KR101345772B1 - Method for Producing Mixed ＶＦＡ from Algae - Google Patents

Abstract

The present invention relates to a method of producing volatile organic acids from algae, and more particularly, to a method of producing volatile organic acids such as acetic acid, butyric acid, propionic acid from algae including brown algae, green algae and red algae through aerobic and anaerobic fermentation. It is about.
Method for producing a volatile organic acid from the algae comprises (a) adding aerobic microorganism Vibrio sp. To the seaweed, and aerobic fermentation; And (b) adding anaerobic microorganisms to the aerobic fermented algae and anaerobic fermentation to produce volatile organic acids.
When using the present invention, it is possible to produce volatile organic acids in high yield from seaweeds, and the organic acids produced may be used as final products themselves or converted to mixed alcohols through chemical hydrogenation reactions and used as gasoline replacement liquid fuels. .

Description

Method for Producing Volatile Organic Acids from Seaweeds {Method for Producing Mixed VFA from Algae}

The present invention relates to a method of producing volatile organic acids from algae, and more particularly, to a method of producing volatile organic acids such as acetic acid, butyric acid, propionic acid from algae including brown algae, green algae and red algae through aerobic and anaerobic fermentation. It is about.

Biofuels, converted from biomass, are attracting global attention as a way to replace fossil fuels and solve greenhouse gas problems. Biofuels, such as bioethanol, biodiesel and biogas, include sugars (sugar cane, beets, sweet sorghum, etc.), starch (grains, corn, potatoes, sweet potatoes, cassava, etc.), and wood (grass, grass, and straw). , Agricultural by-products, etc.). Glucose and starch biomass can produce biofuels in a relatively simple biological conversion process. However, since these sugar and starch systems are used as food or livestock feed, high cost is a problem in addition to food problems. On the other hand, wood-based biomass, which does not compete with food, has about 20% of lignin structurally surrounding cellulose, so lignin must be removed in the conversion process, and pretreatment to facilitate enzyme penetration of crystalline cellulose is easy. Process is required. As such, the conventional biomass has a problem of causing high raw material costs or process costs.

On the other hand, compared to terrestrial plants, algae have low lignin content, have a dense structure, are easy to process, have high productivity per unit area, have great potential as an unexplored field compared to terrestrial resources, and have limited land. It also has advantages such as utilizing the open sea and having no problems with rainfall and soil pollution.

Seaweeds are divided into green algae, brown algae, and red algae according to the color of the algae, and have a compositional characteristic as shown in Table 1 below.

Brown algae Red algae Green algae Representative species Seaweed, kelp, maternity Laver Blue, hearing Moisture content 75-90 wt% 70-80 wt% 70-85 wt% mineral 30-50% by weight 25-35 wt% 10-25% by weight carbohydrate
(chief ingredient) 30-50% by weight
(Alginic acid, fucoidan) 30-60 wt%
(Agar, carrageenan) 25-50% by weight
(Cellulose, starch) protein 7-15% by weight 7-15% by weight 10-15 wt% Fat 2-5% by weight 1-5% by weight 1-5% by weight

However, algae have a lower carbohydrate content and higher protein content than biomass on land. Therefore, the application of the technology to convert from seaweed to ethanol, which is a conventional biofuel, is limited. For this reason, techniques have been proposed to produce volatile organic acids by anaerobic digestion, which can comprehensively use organic components other than carbohydrates instead of ethanol (Korea Patent No. 10-1039432).

Volatile organic acids are low molecular organic acids having a relatively low boiling point and include acetic acid (C2; a molecule containing two carbons), propionic acid (C3), butyric acid (C4), and the like. Volatile organic acids can be recovered in the form of salts and used as eco-friendly snow removers, or can be used as mixed alcohols for fuels through chemical reactions using catalysts.

In the Republic of Korea Patent No. 10-1039432, only thermal treatment, physical treatment, and chemical treatment were applied as pretreatment methods. The possibility of biological pretreatment was mentioned, but no specific methods and effects are presented.

Therefore, the present inventors confirmed that when fermented into an aerobic microorganism that effectively decomposes polysaccharides such as alginic acid, agar and cellulose, which are the main components of seaweed, and then fermented back into anaerobic microorganisms, the productivity of volatile organic acids can be significantly improved. The invention was completed.

An object of the present invention is to provide an effective fermentation method for producing volatile organic acids such as acetic acid, butyric acid, propionic acid, etc. from seaweeds.

