JP3816774B2 - Hydrocarbon-utilized microalgae and bioremediation method using the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The invention of this application relates to a microalgae capable of assimilating hydrocarbons and a method for purifying wastewater using the same. More specifically, the invention of this application relates to a microalgae capable of assimilating hydrocarbons in a wide salt concentration range including a high temperature and a high salt concentration, and further in a wide pH range, and a bioremediation method using the same. .
[0002]
[Prior art and its problems]
Marine pollution, which has become a major social problem in recent years, is known to be caused by harmful chemical substances, oils, radioactive substances, waste dumping, eutrophication products, and the like.
[0003]
In particular, contamination by oils is a problem not only in crude oil production areas but also in the vicinity of factories that use or produce oils, such as food processing plants. In addition, if a tanker accident occurs when transporting crude oil from an oil field, water pollution will occur over a wide area, causing serious damage to the surrounding environment such as seabirds, mammals and seafood. For example, seabirds and marine mammals have a hydrophobic substance on their feathers and skin surface, and maintain body temperature by preventing direct contact with seawater. Loss of body temperature decreases and it cannot survive. In addition, these animals replenish water while discharging excess salt from seawater, but this function is lost due to the adhesion of oil, causing dehydration. In addition, seaweed and seafood are killed by the loss of photosynthesis and respiration due to oil adhering to the surface.
[0004]
Conventionally, crude oil and petroleum that have flowed into water bodies temporarily due to accidents in tankers and oil fields are prevented from spreading by oil fences, and oil is sprayed with an oil treatment agent consisting of surfactants and organic solvents. And mixed with. However, in the method using an oil treatment agent, the spilled oil becomes small oil droplets that can be mixed with water and eventually diluted in the ocean to reduce the scale of the damage, but this is not a fundamental solution. . Moreover, it was not applicable to the treatment of oils contained in a small amount in industrial wastewater.
[0005]
Therefore, a method of decomposing oil by light or heat and a method such as physicochemical adsorption using activated carbon or chemicals have been proposed and used in part. However, these methods use light, heat, or a large amount of reagents, and require mechanical equipment for purification. Therefore, these methods are expensive and difficult to apply to oil treatment in the ocean such as a tanker accident. There were problems such as being unable to cope with widespread pollution.
[0006]
Therefore, in recent years, bioremediation has attracted attention as a method with high environmental adaptability. Bioremediation is a method of emulsifying, decomposing, and assimilating hydrocarbons such as oils and eutrophications in water areas and soils with significant environmental pollution using various microorganisms that have a purification function. Requires little energy to do in Therefore, the cost is low, the treatment can be performed directly at the site of the contamination, the purification treatment can be performed even during the operation of the oil field or the factory, the contamination over a wide range can be purified, etc. Has the advantage of
[0007]
Various microorganisms are known as microorganisms used in such bioremediation. For example, oil-degrading bacteria belonging to the genus Agrobacterium and Rodococcas have been reported to exhibit high hydrocarbon degradability (Biosci. Biotechnol. Biochem., 59, 11, 2159-2161 (1995)). In addition, petroleum degrading yeast (Candida maltosa) has been reported to exhibit high activity even in a low pH range (Wolf, K., ed, Non-conventional Yeast in Biotechnology, pp.426-430 (1996)). . However, these bacteria have a low resolution at a low pH, and yeast has a problem that its activity is significantly reduced at a high temperature of 30 ° C. or higher and at a pH of 6.5 or higher, and therefore cannot be used in a high temperature region.
[0008]
Furthermore, it is reported that Prototheca genus, which is a eukaryotic microorganism showing a yeast-like form, especially green algae such as Prototheca hydrocarbonea and Prototheca zopfii, has oil degradability, but the conditions for their growth and decomposition are pH 3 to 3 9. Sodium chloride concentration of 0.8 to 2.4% by weight, 25 ° C. or less, showing activity only in a limited environment. In particular, at temperatures exceeding 30 ° C., it was impossible to grow and decompose oil components (Appl. Environ. Microb., P.333-336 (1983); System.Appl.Microbiol.5, 119-123). (1984)). Generally, since the salt concentration of seawater is about 3.5% by weight, the conventionally reported algae of the genus Prototheca cannot be used for decomposition and purification of oil components from seawater. Moreover, since the range of the temperature and salt concentration which can be used is limited with respect to soil contamination, it is the fact that it was not able to be used in a coastal area or an area with high temperature.
