Method for producing polyhydroxyalkanoate using microalgal biomass hydrolysate

Abstract

The present invention relates to a method for producing polyhydroxyalkanoate using a microalgal biomass hydrolysate. According to the present invention, PHA productivity can be improved by using a microalgal biomass hydrolysate for PHA production, and the chain length of PHA can be controlled according to the accumulation levels of lipids and carbohydrates in microalgae. Accordingly, the present invention provides the advantage of enabling the customized production of PHA having physical properties suitable for various applications. In addition, the present invention can replace existing biomass and can be effectively applied to PHA production processes requiring cost-effectiveness and eco-friendliness.

Description

Method for producing polyhydroxyalkanoates using microalgae biomass saccharification liquid

[0001] The present invention relates to a method for producing polyhydroxyalkanoates using a saccharified microalgae biomass solution.

[0002] Fossil fuel-based plastics persist in the environment for a long time, causing serious environmental problems such as the generation of microplastics. As an alternative to address these issues, the use of biodegradable bioplastics is gaining attention. Among them, polyhydroxyalkanoates (PHAs) are biodegradable polymers naturally synthesized by microorganisms; possessing high biocompatibility and excellent degradability, they show great potential for application in various industrial fields, including medical supplies, packaging materials, and disposable products.

[0003] In particular, PHA is characterized by physical properties that vary significantly depending on chain length. For example, short-chain PHA, known as Polyhydroxybutyrate (PHB), exhibits high crystallinity and strength, making it suitable for products requiring hardness and durability. On the other hand, medium-chain-length PHA (mcl-PHA), which has long chains, possesses excellent flexibility and elasticity, making it suitable for soft and easily deformable products such as packaging materials and disposable items. As such, technology for controlling the chain length and monomer composition of PHA is a crucial factor in expanding the scope of commercial applications.

[0004] Meanwhile, existing PHA production processes using first and second-generation biomass (corn, lignocellulose, etc.) have problems such as emitting large amounts of carbon dioxide during crop cultivation and pretreatment, significantly increasing energy consumption due to demanding saccharification conditions requiring high temperature, high pressure, and high-concentration acid treatment caused by complex structures like lignin commonly found in second-generation biomass, and causing increased costs due to a lack of nutrients necessary for microbial growth.

[0005] However, existing PHA production processes utilizing first and second-generation biomass (corn, lignocellulose, etc.) have several limitations. Specifically, these materials cause high carbon emissions during crop cultivation and lead to competition with food resources. In particular, second-generation biomass contains complex structures such as lignin, making saccharification conditions demanding, such as requiring high temperature, high pressure, or high-concentration acid treatment. This results in increased process energy consumption and the generation of byproducts that inhibit microbial growth, such as HMF. Additionally, pretreatment solutions derived from these biomass lack essential nutrients, posing a problem that requires the input of additional nutrients during cultivation.

[0006] Accordingly, there is a need to develop new technologies that can replace existing biomass and produce PHA economically and environmentally under milder saccharification conditions.

[0007] Based on the aforementioned technical background, the inventors confirmed that when the biomass of microalgae equipped with carbon fixation capabilities that absorb carbon dioxide is used as a carbon source for PHA-producing microorganisms, the carbohydrate and lipid composition accumulated by the microalgae changes depending on the species and culture conditions, thereby influencing the metabolic pathways of the PHA-producing microorganisms and enabling control of PHA productivity and chain length, and thus completed the present invention.

[0008] The present invention has been devised to solve the aforementioned problems, and aims to provide a method that can control the chain length of PHA and simultaneously improve productivity by using a microalgae biomass saccharification liquid as a carbon source for a PHA-producing strain.

[0009] To solve the above problem, the present invention provides a method for producing polyhydroxyalkanoate comprising: (a) a step of saccharifying microalgae biomass to obtain a saccharified liquid; and (b) a step of culturing a polyhydroxyalkanoate (PHA)-producing strain in the saccharified liquid.

[0010] According to the present invention, the microalgae may be selected from the group consisting of Chlamydomonas reinhardtii, Chlorella sorokiniana, and Scenedesmus.

[0011] According to the present invention, the microalgae can be cultured in a medium containing urea as a nitrogen source.

[0012] According to the present invention, the saccharification can be performed using sulfuric acid as a hydrolysis catalyst.

[0013] At this time, the concentration of the sulfuric acid may be 2~4% (v / v).

