JPH0195793A - Production of propylene by microorganism - Google Patents

Abstract

PURPOSE:To facilitate purification of formed propylene and enhance purity of a product, by precultivating and growing a microorganism having the ability to produce propylene under aerobic condition and aerobically and continuously cultivating the microorganism in the presence of L-valine, etc. CONSTITUTION:A microorganism having the ability to produce propylene is precultivated and grown under aerobic condition. The resultant microorganism is then aerobically and continuously cultivated in the presence of L-valine or L-valine and L-isoleucine to form propylene, which is subsequently collected. Microorganisms belonging to the genus Penicillium, Peacilomyces, Aspergillus or Rhizopus are cited as the microorganism having the ability to produce the propylene. Such microorganisms are preferably cultivated in a culture medium containing <=0.25mM bi- or trivalent iron ions.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an improved method for producing propylene by culturing microorganisms.

[Conventional technology]

Traditionally, propylene has been produced from petroleum cracking gas and natural gas. However, there are natural limits to these reserves on earth.

Regarding the production of propylene by microorganisms, pine mushrooms produce ethane, ethylene, fluoropane, fluoropylene, n-
It was reported that a small amount of butane was produced (E, M, Turner
,M,Wright,T,Ward,andD,J,.
0sborne, J., Gen0M1crobio1.
．． 91, 167-176 (1975)) and anaerobic methane fermentation with mixed bacteria (strains not isolated or identified) in cow dung, and Penicillium digitatum AT.
In the agar plate culture of CCN110080, a small amount of ethane,
Reports that ethylene, propane, and propylene were detected (
J, B, I) avisand R, M, 5quires
, 5science, 119, 881-882 (1954
)). However, in all of these reports, the amount of propylene produced was small, and they were either anaerobic or solid surface cultures.
The microorganisms involved are unknown or the chemical structure of the hydrocarbons produced is unclear.

The applications of the present inventors M regarding "method for producing hydrocarbons having 3 and/or 4 carbon atoms" and "method for producing hydrocarbon mixture" have been published (JP-A-60-84187, JP-A-60-84187,
1-925297), but the types of propylene-producing bacteria and the types of amino acids that should be included in the culture medium are different, and there are also large differences in the amount of propylene produced.

[Problem that the invention seeks to solve]

In all of the above-mentioned conventional reports, the amount of propylene produced is small, and anaerobic and solid surface culture are not necessarily suitable for industrial mass production. Furthermore, since the type of microorganism is not clear, reproducibility is also poor.

Further, in the above-mentioned application by the present inventors, the type of microorganism used, the type of amino acid to be included in the medium, and the culture method are different, and the amount of propylene produced is not necessarily sufficient.

[Means and effects for solving the problem] As a result of various studies conducted by the present inventors on an efficient method for producing propylene using microorganisms with sufficient reproducibility, the present inventors have found that:
Microorganisms that can produce propylene under aerobic culture conditions, such as filamentous fungi belonging to several genera, are grown aerobically and then aerobically cultured in the presence of specific amino acids. It has been found that a surprising amount of propylene is produced.

The present invention is based on this knowledge, and in a method for producing propylene by aerobically culturing a microorganism capable of producing propylene, the microorganism is previously cultured and grown under aerobic conditions, and then L- This is a method for producing propylene using a microorganism, which is characterized by continuously culturing aerobically in the presence of valine, or L-valine and L-isoleucine, and collecting the produced propylene.

In the present invention, microorganisms that can produce propylene by culturing under aerobic conditions are used. Preferred microorganisms are Penicillium, paeci lomyces, Aspergillus (
Aspergillus) or Rhizolapus (l(
These are filamentous fungi belonging to genera such as P. hizopus and their mutant strains. Among these microorganisms, representative strains that produce a significantly large amount of propylene are listed below.

Penicillium Cyclopium pal echinula) Ram (penicillium Cyclopium va)
IFO-7758, Penicillum expansum
expansum) IFQ-7100, Penicillium
Lividum (Penicillium ljvidum)
IFO-6102, Pecilomyces fumoso Oceus (
paecilomyces fumosoroseu
s) IFO-7072, Pecilomyces carneus (
paeci lomyces carneus) IFO
-8292, Pen 0 Fuises Nilegafus (paec
i lomyceselegans) IFO-7060
, Aspergillus clavaras (, Aspergill
us clavatus) IFO-8606, Rhizopus japonicus (Rhizopus japonicu)
s) IFO-4758 etc.