[0009]
Therefore, the invention of this application solves the problems as described above, and a bioremediation method that can be carried out in soils, rivers, lakes, oceans, etc. in a wide range of pH, temperature, and salt concentration, and microorganisms to be applied thereto It is an object of the present invention to provide a microorganism that can assimilate hydrocarbons even in a wide salt concentration range including a high temperature and a high salt concentration, or in a wide pH range.
[0010]
[Means for Solving the Problems]
In order to solve the problems as described above, the invention of this application is firstly a microalgae that assimilate hydrocarbons, and can assimilate hydrocarbons in a temperature range of 30 to 40 ° C. A microalgae characterized by the above is provided.
[0011]
The invention of this application is, secondly, the above-mentioned microalgae that can assimilate hydrocarbons in the salt concentration range of 0-2M, and thirdly, any of the above that can assimilate hydrocarbons in the range of pH 3-9 Fourthly, any one of the above-mentioned microalgae having biosurfactant ability is provided.
[0012]
The invention of this application is that, in the fifth aspect, the microalga is a microalga belonging to the genus Prototheca, and in the sixth, the microalga belonging to the genus Prototheca is a Prototheca zopfii RND-16 (Ferm P-18543) is provided as an embodiment thereof.
[0013]
Furthermore, seventhly, the invention of this application provides a bioremediation method characterized by using at least any one of the above-mentioned microalgae.
[0014]
An eighth aspect of the invention of the present application is a method for purifying a wastewater containing hydrocarbons, comprising at least a step of bringing any one of the microalgae into contact with the wastewater. Ninth, a method for purifying soil contaminated with hydrocarbons, comprising at least a step of bringing any of the above-mentioned microalgae into contact with the contaminated soil. provide.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
The inventors of this application have conducted intensive research on carbon hydrogen in a wide range of pH and salt concentrations in 12 strains of yeast-like microorganisms isolated from 40 ° C and 35.5 ° C drainage of Izu Rendaiji hot spring in Shizuoka Prefecture. The present inventors have found microalgae that assimilate the water and have arrived at the present invention. Specifically, the microalgae of the present invention can assimilate hydrocarbons in a temperature range of 30 to 40 ° C., a pH range of 3 to 9, and a salt concentration range of 0 to 2M.
[0016]
In addition, as will be apparent from the examples described later, when the microalgae of the present invention assimilate hydrocarbons, the microalgae secrete surfactant components, emulsify hydrocarbon components such as petroleum, and form oil droplets. After forming, it enters the oil droplets and proliferates. That is, the microalgae of the present invention has a biosurfactant ability.
[0017]
The present inventors further studied this microalgae, and revealed that the microalgae belong to Prototheca zopfii var. Hydrocaronea from phylogenetic analysis based on the 18S rDNA base sequence. It is also clear that this microalgae forms a single lineage with Prototheca zopfii with a 100% probability and is more closely related to the autotrophic chlorella Auxenochlorella protothecoides than the same heterotrophic Prototheca genus Prototheca wickerhamii Has been.
[0018]
Furthermore, the microalgae of the invention of this application has the following characteristics not found in Prototheca zopfii that has been conventionally reported.
[0019]
(A) assimilate hydrocarbons into ethanol (b) grow in a general medium at a high temperature range of 40 ° C or higher (c) medium with hydrocarbons as a carbon source in a high temperature range up to 40 ° C (D) The maximum assimilation rate of hydrocarbons in stationary culture is more than twice that of conventionally known strains. The inventors of the present application have made a patent deposit on microalgae having the above characteristics. (Accession number: FERM P-18543).
[0020]
Therefore, if the microalgae of the invention of this application is brought into contact with wastewater or soil containing hydrocarbon components such as the ocean, soil, and oil from which crude oil has flowed out, the hydrocarbon components are quickly and efficiently decomposed and purified. Can be processed. In the method for purifying drainage and contaminated soil of the invention of this application, the method for contacting microalgae with drainage or soil is not particularly limited, and the method for spraying the microalgae to the region or water area to be purified, or Various methods such as a method of introducing into a septic tank containing microalgae can be applied. Of course, these purification methods may have various steps such as washing, sedimentation, filtration, stirring, and culturing in addition to the step of bringing wastewater or soil into contact with microalgae.