In order to achieve the above object, the present invention comprises the steps of (a) adding aerobic microorganism Vibrio sp. And (b) adding anaerobic microorganisms to the aerobic fermented algae, and anaerobic fermentation to produce volatile organic acids.

The present invention can improve the economics of organic acid production by providing a method for producing volatile organic acids in high yield from seaweeds. The organic acid thus produced may be used as a final product by itself or may be converted to mixed alcohol through chemical hydrogenation and used as a gasoline substitute liquid fuel.

1 is a graph confirming the production of volatile organic acids according to aerobic fermentation time using a Vibrio sp. According to an embodiment of the present invention.
Figure 2 is a graph confirming the production amount of volatile organic acids according to the inoculum during aerobic fermentation using Vibrio sp. According to an embodiment of the present invention.

In the present invention, polysaccharides such as alginic acid and agar, which are the main components of seaweed, exist in the form of a polymer that is not included in the above-ground plants, and thus, conventional anaerobic microorganisms having microorganisms adapted to decompose cellulose-based biomass or food waste. Taking into account that digestion takes a long time or does not occur in itself, it is expected that the efficiency of the overall process can be improved by introducing an aerobic fermentation process that rapidly decomposes alginic acid in algae into specific aerobic microorganisms. It was.

In one embodiment of the present invention, when the aerobic fermentation with aerobic microorganism Vibrio harveyi before the anaerobic fermentation of seaweed, it was confirmed that the volatile organic acid can be produced in a higher yield.

Accordingly, the present invention in one aspect, (a) adding aerobic microorganism Vibrio sp. To the seaweed, and aerobic fermentation; And (b) adding anaerobic microorganisms to the aerobic fermented algae and anaerobic fermentation to produce volatile organic acids.

Algae used in the present invention is to not be that kind it is limited green algae, brown algae, can be used with all kinds of algae containing red algae, preferably seaweed in (Laminaria), gambling (Pachymeniopis elliptica), room palette (Enteromorpha crinite) may be used wakame (Undaria pinnatifida), Sargassum (Sargassum fulvellum), kkosiraegi (Gracilaria verrucosa), fusiformis (Hizikia fusiforme) or agar (Gelidium amansii).

In the present invention, the Vibrio sp. Is not particularly limited, but Vibrio harveyi is most preferably used.

Aerobic fermentation using the Vibrio species (Vibrio sp.) Is characterized by inoculation of Vibrio species × 10 ⁷ 3.0 per seaweed dry weight of ^{1.0 g ~ 4.0 × 10 7 CFU} (Vibrio sp.) , And 30 to 54 hours of incubation do.

When the inoculation amount and incubation time of the Vibrio sp. Is out of the range, there is a problem that the organic acid production is lowered.

When the aerobic fermentation of algae by Vibrio harveyi is completed, anaerobic fermentation is performed with anaerobic microorganisms, where volatile organic acids are produced.

Anaerobic fermentation refers to the process of decomposing organic matter that can be decomposed in an oxygen-free state with little oxygen, and is finally converted to methane through hydrolysis and acid fermentation. Anaerobic fermentation is currently being applied to recover methane as energy at the same time as wastewater or waste treatment.

In the present invention, the anaerobic microorganism is not limited to a specific microorganism, and any anaerobic microorganism capable of producing an organic acid under anaerobic conditions may be used. Under anaerobic conditions, bacteria that hydrolyze organic matter and produce organic acids include Clostridium sp ., Acetobacterium woodii , Selenomonas rumintium , and Propionibacterium ara. Propionibacterium arabinosum , Butyribacterium methylotrophicum , Lactobacillus amylophilus and the like.

The anaerobic microorganisms can be separated from a high organic matter decomposition activity such as organs or manure of herbivores such as cattle and chlorine, acid fermentation tanks for methane production, methane fermentation tanks, and food waste anaerobic digestion tanks.

In the anaerobic fermentation using the anaerobic microorganism, it is preferable to add an inorganic salt which plays an important role in the control of the pH, osmotic pressure of the culture medium, and examples of the inorganic salts include ammonium salt, calcium salt, magnesium salt, sodium salt and the like. have.

By anaerobic fermentation, organic acids such as acetic acid, propionic acid, butyric acid are produced, and the distribution of the generated organic acids may vary depending on conditions such as pH, temperature, and the type of seaweed used.