[0014] In addition, the above saccharification can be performed for 15 to 30 minutes.

[0015] According to the present invention, the PHA-producing strain may be selected from the group consisting of Pseudomonas putida, Novosphingobium, Halomonas, Bacillus, Cuprianvidus necator, Paracoccus, and Burkholderia.

[0016] According to the present invention, the PHA may be PHB (Polyhydroxybutyrate), mcl-PHA (medium-chain-length PHA), or a combination thereof.

[0017] At this time, as the lipid accumulation amount of the microalgae increases, the ratio of mcl-PHA may increase.

[0018] In addition, as the amount of carbohydrate accumulation in the above microalgae increases, the proportion of PHB may increase.

[0019]

[0020] The features and advantages of the present invention will become more apparent from the following detailed description based on the accompanying drawings.

[0021] Prior to this, terms and words used in this specification and claims shall not be interpreted in their ordinary and dictionary meanings, but must be interpreted in a meaning and concept consistent with the technical spirit of the invention, based on the principle that the inventor may appropriately define the concept of the terms to best describe his invention.

[0022] According to the present invention, by utilizing a saccharified microalgae biomass liquid for PHA production, the productivity of PHA can be improved, and the chain length of PHA can be controlled according to the lipid and carbohydrate accumulation amounts of the microalgae. Accordingly, the present invention provides the advantage of being able to custom-manufacture PHA having physical properties suitable for various applications. Furthermore, the present invention can replace conventional biomass and can be usefully applied to PHA production processes that require economic efficiency and environmental friendliness.

[0023] Figure 1 shows the results of comparing the growth of microalgae (Chlamydomonas reinhardtii CC 2686, Chlamydomonas reinhardtii CC 125, Chlorella sorokiniana: UTEX 2714, Scendesmus obliquus UTEX 393) in TAP-C (conventional medium) and in a modified medium in which the nitrogen source was changed to urea according to the present invention.

[0024] Figure 2 shows the results of measuring the amounts of Carbohydrate and Lipid in each microalgae species during the culture period.

[0025] Figure 3 shows the results of confirming nutrient depletion in the modified medium at the specified recovery time.

[0026] Figure 4 shows the results of the saccharification optimization experiment of the microalgae UTEX 2714 species.

[0027] Figure 5 shows the results of a comparison of the total reducing equivalents in the microalgae saccharification solution.

[0028] Figure 6 shows the results of confirming the fatty acid chain composition in the saccharified liquid.

[0029] Figure 7 shows the results of a comparison of growth in the Cupriavidus necator species cultured in a microalgae saccharification solution.

[0030] Figure 8 shows the results of a comparison of growth in Pseudomonas putida species cultured in a microalgae saccharification solution.

[0031] Figure 9 shows the results of measuring the PHB and mcl-PHA content produced by Cupriavidus necator and Pseudomonas putida species cultured in a microalgae saccharification solution.

[0032] Figure 10 shows the composition of mcl-PHA monomers produced from each microalgae saccharification liquid.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0034]

[0035] A method for producing polyhydroxyalkanoate according to the present invention comprises: (a) a step of saccharifying microalgae biomass to obtain a saccharified liquid; and (b) a step of culturing a polyhydroxyalkanoate (PHA)-producing strain in the saccharified liquid.

[0036] In the present invention, the term "Polyhydroxyalkanoate (PHA)" refers to a natural polyester material that microorganisms accumulate within themselves to store energy and reducing capacity when carbon sources are abundant while elements necessary for growth, such as nitrogen, oxygen, phosphorus, and magnesium, are scarce. Since PHA exhibits biodegradability and biocompatibility while possessing physical properties similar to conventional synthetic polymers derived from petroleum, it can be applied in medical applications such as replacement materials for existing synthetic plastics, wound care, drug delivery, and tissue engineering. Furthermore, PHA is composed of chiral hydroxy acids, which have potential as synthetic materials for anti-human immunodeficiency virus treatments, anticancer agents, antibiotics, and vitamins, and thus can also be applied as a pharmaceutical.

[0037] In the present invention, the term "culture" refers to growing microalgae or PHA-producing strains under appropriately controlled environmental conditions. The culture process of the present invention may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0038] In the present invention, the term "medium" refers to a substance mixed with nutrients as the main component required to culture microalgae or PHA-producing strains, and supplies nutrients and growth factors, including water, which is indispensable for survival and growth. Specifically, the medium and other culture conditions used for the cultivation of the present invention may be any medium used for the cultivation of ordinary microorganisms without special limitations; however, the microalgae or PHA-producing strains of the present invention may be cultured in a conventional medium containing a suitable carbon source, nitrogen source, phosphorus, inorganic compounds, amino acids, and / or vitamins, while controlling the temperature, pH, etc.