[0021]
The bioremediation method using the microalgae of the invention of this application has wider purification conditions such as temperature, salt concentration, pH and the like than the conventional method using microorganisms, so the ocean, high temperature region, low pH soil, coastal region, etc. Applicable in any environment.
[0022]
In addition, it has become clear that the microalgae of the invention of this application not only assimilate hydrocarbons but also perform anaerobic metabolism that generates gas from glucose. Such sugar fermentation (ethanol fermentation) accompanied by the generation of gas is a property that has not been known in conventional Prototheca algae.
[0023]
Hereinafter, the present invention will be described in more detail with reference to examples. Of course, it goes without saying that the invention of this application is not limited to the following examples.
[0024]
【Example】
<Example 1>
The microalgae Prototheca zopfii RND-16 strain (FERM P-18543) isolated from the 40 ° C and 35.5 ° C drainage of Izu Renkoji hot spring in Shizuoka Prefecture is 4% (v / v) n-hexadecane (C ₁₆ : Jun Wako) It was added to YNB medium (manufactured by Yakuhin, # 080-03685) (Difco, # 291940) and cultured at 25, 30 and 35 ° C. for 20 days.
[0025]
From the start of culture, the number of microalgae was counted with a toma hemocytometer.
[0026]
FIG. 1 shows a growth curve at each temperature.
[0027]
From this, it was confirmed that the microalgae of the present application showed good growth even at 35 ° C. in the presence of N-hexadecane, and the rate was clearly higher than 25 ° C.
[0028]
In addition, microalgae actively utilizing and decomposing N-hexadecane were observed with a microscope, and photographs were taken. FIG. 2 shows a microphotograph of the microalgae.
[0029]
From FIG. 2, the microalgae of the present invention secretes a surfactant component, emulsifies n-hexadecane in water to form oil droplets, and then enters the oil droplets to decompose and assimilate n-hexadecane. It was confirmed.
<Example 2>
As in Example 1, the microalgae Prototheca zopfii RND-16 strain (FERM P-18543) was added to tetradecane (C ₁₄ ), pentadecane (C ₁₅ ), hexadecane (C ₁₆ ), and heptadecane (C ₁₇ ), respectively. (1% (v / v)) and cultured at 35 ° C.
[0030]
The concentration of each n-alkane remaining in the culture solution was analyzed by gas chromatography. FIG. 3 (a) shows the result before culturing, and FIG. 3 (b) shows the result after culturing for 10 days.
[0031]
From FIG. 3, it was confirmed that four types of n-alkanes were almost decomposed on the 10th day of culture.
[0032]
From the above test results, it was shown that the microalgae of the invention of this application is a novel microorganism exhibiting completely different properties from the conventionally known Prototheca zopfii. Moreover, it was confirmed that this microalgae can decompose and purify various n-alkanes which are hydrocarbons quickly and efficiently even in a high temperature range.
[0033]
【The invention's effect】
As described in detail above, the invention of this application provides a novel hydrocarbon-utilizing microalgae and a method for purifying hydrocarbon-containing wastewater using the same. This microalgae can decompose and purify hydrocarbon components over a wide range of pH, temperature, and salt concentration, and the ocean and soil from which oil has flowed out due to accidents in oil fields and crude oil tankers, and industrial wastewater containing hydrocarbon components. It is highly useful as a microorganism applicable to bioremediation such as
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a graph showing a growth curve when the microalgae of the present invention is cultured at various temperatures in the presence of n-hexadecane in the examples of the invention of this application.
FIG. 2 is a view showing a photomicrograph showing that the microalgae of the invention of this application assimilate and decompose hexadecane.
FIG. 3 is a diagram showing a gas chromatograph when the microalgae of the present invention is cultured in the presence of various n-alkanes in the examples of the invention of this application. (A: before culture, b: after culture)

Claims (4)

Ru microalgae der assimilates hydrocarbon Prototheca zopfii RND-16 (FERM P -18543). A bioremediation method comprising a step of bringing Prototheca zopfii RND-16 (FERM P-18543) into contact with a hydrocarbon contaminant . The bioremediation method according to claim 2, wherein the contaminant is a wastewater containing hydrocarbons . The bioremediation method according to claim 2, wherein the pollutant is a soil containing hydrocarbons .

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