly washed with water and dried with NaOH.

50 g of pre-treated EFB was added to 500 ml of a mixed solution containing distilled water and enzyme (PEFB added amount of 10% (w / v)) and saccharified for 3 days with stirring at 45 rpm at 150 rpm. In this case, 40 FPU (Filter Paper Assay Unit) was used for 1 g of dry weight of EFB pretreated by the above-described method.

As a result of analyzing the components of the PEFB saccharification solution, glucose and xylose were present as 68 g / L, 22 g / L, respectively, HMF and furfural was not detected (Fig. 1).

The oil content in the microalgal KRS101 strain was analyzed using a modified Bligh-Dyer method (Burja et al ., 2007). Chlorroform 6.25mL, methanol 12.5mL, 50mM K ₂ HPO ₄ buffer (pH 7.4) was added to 125mg of dry cell weight, and the mixture was reacted at 200rpm at 28 ℃ for 1 hour, followed by adding Chloroform 6.25mL and K2HPO4 buffer 6.25mL. After mixing, the mixture was left for 30 minutes to separate into an aqueous solvent and an organic solvent layer containing oil. The chloroform layer was carefully transferred to a pre-weighed aluminum dish, dried at 80 ° C. for 30 minutes, and then weighed with oil. 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: Aluminum Plate + Weight of Geology

VC: total volume of chloroform

VP: Volume of Chloroform transferred to aluminum dish

WS: weight of 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 methanol-sulfuric acid solution (96: 4 (v / v)%) and reacted at 90 ° C. for 1 hour to produce a fatty acid ester, followed by extraction with 0.3 mL of nucleic acid, followed by gas chromatography. It was.

As a result of examining the optimum culture conditions of microalgae KRS101 through the reaction surface analysis method (Fig. 2), PEFB saccharified solution glucose concentration 53.8 g / L, Yeast extract concentration 6.03 g / L, sea salt concentration 20.1 The highest oil content (15.35 g / L) and DHA content (5.28 g / L) were shown at g / L and initial culture solution pH 5.7 (Table 1).

Review of microalgal KRS101 culture conditions using PEFB saccharification solution by Response Surface Methodology Std F1 (Saccharification 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

PEFB Saccharification Microalgae using KRS101 Strain Culture and Oil Production

Fermentation culture of microalgal KRS101 strain using PEFB saccharification liquid was carried out as the culture conditions of Example 1. As shown in FIG. 2, after 36 hours of incubation, all of the glucose in the culture medium was consumed, and then xylose was consumed. In culture, the maximum oil yield was 12.53 g / L (% oil g / g glucose), with a DHA content of 5.39 g / L, 43% of total fatty acids.

PEFB of Simultaneous saccharification Oil production by cultivation

As a culture condition of Example 1, co-glycosylation of microalgae KRS101 strain using PEFB was carried out. The glycosylase was 40 FPU (Filter Paper Assay Unit) was used for 1 g of dry weight of EFB pretreated by the above method. First, glucose in the culture solution was completely consumed from the 3rd day of culture in co-glycosylation culture with PEFB 5%, and the use of xylose began to be started (FIG. 3A). The total amount of carbon source produced by 5% PEFB injected through the control experiment without inoculation with microalgae strains was 33.7 g / L and 10.8 g / L, respectively. The maximum yield of oil produced therefrom was 1.7 g / L (0.57 g / L day), and the content of DHA in total fatty acids was 45% or more (FIG. 3C).

Meanwhile, as shown in FIG. 3B, 57.3 g / L of glucose and 26.2 g / L of xylose were produced in the co-glycosylation experiment with 10% PEFB, and only 31.1 g / L of glucose was consumed until the 5th day of culture. Appeared to be rarely used. However, oil production was twice as high as with 10% PEFB compared with 5% PEFB (3.4 g / L, 0.68 g / L day).

PEFB Simultaneous Glycation Fermentation Effect of Microalgae Inoculation

In this example, the microalgae inoculation amount was changed to 2%, 5%, and 10%, and a simultaneous glycosylation experiment with 10% PEFB was performed. As shown in FIG. 4A, the PEFB glycosylated solution was used in proportion to the inoculation amount, and especially in the coglycosylated culture using 10% inoculation, all glucose in the PEFB glycosylated solution was consumed and xylose was used. Appeared to begin to be. Correspondingly, the oil production increased by about four times in the co-glycosylation culture using microalgal strain inoculation (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

Cultivation method of microalgae using fiber based palm oil industry by-products comprising the following steps:
(A) after the fibrous palm oil industry by-products are immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pre-treat;
(b) adding a distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-products and stirring to prepare a fibrous palm oil industry by-product saccharification liquid; And
(c) inoculating the microalgae into the fibrous palm oil industry by-product saccharification solution and culturing.

The method of claim 1, wherein the microalgae are Thraustochytrid microalgae.

Cultivation method of microalgae using fiber based palm oil industry by-products comprising the following steps:
(A) after the fibrous palm oil industry by-products are immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pre-treat; And
(b) inoculating the pretreated fibrous palm oil industry by-products with distilled water, saccharase and microalgae, to simultaneously perform saccharification and culture.

4. The method of claim 3, wherein the microalgae are Thraustochytrid microalgae.

Method for producing bio-oil, characterized in that using the fibrous palm oil industry by-products comprising the following steps as a carbon source:
(A) after the fibrous palm oil industry by-products are immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pre-treat;
(b) adding a distilled water and a saccharifying enzyme to the pretreated fibrous palm oil industry by-products and stirring to prepare a fibrous palm oil industry by-product saccharification liquid;
(c) inoculating the cultured palm oil industry by-product saccharification solution by inoculating Thraustochytrid microalgae; And
(d) obtaining bio-oil in cultured Thraustochytrid microalgae.

Method for producing bio-oil, characterized in that using the fibrous palm oil industry by-products comprising the following steps as a carbon source:
(A) after the fibrous palm oil industry by-products are immersed in an alkaline solution, after 30 minutes to 3 hours at 100 ~ 130 ℃, washed with water and dried to pre-treat;
(b) inoculating the pretreated fibrous palm oil industry by-products with distilled water, saccharase and Thraustochytrid microalgae, and simultaneously saccharifying and culturing; And
(c) obtaining bio-oil from cultured Thraustochytrid microalgae.