[0039]

[0040] First, the above step (a) is a step of saccharifying microalgae biomass to obtain a saccharified liquid.

[0041] At this time, the above microalgae may be any type capable of providing carbohydrates, lipids, etc. upon cultivation, but, for example, as can be seen from the results of the following examples, it may be at least one selected from the group consisting of Chlamydomonas reinhardtii, Chlorella sorokiniana, and Scenedesmus, but is not necessarily limited thereto.

[0042] In addition, it is preferable to inoculate the above microalgae into a medium supplied with carbon dioxide and photoculture them. For example, 150 to 350 μEm -2 s -1 Microalgae can be photocultured under conditions of light intensity, a temperature of 20 to 30°C, a pH of 6.5 to 8, and 3 to 8% CO2.

[0043] In addition, it is preferable to culture the microalgae in a medium containing urea as a nitrogen source. As can be seen from the results of the following examples, urea improves both the growth potential and carbohydrate / lipid accumulation of the microalgae, which can be an important factor in controlling the PHB or mcl-PHA composition ratio in the subsequent PHA production stage.

[0044]

[0045] In addition, the above saccharification can be performed using a hydrolysis catalyst.

[0046] At this time, the hydrolysis catalyst may include various hydrolysis catalysts known in the art, and may use one or more acid catalysts selected from, for example, sulfuric acid, hydrochloric acid, bromic acid, nitric acid, acetic acid, perchloric acid, phosphoric acid, p-toluene sulfonic acid (PTSA) and solid acids (e.g., citric acid, oxalic acid, various amino acids, maleic acid, phthalic acid, fumaric acid and sulfamic acid) or one or more alkali catalysts selected from potassium hydroxide, sodium hydroxide, calcium hydroxide and aqueous ammonia solution, and preferably sulfuric acid may be used.

[0047] When using sulfuric acid as a hydrolysis catalyst, it is preferable that the concentration of the sulfuric acid be 2 to 4% (v / v). When the concentration of sulfuric acid is controlled within the above numerical range, as can be seen from the results of the following examples, the total reducing equivalent in the saccharification solution can be significantly improved, and at the same time, the toxic substance Hydromethylfurfural (HMF) is not produced. On the other hand, if the concentration of sulfuric acid is below the above lower limit, there is a problem that the total reducing equivalent in the saccharification solution is low, and if the concentration of sulfuric acid exceeds the above upper limit, not only is the effect of the total reducing equivalent in the saccharification solution reduced, but the toxic substance HMF is produced, which is undesirable.

[0048] In addition, it is preferable to perform the saccharification for 15 to 30 minutes. When the saccharification time is controlled within the above numerical range, the total reducing sugar in the saccharification solution can be significantly improved, as can be seen from the results of the following examples. On the other hand, if the saccharification time is below the above lower limit, there is a problem that the total reducing sugar in the saccharification solution is low, and if the saccharification time exceeds the above upper limit, there is a problem that the total reducing sugar in the saccharification solution may actually decrease, which is undesirable.

[0049]

[0050] Next, step (b) above is the step of culturing a polyhydroxyalkanoate (PHA) producing strain in the saccharification solution.

[0051] At this time, the above-mentioned PHA-producing strain is not specifically limited as long as it is a strain capable of producing PHA, and, for example, may be at least one selected from the group consisting of Pseudomonas putida, Novosphingobium, Halomonas, Bacillus, Cuprianvidus, Paracoccus, and Burkholderia, but is not necessarily limited thereto.

[0052] In addition, when culturing the above PHA-producing strain, the culture temperature can be maintained at 20 to 45°C, specifically 25 to 40°C, and culture can be performed for about 10 to 160 hours, but is not limited thereto.

[0053] The PHA produced by the culture of the present invention may be secreted into the culture medium or remain within the cell.

[0054] The PHA production method of the present invention may additionally include, for example, the step of preparing a PHA-producing strain, the step of preparing a medium for culturing said strain, or a combination thereof (in any order), prior to the culturing step.

[0055] In the present invention, the PHA may be PHB (Polyhydroxybutyrate), mcl-PHA (medium-chain-length PHA), or a combination thereof.