The medium for culturing these microorganisms may contain carbon sources, nitrogen sources, inorganic salts, and other nutrients.
For the microorganisms exemplified above, a normal mold culture medium can be used.

As carbon sources, carbohydrates such as glucose, sucrose, maltose, starch, xylose, and sorbitol, alcohols such as glycerin and ethanol, organic acids such as acetic acid and fatty acids, and crude raw materials containing these are used. In particular, renewable biomass produced naturally and artificially, such as agricultural, forestry, fisheries,
Waste food sources and waste generated from livestock farming, factory wastewater discharged from various manufacturing plants, industrial waste, sludge by-products from productive treatment such as public sewage and various industrial wastewater, or Human waste and the like are used as main raw materials useful for this invention.

These main ingredients vary depending on the various bacterial strains used, but
If necessary, pretreatment of dissolution or decomposition may be performed in advance.

As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, etc. are preferable. Note that when biomass as described above is used as the main raw material, it may not be necessary to add these nitrogen sources.

Inorganic salts are common, such as phosphates, potassium salts, magnesium salts, sodium salts, calcium salts, etc., and may not be necessary in the case of biomass.

Vitamins, amino acids, and yeast extracts, peptones, meat extracts, cornstarch liquor, etc. containing these may contribute to promoting the growth of this strain or producing propylene.

In particular, in the method of the present invention, it is preferable to use a medium containing 0.25 mM or more of divalent or trivalent iron ions and 0.08 to 0.48 μM of divalent copper ions. The details will be explained in Examples below.

In culturing propylene-producing microorganisms, the microorganisms grow (proliferate) at the above-mentioned stage, and then if the culture is continued, propylene is produced.

In the present invention, continuous culturing is particularly carried out in the presence of L-valine, or L-valine and L-inleucine. If these amino acids are added to the medium during the growth stage of microorganisms, they may be consumed by the composition of the microbial cells and may not have any effect. It is preferable to carry out the addition.

The preferred amount of L-valine or L-valine and L-inleucine added is 0.1 to 3 da/e to the medium, respectively.
, preferably 1 to 21/l. However, it goes without saying that if the microorganism has the property of being able to self-supply these amino acids in excess of the amount necessary for its cell structure, there is no need to externally add these amino acids to the culture medium.

Cultivation is carried out under aerobic conditions, such as aerated agitation culture, or
Perform static culture. Preferably, 1)H of the medium is 3 to 8
The culture temperature is controlled at 20-35°C, and the best conditions are set for each strain.

The amount of each hydrocarbon in the produced biogas can be measured as follows. The test solution x = 1 to 5 ml during or at the end of the culture is sterilized in advance, and the whole volume is v = i.
The sample was collected in a 50ml test tube, sealed with a sterile rubber cap, and shaken back and forth at 20-35°C for t-1 to 24 hours. Since the respiration rate differs depending on the strain used, conditions must be set so that oxygen is not depleted during shaking, i.e., V, x, t.
It is preferable to change the level as necessary.

After reciprocating shaking, remove V = 0.1 to 2 yttl of gas from the space above the test tube with a gas syringe, and
ID method (column temperature may vary depending on the type of column packing material)
Change to optimal temperature of ~120°C. The injection temperature also varies depending on the type of filler. ) and then subjected to conventional gas chromatography using nitrogen gas as a carrier gas. In addition,
Preferred examples of column packing materials include porapak Q,
X-28, Bond-(rC/PIC.

Activated Alumina, etc., and should be selected and used as appropriate depending on the type of hydrocarbon to be measured.

Separately, use standard substances for various hydrocarbons, perform gas chromatography under the same conditions and in the same manner as above, and measure the residence time of each hydrocarbon on the recording paper. In addition, a calibration curve is determined for each hydrocarbon using standard substances for each type of hydrocarbon.

Regarding the gas chromatography of the above-mentioned gas to be detected, the residence time of each peak portion on the recording paper is measured and compared with that of the above-mentioned standard gas to determine the type of the corresponding hydrocarbon. Next, the area of the corresponding portion of each hydrocarbon is measured, and the amount Einl of each hydrocarbon is determined using the calibration curve for the standard gas.

