TWI796053B - Method of producing polyhydroxyalkanoates - Google Patents

Abstract

The present invention provides a method of producing polyhydroxy- alkanoates, the method comprises (a) providing bacteria with alkaline tolerance under an alkaline environment with pH value being equivalent to pH 8 or above pH8; and (b) adding the bacteria with alkaline tolerance into alkaline medium containing carbon source and nitrogen source whereby the bacteria with alkaline tolerance ferment the carbon source and nitrogen source in the alkaline medium, thereby producing polyhydroxyalkanoates. By the above method of producing polyhydroxyalkanoates, a method of producing polyhydroxyalkanoates which is unnecessary to sterilize the ferment environment is provided.

Description

Production method of polyhydroxyalkanoate

The invention relates to a method for producing polyhydroxyalkanoate, in particular to a method for producing polyhydroxyalkanoate without sterilization in the fermentation environment.

Polyhydroxyalkanoates (PHA), a type of biodegradable plastic, have similar material properties to traditional petrochemical plastics, and thus can be used as a substitute for traditional petrochemical plastics (eg, polyethylene or polypropylene). Since PHA plastics can be decomposed by marine microorganisms in the marine environment, it can reduce the pollution of the marine ecological environment caused by traditional petrochemical plastics.

However, in the traditional PHA fermentation production process, before fermentation, the medium and the fermentation tank usually need to be sterilized to avoid contamination of the tank by bacteria during the fermentation process. The aforementioned sterilization steps consume a large amount of energy, which increases the additional cost of the PHA fermentation production process. Therefore, how to develop a fermentation environment that can ferment and produce PHA without sterilization, thereby reducing the production of the PHA fermentation production process. Cost, remains an unresolved issue.

The purpose of the present invention is to address the above-mentioned problems, and to provide a method for producing polyhydroxyalkanoate, which comprises the following steps: (a) providing alkali-tolerant bacteria, which has the ability to withstand an alkaline environment, and the pH of the alkaline environment is pH 8 or above pH 8; and (b) adding the alkaline tolerant bacteria to an alkaline medium having a pH of pH 8 or above pH 8 containing nitrogen source and carbon source, making the alkaline tolerant bacteria use the nitrogen source and carbon source in the alkaline medium to ferment, thereby producing polyhydroxyalkanoate.

As mentioned above, in step (a), the alkali-tolerant bacteria is Raoultella.

As mentioned above, in the step (b), the alkaline medium contains elemental phosphorus, elemental magnesium, elemental iron and elemental copper.

As mentioned above, in step (b), the weight percentage of carbon source and nitrogen source in the alkaline medium is 5:1-50:1.

As mentioned above, in step (b), the weight ratio of carbon source and nitrogen source in the alkaline medium is 10:1.

By the method for producing polyhydroxyalkanoate as described above, a method for producing polyhydroxyalkanoate without sterilization in the fermentation environment is provided.

Figure 1 shows the growth of miscellaneous bacteria in the fermentation broth of the embodiment of the present invention under different pH conditions.

FIG. 2 shows the OD values of the strains used in the production method of polyhydroxyalkanoate in the embodiment of the present invention grown under different pH conditions.

FIG. 3 shows the possible production efficiency of PHA under different pH conditions of the strains used in the production method of polyhydroxyalkanoate according to the embodiment of the present invention.

FIG. 4 shows the H-NMR spectrum of PHA produced by the polyhydroxyalkanoate production method of the embodiment of the present invention.

In order to fully understand the purpose, characteristics and effects of the present invention, the present invention will be described in detail through the following specific examples in conjunction with the accompanying drawings, as follows: Polyhydroxyalkanoic acid in this example The ester production method is outlined below. First, an alkali-tolerant bacterium is provided, which has the ability to tolerate an alkaline environment whose pH is pH 8 or higher. Next, add the alkaline tolerant bacteria into the alkaline medium, which contains nitrogen source and carbon source, so that the alkaline tolerant bacteria can use the nitrogen source and carbon source in the alkaline medium to ferment, by This yields polyhydroxyalkanoates. The specific process of the polyhydroxyalkanoate production method in this embodiment is as follows.

Screening of Alkaline Tolerant Bacteria

The alkaline tolerant bacteria used in this example is Raoultella sp. The screening procedure for strain INER-Nt8 is described below. First, samples were collected in a sewage treatment plant, and several strains were isolated from the samples by using the pH 8 used in the following "Observation of the Growth of Miscellaneous Bacteria in Fermentation Broth Under Different pH Conditions" culture medium, and screen out the strains that can tolerate the alkaline environment, and then use the following Nile red staining method and NMR analysis to further screen the alkaline-tolerant bacteria that can produce PHA, and finally pass the screened alkaline-resistant bacteria The 16S ribosomal RNA gene of the bacterium was compared with other bacterial species, and the Raoultella strain used in this embodiment was found, and it was named INER-Nt8. The strain INER-Nt8 is deposited in the Food Industry Development Institute of the Foundation, and the deposit number is BCRC 911082.