[0056] At this time, as the lipid accumulation of the microalgae providing the microalgae biomass saccharification liquid increases, the ratio of mcl-PHA may increase.

[0057] In addition, as the amount of carbohydrate accumulation in the microalgae providing the microalgae biomass saccharification liquid increases, the proportion of PHB may increase.

[0058]

[0059] The PHA production method of the present invention may further include a step of recovering PHA from the medium or strain cultured by step (b).

[0060] The above recovery may involve collecting the desired PHA using a suitable method known in the art according to the culture method of the microorganism of the present invention, such as a batch, continuous, or fed-batch culture method. For example, various chromatographs such as centrifugation, filtration, treatment with a crystallizing protein precipitating agent (salting out method), extraction, ultrasonic disruption, ultrafiltration, dialysis, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, HPLC, or a combination thereof may be used, and the desired PHA can be recovered from the culture medium or microorganism using a suitable method known in the art.

[0061] In addition, the PHA production method of the present invention may additionally include a purification step. The purification may be performed using a suitable method known in the art. In one example, where the PHA production method of the present invention includes both a recovery step and a purification step, the recovery step and the purification step may be performed continuously or discontinuously regardless of the order, or simultaneously or integrated into a single step, but are not limited thereto.

[0062]

[0063] The present invention also provides a composition for producing PHA comprising a microalgae biomass saccharification liquid.

[0064] The terms used herein and the details related to the microalgae biomass saccharification liquid are as described above.

[0065] [Example]

[0066] The present invention will be described in more detail below through examples. These examples are solely for illustrating the present invention, and it will be obvious to those skilled in the art that the scope of the present invention is not to be interpreted as being limited by these examples. Accordingly, the actual scope of the present invention is defined by the appended claims and their equivalents.

[0067]

[0068] Microalgae strain selection

[0069] Microalgae strains were selected to secure a saccharification solution suitable for PHA-producing strains (in this example, Cupriavidus necator and Pseudomonas putida were used).

[0070] First, Chlamydomonas reinhardtii CC 125 (wild type) and the starchless mutant Chlamydomonas reinhardtii CC 2686 (starchless mutant, characterized by lipid accumulation) were cultured. In particular, since the starchless mutant has a relatively high lipid accumulation, it was used to verify whether the ratio of medium-chain polyhydroxyalkanoates (mcl-PHA) is enhanced through beta-oxidation of the PHA-producing strain Pseudomonas putida. Additionally, to confirm whether bacterial growth improves as the sugar content in the saccharified liquid increases, thereby improving the ratio of polyhydroxybutyrate (PHB), Chlorella sorokiniana UTEX 2714 and Scenedesmus obliquus UTEX 393, which have high carbohydrate accumulation, were used.

[0071]

[0072] Experimental method

[0073] Microalgae biomass production

[0074] All four of the aforementioned microalgae were cultured in an incubator supplied with 5% CO2. Carbohydrate and lipid content were measured daily, and as shown in the results below, Chlamydomons reinhardtii was harvested on day 11, when lipid accumulation was highest, while Chlorella and Scendesmus sp. were harvested on days 11 and 12, when carbohydrate accumulation was highest. After adjusting the pH to 7.0–7.5, the light intensity was set to 250 μE m⁻¹. -2 s -1 Photoculture was performed by setting the temperature to . After the culture was completed, the biomass was recovered by centrifugation at 3000 rpm for 10 minutes, freeze-dried at -50℃ for 24 hours, and stored at -20℃ until use in the experiment.

[0075]

[0076] Preparation of culture medium for enhancing carbohydrate / lipid content accumulation in microalgae

[0077] As the culture medium, we used a TAP-C medium, which has been widely used for conventional microalgae cultivation, with the nitrogen source replaced by urea. It was determined that ammonium chloride, the nitrogen source in TAP-C medium, is unsuitable for long-term cultivation because it causes oxidation of the culture solution as the cultivation time increases. On the other hand, it was determined that urea is advantageous for lipid and carbohydrate accumulation because it causes temporary nitrogen depletion during the process of uptake by microalgae. As explained in the results below, it was confirmed that not only was the growth of microalgae improved, but carbohydrate and lipid accumulation was also increased.