In general, anaerobic fermentation is known to produce organic acids and carbon dioxide and hydrogen together as by-products. In the case of generated hydrogen, impurities can be removed with an adsorbent such as activated carbon or zeolite or separated with high purity using an appropriate separator, and the separated hydrogen can be used in a hydrogenation reaction described later. However, the methane production process in which the organic acid is decomposed into methane and water by the methane-producing bacteria after the production of the organic acid may proceed, but it is preferable to suppress the further methane production process from the organic acid to methane because the present invention is to obtain an organic acid. The methane production process can be suppressed by adding a methane fermentation inhibitor to the medium composition, the methane fermentation inhibitor may be used, such as iodoform (bromoform), bromoethanesulfonic acid (bromoethanesulfonic acid) have.

[Example]

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.

Comparative Example 1 Production of Organic Acid by Anaerobic Fermentation

In order to confirm the degree of organic acid generation by anaerobic fermentation of each seaweed, the experiment was carried out as follows. Seaweeds dried in air (kelp, gambling, green seaweed) were ground to a size of about 1 mm. Into a 250mL Erlenmeyer flask, 1.0g (dry weight) of crushed seaweed was added, 10mL of an inorganic salt solution having the composition shown in Table 2 was added, and the final volume was made to 100mL with distilled water, followed by calcium carbonate (CaCO ₃ ) 1.0 g was added. Finally, the oxygen in the solution was removed by aeration with nitrogen gas for 10 minutes.

After adding 10 mL of anaerobic digester sludge supernatant from Seongnam wastewater treatment plant, the total amount of organic acid including acetic acid, acetic acid, propionic acid, butyric acid, etc., produced by incubating for 5 days at 35 ° C. in a stirred incubator adjusted to 100 rpm was measured. It was measured by HPLC using HPX-87H column.

The microorganism included as a dominant species in the sludge supernatant of the anaerobic digester used was Clostridium thermoaceticum , Clostridium formicoaceticum , Clostridium thermoautotropium ), Acetobacterium woodii and Selenomonas rumintium . The results of measuring the amount of organic acid produced are shown in Table 3 below.

ingredient Concentration (g / L) ingredient Concentration (g / L) NH ₄ HCO ₃ 20 Na ₂ MoO ₄ .2H ₂ O 0.1 KH ₂ PO ₄ 10 CaCl ₂ .2H ₂ O 0.1 MgSO ₄ .7H ₂ O One MnSO ₄ .7H ₂ O 0.15 NaCl 0.1 FeCl ₂ 0.028 Iodoform 0.1

1 day (g / L) 3 days (g / L) 5 days (g / L) Acetic acid Total Organic Acid Acetic acid Total Organic Acid Acetic acid Total Organic Acid Kelp 0.70 1.29 1.12 1.81 1.50 2.20 gambling 0.32 0.43 0.75 1.14 1.00 1.43 Blue 0.70 0.90 1.05 1.27 1.23 1.33

Example 1 Organic Acid Production by Aerobic Fermentation and Anaerobic Fermentation

First, Vibrio harveyi (ATCC 14126) was incubated at 25 ° C. for 48 hours in aerobic conditions. The composition of the culture was as follows: Bactotryptone 5g / L, yeast extract 2.5g / L, glycerol phosphate 23.5g / L, NH ₄ Cl 0.3g / L, MgSO ₄ 7H ₂ O 0.3 g / L, FeCl ₃ 0.01g / L, CaCO ₃ 1.0g / L, KH ₂ PO ₄ 3.0g / L, NaCl 30g / L.

The dried seaweeds (Kashima, gambling, green seaweed) dried in air are crushed to a size of about 1 mm, and 1.0 g (dry weight) of crushed seaweeds and 100 mL of distilled water are added to a 250 mL Erlenmeyer flask, and about 3.3 × 10 ⁷ CFU Vibrio harveyi (ATCC 14126) was inoculated and fermented under aerobic conditions for 48 hours.

After aerobic fermentation, anaerobic digestion sludge generated in Seongnam sewage treatment plant was inoculated at 10% by volume with respect to aerobic fermentation broth, and anaerobic fermentation was carried out in the same manner as in Comparative Example 1. The organic acid concentration of the culture medium was measured by HPLC using a Biorad Aminex HPX-87H column according to the incubation time, and the results are shown in Table 4.

1 day (g / L) 3 days (g / L) 5 days (g / L) Acetic acid Total Organic Acid Acetic acid Total Organic Acid Acetic acid Total Organic Acid Kelp 3.50 4.08 3.70 3.39 3.18 4.20 gambling 1.53 1.89 2.23 3.04 2.00 2.20 Blue 1.42 1.76 1.24 1.92 1.17 1.84

From Table 4, it was confirmed that when aerobic fermentation was first performed before anaerobic fermentation of seaweeds, the productivity of organic acids increased by about two times depending on the sample.