Strain INER-Nt8 can survive in an alkaline environment with pH 8 or higher. In this embodiment, the Raoultella strain is used to ferment and produce PHA, but in other embodiments, other bacterial strains that can survive in an alkaline environment and can ferment and produce PHA can also be used instead of the above-mentioned bacterial strains. limit. In addition, the term "alkaline environment" or "alkaline" in this embodiment includes a range of pH 8 or higher than pH 8.

Preservation of Alkaline Tolerant Bacteria

Nutrient Broth culture fluid (purchased from BD 234000 (Culture Media Nutrient Broth 500g)) was provided, 1 mL of Nutrient Broth culture fluid was packed in a 5 mL test tube, and 1 mg of bacterial strain INER-Nt8 bacterial powder was inoculated in Nutrient Broth culture fluid as Preliminary culture bacterium liquid, the glucose of 10g/L is contained in Nutrient Broth nutrient solution, and the pH value of Nutrient Broth nutrient solution is adjusted to pH 8 or higher with lye, preliminary culture bacterium liquid is in incubator with 30 ℃ (culture The temperature can be controlled between 18°C and 36°C) and cultured for 24 hours. Then, transfer the primary culture liquid that has completed the cultivation to 200mL of Nutrient Broth culture liquid (packed in a 1000mL shake flask) for scale-up culture, as the scale-up culture culture liquid, the Nutrient Broth culture liquid in this scale-up culture step The composition is the same as that of the Nutrient Broth culture solution in the initial cultivation step. The amplified culture solution is cultivated in an incubator at 30°C, and samples are taken from the amplified culture solution every hour after the start of the culture to analyze Measure the OD ₆₀₀ value of the amplified culture solution with a photometer, stop the culture when the measured OD ₆₀₀ value is close to 2, and the culture time can be from 8 hours to 24 hours. Finally, the cultured amplified culture liquid was mixed with glycerol to make a strain preservation sample (mixing ratio of 80% (v/v) bacterial liquid and 20% (v/v) glycerol), and stored at -80 ℃ in the refrigerator.

Observation test on the growth of miscellaneous bacteria in fermentation broth under different pH conditions

First, prepare six 500mL Erlenmeyer flasks, and pour 200mL of liquid medium for fermentation of the strain INER-Nt8 into each conical flask, which contains 50g/L of glucose and 3.75g/L of yeast extract , 6.25g/L protein, 3.7g/L potassium dihydrogen phosphate (phosphorus element required for bacterial growth and replication, not an essential component for strain fermentation), 5.8g/L dipotassium hydrogen phosphate (for bacterial growth and replication The required phosphorus element is not an essential component for bacterial strain fermentation), 0.02g/L magnesium chloride (magnesium element required for bacterial growth and replication, not an essential component for bacterial strain fermentation) and 0.1% trace elements (iron in trace elements and copper are mainly used as catalysts in the enzyme system, and it is not an essential component for strain fermentation. In this embodiment, the formula of trace elements is 2.78 grams of FeSO ₄ .7H ₂ O, 1.98 grams of MnCl ₂ .H ₂ O , 2.81 grams of CoSO ₄ .7H ₂ O, 1.67 grams of CaCl ₂ .2 H ₂ O, 0.17 grams of CuCl ₂ .2 H ₂ O, 0.29 grams of ZnSO ₄ .7 H ₂ O were all dissolved in 1 L of 1M HCl In, when adding the liquid culture medium, the formula of the above-mentioned trace elements is first diluted 1000 times and then used). The culture media in the above six Erlenmeyer flasks are named as the first group to the sixth group respectively. Next, adjust the pH value of the first group of culture medium to pH 5; adjust the pH value of the second group of culture medium to pH 6; adjust the pH value of the third group of culture medium to pH 7; adjust the pH value of the fourth group of culture medium to pH 8; adjust the pH value of the culture medium of the fifth group to pH 9; adjust the pH value of the culture medium of the sixth group to pH 10. After the pH value adjustment of the above-mentioned six groups of culture media was completed, they were placed in a room temperature environment for 14 days without sterilization, and the growth of miscellaneous bacteria was observed.