[0078]

[0079] Measurement of lipid content in microalgae

[0080] The lipid content of microalgae was measured using the Bligh & Dyer method. First, 1 g of dried microalgae sample was taken, and 20 mL of a solvent mixture of methanol and chloroform in a 2:1 ratio was added to homogenize it. The homogenized mixture was shaken for 30 minutes to elute the lipids within the microalgae. Subsequently, the supernatant was recovered, and distilled water was added to separate it into upper and lower layers. The lower chloroform layer was separated, the solvent was evaporated using a rotary evaporator, and the residue was dried to measure the amount of lipids. The final lipid content was calculated as a percentage of the dry weight of the microalgae.

[0081]

[0082] Measurement of carbohydrate content in microalgae

[0083] The carbohydrate content of microalgae was measured using the phenol-sulfuric acid method. 100 mg of dried microalgae sample was taken and hydrolyzed with 10 mL of 2.5 N hydrochloric acid. This mixture was heated at 100°C for 1 hour and then cooled. The hydrolyzed solution was filtered to recover the supernatant, and 1 mL of phenol solution and 5 mL of sulfuric acid were added to react with it. After confirming that the color of the reaction mixture changed from yellow to orange, the absorbance was measured at 490 nm using a spectrophotometer. The carbohydrate content in the sample was calculated based on a standard glucose curve, and the results were expressed as a percentage of the dry weight of the microalgae.

[0084]

[0085] Microalgae saccharification process

[0086] Freeze-dried microalgae biomass was saccharified using sulfuric acid. The sulfuric acid concentrations were set to 1%, 2%, 3%, 4%, and 8% (v / v), and the hydrolysis times were set to 10, 20, and 30 minutes. Hydrolysis was carried out using an autoclave at 121°C and 1 bar. Saccharification efficiency was measured using the 3,5-dinitrosalicylic acid method (DNS), which represents the total reducing equivalent.

[0087] After hydrolysis was complete, the remaining biomass was removed by centrifugation at 3000 rpm for 10 minutes, and the supernatant was adjusted to a pH of 7.0 to 7.2 using 10M NaOH. Subsequently, it was filtered through a 0.22 μm filter immediately before culturing the bacteria and then used.

[0088]

[0089] Culture of PHA-producing strains

[0090] Cupriavidus necator and Pseudomonas putida were pre-cultured (24 hours) in Lysogeny Broth (LB). Each microalgae saccharification broth was inoculated with the strains at an OD of 0.1 and cultured at 30°C under shaking incubator conditions.

[0091] OD was measured at 4-hour intervals, and cells were collected at three time points: stationary, decreasing, and intermediate.

[0092]

[0093] PHA production measurement

[0094] The recovered cells were analyzed by GC-FID after methanolysis. The PHA component was quantified using a flame ionization detector (FID), and the weight ratio and monomer ratio were calculated by comparing the composition of PHB and mcl-PHA monomers with a standard.

[0095]

[0096] Results and Discussion

[0097] Comparison of microalgae growth

[0098] Figure 1 shows the results of comparing the growth of microalgae (Chlamydomonas reinhardtii CC 2686, Chlamydomonas reinhardtii CC 125, Chlorella sorokiniana: UTEX 2714, Scendesmus obliquus UTEX 393) in TAP-C (conventional medium) and Modified medium in which the nitrogen source was changed to Urea.

[0099] As shown in Figure 1, growth in Chlamydomonas reinhardtii CC 2686 & Chlamydomonas reinhardtii CC 125 increased by approximately twofold, and in Chlorella sorokiniana UTEX 2714 and Scendesmus obliquus UTEX 393 increased by approximately fourfold. In addition, while the growth period in the existing medium was short at about 12 days, it was confirmed that microalgae could be cultured for more than 25 days in the modified medium.

[0100]

[0101] Measurement of lipid and carbohydrate accumulation in microalgae

[0102] Figure 2 shows the results of measuring the amounts of Carbohydrate and Lipid in each microalgae species during the culture period.

[0103] As shown in Figure 2, it was confirmed that UTEX 2714 exhibited the highest carbohydrate content compared to other microalgae species (favorable for PHB production in Cupriavidus necator), and CC 2686 (starchless mutant) exhibited the highest lipid content compared to other microalgae species (favorable for PHB production in Pseudomonas putida). Based on these results, the 12th day, when carbohydrate productivity was highest, was determined as the microalgae biomass recovery time for UTEX 2714 and UTEX 393, which had high carbohydrate content (0.166 g / L / day and 0.132 g / L / day, respectively), and for comparison of lipid content, the 11th day, when lipid productivity was highest, was determined as the microalgae biomass recovery time for CC 125 and CC 26856 (0.178 g / L / day and 0.065 g / L / day, respectively).