The production rate Pi''/ml・hr of each hydrocarbon in the test gas can be calculated using the following formula. This shows that it varies depending on the type of

In order to separate and collect the target propylene from the generated biogas, the generated biogas can be adsorbed directly onto a suitable adsorbent such as zeolite or activated carbon, separated from impure gases, and then desorbed. It is also possible to adsorb and desorb onto the above-mentioned adsorbent after contacting with a caustic soda solution to remove by-product carbon dioxide gas. As zeolites, molecular sieves 8A, 4A, 5
A, and IOX [Union Showa (manufactured by Candle)], Zeorum A
-3, A-4, A-5, and F-9 (manufactured by Toyo Tsudani Gyogyo), etc. are used. Furthermore, as the activated carbon, molecular sieving carbon (manufactured by Takeda Pharmaceutical Co., Ltd. (Kasei)) is used.

〔Effect of the invention〕

A feature of the present invention is that the main raw material used is biomass that is easily available and reproducible, especially waste resources and waste generated from agriculture, forestry, fisheries, livestock, etc., or from various manufacturing plants. Factory wastewater discharged from the factory, industrial waste, or sludge or human waste produced by-products from the productive treatment of public sewage and various factory wastewater can be advantageously used, and the method of the present invention can be carried out. By doing so, it can be said that this corresponds to a kind of microbiological waste liquid treatment or waste treatment of the biomass used as the main raw material. Furthermore, compared to current production methods for crude oil and natural gas, the main raw material is reproducible biomass, so there is no risk of depletion, and since the reaction uses the action of microorganisms, it requires relatively low temperatures and low pressure. The process of the present invention can be produced under suitable conditions, most of the impurity gas produced by the method of the present invention is carbon dioxide, and therefore the target propylene can be easily purified and the product has a high purity. Features include:

Hereinafter, the present invention will be explained in more detail with reference to Examples.

[Example 1] In an 800 ml Erlenmeyer flask, 30% of the synthetic medium (Tazoshi carboxy methyl cellulose (CMC)) shown in Table 1 was added.
Add 100 ml of 'j/l culture solution), autoclave sterilize at 120°C for 15 minutes, cool,
Two platinum loops of each type of slant-cultured bacterial strain were inoculated and incubated at 25°C for 3 days in a rotary shaking culture machine (rotation radius 7).
cm, 180 rpm).

Add the synthetic medium (C
After sterilizing and cooling as above, 5 ml of the above preculture solution was transplanted, and the main culture was carried out in the above rotary shaking culture machine at 25°C for 52 hours as usual. .

One volume of the culture solution obtained in this way was 84 ml (18
wl〆) into sterilized test tubes, and add 1 part of sterile water.
ml 1 or L-valine (L-vag containing liquid 1
ml (concentration of 11/l in terms of the original culture solution (7
) L-Val solution) or 1 ml of a solution containing L-Va# and L-isoleucine (L-Ice) (solutions with a concentration of 1y/4 each in terms of the original culture solution) were dispensed. Immediately, the tube was sealed tightly and cultured in a sealed state using a reciprocating shaker (IO times/min, amplitude 3.5a) at 25°C for 15 hours to generate biogas. Note that, as mentioned above in the main text, the respiration rate differs depending on the strain used, so it is important to set conditions so that oxygen is not deficient during the seal culture period.

After the seal culture is completed, one portion of the generated biogas is extracted from the gas phase above the test tube using a gas syringe, and subjected to gas chromatography using the method described in the text.
The production rate of the target propylene was calculated.

The results are shown in Table 2. L-Va#, or/
And when TJ-Van and L-Iffe are not added,
Although neither strain produces propylene, the addition of these amino acids produces propylene, especially L
Simultaneous addition of -Val and L-11e produced significant amounts of propylene. P, cyclopium IFO7
Regarding 758, an experiment in which L-Ile was added alone was added and the organized results are shown in Table 3. L-van
The simultaneous addition of both L-Ice and L-Ice clearly shows a synergistic effect.

Although the details are unknown at present, it is thought that L-Va# acts as a precursor and L-angle e acts as a promoter for the production of propylene.

Margin note 1 below. Number of bacteria used: p, cyclopium IFO7
758 Note 2. Synthetic medium used, Tazoshi, L in the above table
The amount of V a lNL -Ile added was 1 g/
4 Culture solution.