The growth situation of each group of miscellaneous bacteria is shown in Figure 1. In the first group to the third group of culture medium with a pH value of pH 5-7, the miscellaneous bacteria can grow in the culture medium, but in the pH value of pH 8-10. In the four to sixth groups of media, no growth of miscellaneous bacteria was observed. From the above results, it can be seen that the alkaline environment can inhibit the growth of bacteria in the general natural environment.

In this example, the medium used contains 50g/L of glucose, 3.75g/L of yeast extract, 6.25g/L of protein, 3.7g/L of potassium dihydrogen phosphate, 5.8g/L of dihydrogen phosphate Potassium, 0.02g/L magnesium chloride and 0.1% trace elements. But in other embodiments, the concentration of each component in the above-mentioned medium can be adjusted according to requirements, for example, the medium used can contain 40-100g/L glucose, 1-5g/L yeast extract, 2-8g/L protein, 2~5g/L potassium dihydrogen phosphate, 4~7g/L dipotassium hydrogen phosphate, 0.01~0.05g/L magnesium chloride and 0.01~0.5% trace elements. In other embodiments, the culture medium used by the bacterial strain INER~Nt8 can be adjusted to only contain 40-100 g/L of glucose, 1-5 g/L of yeast extract and 2-8 g/L of protein as required, as long as the culture medium Adjust the pH value to be above pH 8. In yet another embodiment, the culture medium used by the bacterial strain INER-Nt8 may only contain a carbon source and a nitrogen source, wherein the weight ratio of the carbon source to the nitrogen source (hereinafter referred to as the carbon-nitrogen ratio) may be 5:1 to 50:1, And as long as the pH value of the medium is adjusted to be above pH 8; here, the source of carbon source can be multiple carbon source, the concentration of multiple carbon source is between 2-10%, and the type of multiple carbon source can be oil , glucose, starch hydrolyzate, cellulose hydrolyzate, fruit juice, crop juice or five-carbon sugar or six-carbon sugar derived from sugar; the type of nitrogen source used can be yeast extract, protein, ammonium nitrate, ammonium sulfate , ammonium chloride, potassium nitrate or urea.

Determination of OD value of strain INER-Nt8 growing under different pH conditions

First, six 500 mL Erlenmeyer flasks were prepared, and 200 mL of the liquid culture medium used in the aforementioned miscellaneous bacteria growth observation test was poured into each Erlenmeyer flask. The culture media in the above six Erlenmeyer flasks are named as the first group to the sixth group respectively. Under non-sterile conditions, adjust the pH value of the first group of medium to pH 5; adjust the pH of the second group of medium to pH 6; adjust the pH of the third group of medium to pH 7; adjust the pH of the fourth group of medium The pH value of the medium was adjusted to pH 8; the pH value of the medium in the fifth group was adjusted to pH 9; the pH value of the medium in the sixth group was adjusted to pH 10.

Next, inoculate the INER-Nt8 bacterium solution prepared in the aforementioned strain preservation step into the culture medium of the first group to the sixth group respectively with an inoculation ratio of 10% (v/v). The initial OD ₆₀₀ values of each group of fermentation broth were measured respectively. Then, the culture media of the first group to the sixth group containing the strain INER-Nt8 were placed in an incubator and cultured at 30° C. for 48 hours for fermentation. After culturing for 24 hours and 48 hours respectively, the fermentation broths of groups 1 to 6 were taken out, and the post-fermentation OD ₆₀₀ values of the fermentation broths of each group were measured with a spectrophotometer.

Result as shown in Figure 2, the OD value of the first group is initial OD ₆₀₀ value 0.057, rises to OD ₆₀₀ value 1.423 after fermentation 24 hours, rises to OD ₆₀₀ value 1.726 after fermentation 48 hours; The OD value of the second group The initial OD ₆₀₀ value was 0.079, which rose to OD ₆₀₀ value of 1.518 after 24 hours of fermentation, and rose to OD ₆₀₀ value of 1.755 after 48 hours of fermentation; the OD value of the third group was 0.126 in the initial OD ₆₀₀ value, and increased after 24 hours of fermentation to _OD600 value of 1.548, and rose to _OD600 value of 1.857 after 48 hours of fermentation; the OD value of the fourth group was 0.178 at the initial _OD600 value, rose to _OD600 value of 1.606 after 24 hours of fermentation, and rose to OD600 value of 48 hours after fermentation The OD ₆₀₀ value was 1.904; the OD value of the fifth group was 0.223 at the initial OD ₆₀₀ value _, which rose to 1.678 after 24 hours of fermentation and _1.956 after 48 hours of fermentation; the OD value of the sixth group was initial The OD ₆₀₀ value was 0.263, which rose to _1.649 after 24 hours of fermentation and _2.003 after 48 hours of fermentation. The above experimental results show that INER-Nt8 can grow well in the alkaline environment of pH 8-10.