[0104]

[0105] Determination of nutrient depletion point in a urea-containing medium

[0106] Figure 3 shows the results of confirming nutrient depletion in the modified medium at the specified recovery time.

[0107] As shown in Figure 3, Phosphate (left) decreased by 50–60% for each microalgae species, and Nitrogen (right) decreased by 80–90% for each microalgae species. Since the carbohydrate and lipid content increases only when nutrients (phosphate, nitrogen) within the microalgae are depleted, it was confirmed that nutrient depletion occurred during the recovery period when carbohydrate and lipid productivity was highest.

[0108]

[0109] Confirmation of optimal saccharification conditions

[0110] Figure 4 shows the results of the saccharification optimization experiment of the microalgae UTEX 2714 species.

[0111] Through the results of Figure 4, it was confirmed that the total reducing equivalent in the saccharified solution can be significantly improved when the sulfuric acid concentration is 2 to 4% (v / v) and the saccharification time is 20 to 30 minutes.

[0112] Next, the amount of HMF produced according to sulfuric acid concentration was measured, and the results are shown in Table 1 below.

[0113]

[0114] As shown in Table 1 above, it was confirmed that when the concentration of sulfuric acid is 2 to 4% (v / v), the toxic substance Hydromethylfurfural (HMF) is not produced at all.

[0115]

[0116] Figure 5 shows the results of a comparison of the total reducing equivalents in the microalgae saccharification solution.

[0117] Through the results of Figure 5, the proportionality between the carbohydrate content accumulated by microalgae and the total reducing equivalent of the microalgae saccharification liquid was confirmed (the UTEX 2714 species, which had the highest carbohydrate content, had the highest reducing equivalent).

[0118] Next, each type of microalgae reducing sugar was analyzed, and the results are shown in Figure 2 below.

[0119]

[0120] As shown in Table 2 above, it was confirmed that the PHA-producing strains used in the present invention, Cupriavidus necator and Pseudomonas putida, account for the majority of glucose, which is known as the most preferred sugar.

[0121]

[0122] Confirmation of fatty acid chain composition in saccharified solution

[0123] Figure 6 shows the results of confirming the fatty acid chain composition in the saccharified liquid.

[0124] Through the results of Figure 6, it was confirmed that CC 2686 is composed mainly of long chains of C 10-C 18, while CC 125 is composed of shorter chains of C 6-C 10.

[0125]

[0126] Comparison of the growth potential of PHA-producing strains

[0127] Figure 7 shows the results of a comparison of growth in the Cupriavidus necator species grown in a microalgae saccharification solution.

[0128] Conventional medium: Minimal Salt Medium (MSM) + 10 g / L glucose

[0129] Through the results of Figure 7, it was confirmed that the growth of Cupriavidus necator grown in all microalgae saccharification solutions was superior to that of the control medium, and in particular, the growth was highest in the UTEX 2714 saccharification solution, which had the highest reducing sugar content.

[0130] In addition, since the total reducing equivalents differed depending on the microalgae species, the yield was calculated according to Equation 1 below (black box), and

[0131] [Equation 1]

[0132]

[0133] As a result, it was confirmed that the bacterial growth yield was highest in the UTEX 2714 species, which had a high total reducing sugar content in the saccharified solution.

[0134]

[0135] Figure 8 shows the results of a comparison of growth in Pseudomonas putida species grown in a microalgae saccharification solution.

[0136] Conventional medium: Minimal Salt Medium (MSM) + 10 g / L glucose

[0137] Through the results of Figure 8, it was confirmed that the growth of Pseudomonas putida grown in all microalgae saccharification solutions was superior to that of the control medium, and in particular, the growth was highest in the UTEX 2714 saccharification solution, which had the highest reducing sugar content.

[0138] In addition, since the total reducing sugars varied depending on the microalgae species, the yield was calculated according to Equation 1 above (black box), and as a result, it was confirmed that the bacterial growth yield was highest in the UTEX 2714 species, which had a high total reducing sugar in the saccharification solution.

[0139]

[0140] PHA Production and Composition Analysis

[0141] Figure 9 shows the results of measuring the PHB and mcl-PHA content produced from Cupriavidus necator and Pseudomonas putida species grown in a microalgae saccharification solution.