Note 3. As shown in the synthetic medium composition in Table 1, the Fe3+ concentration was 0.05mM, and the Cu concentration was 0.08μM0 [Example 2] Synthetic medium 1 shown in Table 1 was added to a 300 ml Erlenmeyer flask.
00m/, preculture, main culture, and
Seal culture was performed. Tazoshi, P.

cyclopium IFO7758 was used. In addition, Fe2(SO4)a in the synthetic medium components listed in Table 1,
Instead of 0.0111/l, the iron salt listed in Table 4 was used, and the amount of CuSO4.
μM).

The results are shown in Table 4. From this result, the concentration of iron ions contained in the medium is preferably 0.25 mM or more.

Table 4 Effect of iron ion concentration Note 29 Use of synthetic medium, Tazo, Fe2 (80
4) 8 as shown in the table above, and the OL+ concentration as 0.
The concentration was changed to 12 μM.

Note I L-VaA and L-Ice at the same time 1 f
//1 culture solution was added.

[Example 3] Synthetic medium 1 shown in Table 1 was placed in an 800 ml Erlenmeyer flask.
00 ml was prepared, and preculture, main culture, and seal culture were performed in the same manner as in Example 1. Tazoshi, P.

cyclopium IFO7758 was used. In addition, CuSO4・5H20 in the synthetic medium components listed in Table 1
2ml/1 inorganic salt solution (0.02~/l culture solution, Cu
(0.08 μM) was changed to the copper concentration listed in Table 5, and the amount of Fe2(SO4)s was changed to 0.08 μM).
．． 01g/l to 0.25fl/Ic Fe3+ in medium
The concentration was changed to 1.25mM).

The results are shown in Table 5. The concentration of copper ions contained in the medium is preferably 0.08 to 0.48 μM.

Table 5 Effect of copper ions Note 1. Strain used: p, cyclopium IFO77
58Note 20 Use of synthetic medium, Tazo, CuSO in Table 1
The concentration of Fe2(804)8 was changed to 1.25 mM as shown in the table above (calculated per 14 culture fluids).

Note 8. 1g of L-Fe6 and L-Ile at the same time
/4 culture solution was added.

[Example 4] Residue after squeezing Satsuma mandarin oranges to extract juice (moisture near 7%, solid content = 23%, total sugar: 19.4%) 40
Add about 500 vrl of water to 0 g, homogenize with a mixer, add 1 g of sodium nitrate, adjust the liquid volume to 11, and place in a 2.61 mini jar fermenter.
Sterilize in an autoclave at 0°C for 120 minutes, cool, and culture with shaking in the same medium.
um IFO775B preculture solution 50M/transplanted,
0. I Aerated sterile air in VVM and stirred at 40 rpm.
Orpm and cultured at a culture temperature of 25°C for 10 days. In addition, the L-Val content in the medium is approximately 0.3 (//IIX
The L-Ile content was approximately 0.15 g/l.

Throughout this entire cultivation period, the exhaust gas is sequentially introduced into a 10% caustic soda tank, a water washing tank, and a water separation tank to remove impurity gases.
Next, Zeorum A-3 [manufactured by Toyo Tsudani Gyo (+411)]
The impurity gas was further adsorbed and removed, and the passing gas was introduced into a Zeorum A-4 (Toyo Tsudani Industry (Interior)) filling tube, and the adsorbed propylene was vacuum-suctioned and desorbed and recovered.

The propylene obtained was about 1.7~.

Claims (1)

[Claims] 1. In a method for producing propylene by aerobically cultivating a microorganism capable of producing propylene, the microorganism is previously cultured and grown under aerobic conditions, and then L-valine or A method for producing propylene using a microorganism, which comprises continuously culturing aerobically in the presence of L-valine and L-isoleucine and collecting the produced propylene. 2. The production method according to claim 1, wherein the microorganism belongs to the genus Penicillium, Pecilomyces, Aspergillus, or Rhizopus. 3. The production method according to claim 1, wherein the microorganism is cultured in a medium containing a trace amount of divalent or trivalent iron ions, preferably 0.25 mM or more. 4 Microorganisms in trace amounts, preferably 0.08-0.48μ
2. The production method according to claim 1, which comprises culturing in a medium containing divalent copper ions of M.