Determination of PHA Production Efficiency of Strain INER-Nt8 under Different pH Conditions

First, three 500 mL Erlenmeyer flasks were prepared, and 200 mL of the liquid culture medium used in the aforementioned miscellaneous bacteria growth observation test was poured into each Erlenmeyer flask. The culture media in the above three Erlenmeyer flasks were named as pH 8 group, pH 9 group and pH 10 group respectively. Under non-sterilized conditions, the pH 8 group medium The pH value was adjusted to pH 8; the pH value of the medium in the pH 9 group was adjusted to pH 9; the pH value of the medium in the pH 10 group was adjusted to pH 10.

Next, the INER-Nt8 bacterial solution prepared in the aforementioned strain preservation step was inoculated in the media of pH 8, pH 9, and pH 10 groups at an inoculation ratio of 10% (v/v). Then, the medium containing the pH 8 group, the pH 9 group and the pH 10 group containing the strain INER-Nt8 was placed in an incubator and cultured at 30° C. for 48 hours for fermentation. After cultivating for 48 hours, take out the fermentation liquid of pH 8 group, pH 9 group and pH 10 group, and use Nile Red (Nile Red) staining method to evaluate the possible production efficiency of PHA of strain INER-Nt8 under different pH conditions .

The Nile Red staining method was performed as follows. Dissolve the mother liquor of Nile red dye in DMSO first, the concentration of Nile red is 0.25 mg/mL, filter the Nile red dye through a filter of 0.22 μm and store it in the dark. When staining, dilute with sterile water to a concentration of 2.5 μg/ml.

Take 20 μL of the fermented bacteria liquid of the above pH 8 group, pH 9 group and pH 10 group respectively and add them to the opaque 96-well plastic black plate, and then add 100 μL of Nile red dye (2.5 μg /ml) was used as the experimental group, and 100 μL of sterilized water was added as the control group. After the 96-well plastic black plate was allowed to stand for 30 minutes, it was analyzed by a disc fluorescence analyzer. The conditions for using the filter set were: excitation: 480±10nm , emission: 580±10nm.

Since the Nile red stain can detect the PHA particles in the bacteria, the possible production efficiency of PHA of the strain INER-Nt8 under different pH conditions can be evaluated by the Nile red staining method. The results are shown in Figure 3. Under the wavelength detection of OD ₆₀₀ , the OD ₆₀₀ value of the pH 8 group was 3694, the OD ₆₀₀ value of the pH 9 group was 2707, and the OD ₆₀₀ value of the pH 10 group was 1358. From the results in Figure 3, it can be seen that in the fermentation environment of pH 8~pH 10, bacteria can produce PHA.

Proton Magnetic Resonance Spectral Analysis of PHA Produced by Strain INER-Nt8

First, prepare a 500mL Erlenmeyer flask, and pour 200mL of liquid medium for the fermentation of the strain INER-Nt8 into the Erlenmeyer flask. The medium contains 50g/L of glucose, 1.875g/L of yeast extract, 3.125 g/L protein, 3.7g/L potassium dihydrogen phosphate, 5.8g/L dipotassium hydrogen phosphate, 0.02g/L magnesium chloride and 0.1% trace elements, the carbon-nitrogen ratio of the above medium is set to 10: 1 (for example, glucose as a carbon source is 50 g, yeast extract as a nitrogen source is 1.875 g, and protein as a nitrogen source is 3.125 g, so that the weight ratio of carbon source to nitrogen source is 10:1). Adjust the pH of the medium to pH 9 without sterilization.

Next, inoculate the INER-Nt8 bacterium solution prepared in the aforementioned strain preservation step into the aforementioned culture medium at an inoculation ratio of 10% (v/v). Then, the medium containing the strain INER-Nt8 was placed in an incubator and cultured at 30°C for 72 hours for fermentation. After culturing for 72 hours, the fermentation broth was taken out. The fermentation broth was placed in a centrifuge and centrifuged at a speed of 4000rpm for 15 minutes, thereby separating into bacterial sediment and fermentation broth.