Example 2: Screening of Aerobic Fermentation Strains

Vibrio harveyi, instead of (ATCC 14126), except that using a Vibrio alginolyticus (ATCC 17749) was fermented seaweed in the same manner as in Example 1.

Vibrio alginolyticus (ATCC 17749) contains Peptone, 5.0g / L, yeast extract 1.0g / L, ferric citrate 0.1g / L, NaCl 19.45g / L, MgCl ₂ 8.8g / L, Na ₂ SO ₃ before aerobic fermentation of seaweed. 3.24 g / L, CaCl ₂ · 2H ₂ O 2.38 g / L, KCl 0.55 g / L, NaHCO ₃ 0.16 g / L, KBr 80 mg / L, SrCl ₂ · 6H ₂ O 57 mg / L, H ₃ BO ₃ 22 mg / L, Na ₂ SiO ₃ · 5H ₂ O 2.4 mg / L, NaF 57mg / L, NH ₄ NO ₃ 1.6mg / L, Na ₂ HPO ₄ · 12H ₂ O 20mg / L in a culture medium containing 30 mg 48 The cells were incubated in aerobic conditions for a period of time.

After aerobic fermentation, anaerobic digestion sludge generated in Seongnam sewage treatment plant was inoculated at 10% by volume with respect to aerobic fermentation broth, and anaerobic fermentation was carried out in the same manner as in Comparative Example 1. The concentration of the organic acid in the culture medium was measured by HPLC using a Biorad Aminex HPX-87H column according to the incubation time, and the results are shown in Table 5.

1 day (g / L) 3 days (g / L) 5 days (g / L) Acetic acid Total Organic Acid Acetic acid Total Organic Acid Acetic acid Total Organic Acid Kelp 2.02 3.96 2.59 3.20 3.28 3.96 gambling 0.30 0.47 1.14 1.45 1.28 1.38 Blue 1.01 1.19 1.50 1.59 1.48 1.84

From Table 5, when using the Vibrio harveyi than Vibrio alginolyticus, it was found that the organic acid produced a further increase. That is, even in the same Vibrio genus, it was confirmed that there is a difference in the aerobic fermentation capacity of seaweed according to species.

Example 3: Optimization of Aerobic Fermentation Conditions

In order to optimize aerobic fermentation conditions, the effect of fermentation time and volume fraction of inoculum was investigated. Experimental method was carried out in the same manner as in Example 1, confirming the production of volatile organic acids according to the aerobic fermentation time is shown in Figure 1, and the production of volatile organic acids according to the inoculum during aerobic fermentation is shown in Figure 2.

1 and 2, Vibrio harveyi (ATCC 14126) was inoculated at about 3.3 × 10 ⁷ CFU and fermented for 48 hours, it was confirmed that the highest organic acid was produced.

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 thereto will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (7)

(A) adding aerobic microorganism Vibrio sp. to the seaweed, and aerobic fermentation; And
(b) adding anaerobic microorganisms to the aerobic fermented algae, and anaerobic fermentation to produce volatile organic acids.
The method of claim 1, wherein the Vibrio sp. Is Vibrio harveyi .
3. The method of claim 2, wherein Vibrio Harvey (Vibrio harveyi) is a method of producing volatile organic acids from algae, characterized in that the Vibrio Harvey (Vibrio harveyi) ATCC 14126.
According to claim 1, wherein the aerobic fermentation is inoculated with Vibrio sp. ( Vibrio sp. ) Of 3.0 × 10 ⁷ ~ 4.0 × 10 ⁷ CFU per 1 g dry weight of seaweed, volatile from seaweeds, characterized in that incubated for 30 to 54 hours How to produce organic acid.
The method of claim 1, wherein the anaerobic microorganisms are Clostridium thermoaceticum ( Clostridium thermoaceticum ), Clostridium formicoaceticum ( Clostridium formicoaceticum ), Clostridium thermoautotropium (Acetobacterium) Acetobacterium woodii , Selenomonas rumintium , Propionibacterium arabinosum , Butyribacterium methylotrophicum and Lactobacillus amylocillus amylous A method for producing volatile organic acids from seaweeds, characterized in that the dominant species is one or two or more microorganisms selected from the group consisting of.
The method of claim 1, wherein the algae is brown algae, red algae or green algae.
The method of claim 1 wherein the organic acid comprises acetic acid, propionic acid and butyric acid.

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