[0142] As shown in Figure 9, PHB (left) showed the highest content at 26% DCW (Dry Cell Weight) in UTEX 2714, which had the highest total reducing equivalent (black box indicates PHB in g / L), and mcl-PHA (right) showed the highest content at 34% in CC 2686, which had the highest lipid accumulation (black box indicates mcl-PHA in g / L). Through this, it was confirmed that when using a microalgae species with high carbohydrate accumulation (UTEX 2714), the total reducing equivalent in the saccharification solution was high, which can increase the accumulation of PHB, and conversely, when using a microalgae species with high lipid accumulation (CC 2686), the accumulation of mcl-PHA can be increased.

[0143] In addition, Figure 10 shows the composition of mcl-PHA monomers produced from each microalgae saccharification liquid.

[0144] Through the results of Figure 10, it can be seen that the composition of mcl-PHA monomers varies depending on the fatty acid composition in the microalgae saccharification solution, and consequently, the composition of the mcl-PHA monomers also changes. It was confirmed that the mcl-PHA produced in the CC 2686 saccharification solution, in which the fatty acid composition was mainly composed of long chain lengths, consisted of C12-C14 monomers, which are mainly long chains, whereas the mcl-PHA produced in the CC 125 saccharification solution, in which the fatty acid composition was mainly composed of short chain lengths, had a C8 short chain mcl-PHA monomer composition. In addition, UTEX 2714 and UTEX 393 had similar fatty acid compositions, and their mcl-PHA monomer compositions were also similar. Through this, it was confirmed that the lipid content accumulated by each microalgae differed, and consequently, the fatty acid composition in the saccharification solution varied; and because the fatty acid composition in the saccharification solution differed, Pseudomonas putida, which produces mcl-PHA using fatty acids as substrates, produced mcl-PHA with different monomer compositions.

[0145]

[0146] Based on the above results, it was confirmed that according to the present invention, the productivity of PHA can be improved by utilizing microalgae biomass saccharification liquid for PHA production, and the chain length of PHA can be controlled according to the lipid and carbohydrate accumulation amounts of the microalgae. Therefore, the present invention provides the advantage of being able to custom-manufacture PHA with physical properties suitable for various applications. Furthermore, the present invention can replace existing biomass and can be usefully applied to PHA production processes that require economic efficiency and environmental friendliness.

[0147]

[0148] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0149] According to the present invention, PHA having properties suitable for various applications can be customized and productivity is also improved, so it can be usefully utilized in the field of PHA production requiring economic efficiency and eco-friendliness.

Claims

1. (a) a step of saccharifying microalgae biomass to obtain a saccharified liquid; and (b) a step of culturing a polyhydroxyalkanoate (PHA) producing strain in the above saccharification solution; a method for producing polyhydroxyalkanoate comprising.

2. In Paragraph 1, A method for producing polyhydroxyalkanoates, characterized in that the microalgae are selected from the group consisting of Chlamydomonas reinhardtii, Chlorella sorokiniana, and Scenedesmus.

3. In Paragraph 1, A method for producing polyhydroxyalkanoate, characterized in that the above-mentioned microalgae are cultured in a medium containing urea as a nitrogen source.

4. In Paragraph 1, A method for producing polyhydroxyalkanoate characterized in that the above saccharification is performed using sulfuric acid as a hydrolysis catalyst.

5. In Paragraph 4, A method for producing polyhydroxyalkanoate characterized by the concentration of the sulfuric acid being 2~4% (v / v).

6. In Paragraph 4, A method for producing polyhydroxyalkanoate characterized by the above saccharification being performed for 15 to 30 minutes.

7. In Paragraph 1, A method for producing polyhydroxyalkanoates, characterized in that the above-mentioned PHA-producing strain is selected from the group consisting of Pseudomonas putida, Novosphingobium, Halomonas, Bacillus, Cuprianvidus necator, Paracoccus, and Burkholderia.

8. In Paragraph 1, A method for producing a polyhydroxyalkanoate characterized in that the above PHA is PHB (Polyhydroxybutyrate), mcl-PHA (medium-chain-length PHA), or a combination thereof.

9. In Paragraph 8, A method for producing polyhydroxyalkanoate characterized by the fact that the ratio of mcl-PHA increases as the lipid accumulation amount of the microalgae increases.

10. In Paragraph 8, A method for producing polyhydroxyalkanoate characterized by the ratio of PHB increasing as the amount of carbohydrate accumulation in the above microalgae increases.

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