Finally, the bacterial sediment was taken for bacterial destruction and extraction. Dissolve the bacterial cell sediment in chloroform first to destroy the bacteria, and dissolve the PHA in the bacterial cell, then add deionized water to the bacterial cell/chloroform mixture, and mix the above bacterial cell/chloroform/deionized water again The liquid was placed in a centrifuge and centrifuged at a speed of 4000rpm for 15 minutes. After the centrifugation was completed, the organic phase liquid of the lower layer was taken out, and a hydrogen nuclear magnetic resonance spectrometer (Varian Mercury Plus, purchased from Agilent) was used and according to the product of the hydrogen nuclear magnetic resonance spectrometer. In the operation manual, the aforementioned organic phase liquid is subjected to hydrogen nuclear magnetic resonance (NMR) spectroscopic analysis to determine the type of PHA produced through the above steps.

The analysis results are shown in Figure 4, as can be seen from the NMR spectrum, the PHA species produced is 3-hydroxybutyric acid and 3-hydroxyvaleric acid copolymer (PHBV). However, in other embodiments, different types of PHA, such as poly-3-hydroxybutyrate, can be produced through the adjustment of the culture material source and the selection of strains (PHB), 3-hydroxybutyric acid and 4-hydroxybutyric acid copolymer (P3HB4HB), 3-hydroxybutyric acid and 3-hydroxyhexanoic acid copolymer (PHBHHx) and medium chain length polyhydroxyalkanoate (PHA), and It is not limited to this embodiment.

In this embodiment, the bacterium is destructed through chloroform, but in other embodiments, high pressure bacteriostasis, shock bacteriostasis, ultrasonic bacteriostasis, osmotic pressure bacteriostasis, and chemical solvent bacteriostasis can also be used Or freeze-thaw method, but not limited to the present embodiment.

In this embodiment, PHA is extracted through chloroform, but in other embodiments, the method of extracting PHA can also be selected from organic solvent extraction, physical mechanical extraction or supercritical extraction. In organic solvent extraction, organic The extraction solvent of the solvent extraction method may also be alcohol, hexane, n-heptane or other suitable solvents, and is not limited to this embodiment.

From the above experimental results, it can be seen that the PHA production method in this example uses an alkaline fermentation environment that can inhibit the growth of miscellaneous bacteria, and an alkaline-tolerant strain that can ferment and produce PHA in an alkaline environment, so that the fermentation environment does not need to be sterilized Treatment can produce the effect of PHA. In addition, in the traditional PHA fermentation production process, bacterial fermentation is usually carried out in an environment with a neutral pH value. However, in the process of obtaining fermentable sugars from biomass raw materials, acid treatment or alkali treatment is usually required to further Obtain fermentable sugars, that is, the fermentable sugars initially obtained are acidic or alkaline, so before fermentation, the pH of the fermentable sugars needs to be adjusted to neutral, so that the pH adjustment step will also be Add another production cost. Since the PHA production method in this example is to directly use the alkaline fermentation broth to ferment and produce PHA, the PHA production method in this example saves the cost of sterilization of the fermentation environment and adjusts the fermentation process compared with the traditional PHA production method. Ambient pH cost.

As mentioned above, with the above polyhydroxyalkanoate production method, the fermentation environment can produce PHA without sterilization treatment, and there is no need to adjust the pH value of the fermentation environment, which greatly saves the production of PHA compared with the traditional PHA production method cost.

The present invention has been disclosed above with preferred embodiments, but those skilled in the art should understand that the embodiments are only used to describe the present invention, and should not be construed as limiting the scope of the present invention. It should be noted that all changes and substitutions equivalent to the embodiment should be included in the scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the scope of the patent application.

【Biological Material Storage】

Domestic storage information

ROC Food Industry Development Institute Bioresource Collection and Research Center (BCRC)

November 16, 2021

BCRC 911082

Claims (4)

A method for producing polyhydroxyalkanoate, which comprises the following steps: (a) providing an alkaline-tolerant bacterium, the alkaline-tolerant bacterium is Raoultella, which is deposited in the Food Industry Development Research Institute, and the deposit number is BCRC 911082, which has the ability to tolerate an alkaline environment having a pH higher than pH 9; and (b) adding the alkaline-tolerant bacteria to an alkaline medium whose pH is pH 8 or higher than pH 8, the alkaline medium contains a nitrogen source and a carbon source, and the alkaline tolerant bacteria use the nitrogen source and the carbon source in the alkaline medium to ferment, thereby producing polyhydroxyalkanoate, The weight ratio of carbon source and nitrogen source in the alkaline medium is 5:1-50:1. The method according to claim 1, wherein, in step (b), the weight ratio of carbon source and nitrogen source in the alkaline medium is 10:1. The method according to claim 1, wherein, in step (b), the alkaline medium contains elemental phosphorus, elemental magnesium, elemental iron and elemental copper. The method according to claim 3, wherein, in step (b), the weight ratio of carbon source and nitrogen source in the alkaline medium is 10:1.

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