Method for processing carboxylic acid-containing separation product of separation process

Abstract

The invention relates to a method for processing a carboxylic acid-containing separation product of a separation process, in particular a process for separating fish oil and / or algal oil, wherein polyunsaturated hydrocarbons are produced at least from a portion of the carboxylic acid by means of a microorganism. Suitably, the polyunsaturated hydrocarbons are or include omega-3 fatty acids and / or omega-6 fatty acids, preferably EPA, DHA and / or DPA. In one embodiment of the invention, the microorganisms are or include yeasts, fungi, bacteria and / or in particular marine protists, preferably microalgae. The yeast is preferably a Yarrowia lipolytica-based yeast, and the marine protists are preferably microalgae of Schizochytrium limacinum, preferably Schizochytrium limacinum SR21. The invention also relates to a biomass containing polyunsaturated hydrocarbons produced by microorganisms.

Description

Technical Field

[0001] The present invention relates to a process for processing carboxylic acids. Background Art

[0002] US2010 / 0166620 A1 discloses a system and method for preparing fatty acid methyl esters, commonly known as "biodiesel", which are obtained from a fat mixture of unknown composition, which contains triglycerides, proteins or any other organic form of matter that contains enough fatty acids to be converted into biodiesel. Summary of the invention

[0003] The object of the present invention is to enable carboxylic acids to be utilized better.

[0004] The invention is achieved by a process for processing carboxylic acid-containing separation products of preferably industrial separation processes, in which polyunsaturated hydrocarbons are produced at least from a portion of the carboxylic acids with the aid of microorganisms.

[0005] Surprisingly, microorganisms can be used for processing separation products particularly well. By means of this method, by-products that were usually discarded before in particular in industrial separation processes can also be further processed. Advantageously, not only the expense of discarding and waste is avoided, but also the output of separation processes can be increased. This is particularly applicable to the situation where the purpose of separation processes is to produce products that comprise polyunsaturated hydrocarbons. When processing fish oil and / or algae oil in separation processes, the present invention has been shown to be particularly advantageous.

[0006] The fish oil to be treated by means of the separation process, preferably the pretreated fish oil intermediate, suitably has an omega-3 fatty acid content of 30-80% by weight, preferably at least one of the following components: 0-80% by weight of eicosapentaenoic acid (EPA), 0-70% by weight of docosahexaenoic acid (DHA), and / or 0-10% by weight of docosapentaenoic acid (DPA).

[0007] The algae oil to be processed by the separation process, preferably a pretreated algae oil intermediate, suitably has an omega-3 fatty acid component of 30-90% by weight, preferably at least one of the following components: 0-85% by weight of eicosapentaenoic acid (EPA), 0-85% by weight of docosahexaenoic acid (DHA), and / or 0-10% by weight of docosapentaenoic acid (DPA).

[0008] In one embodiment of the present invention, the raw fish oil from which the fish oil intermediate is produced has an omega-3 fatty acid component of 10-50% by weight, preferably at least one of the following components: 0-25% by weight EPA, 0-25% by weight DHA, and / or 0-7% by weight DPA.

[0009] In one embodiment of the present invention, the raw algae oil from which the algae oil intermediate is produced has an omega-3 fatty acid component of 10-80% by weight, preferably at least one of the following components: 0-70% by weight EPA, 0-70% by weight DHA, and / or 0-5% by weight DPA.

[0010] Suitably, the raw fish oil and / or raw algae oil is produced by distillation, deacidification, ethylation / esterification, saponification, winterization, mechanical separation and / or supercritical fluid extraction, preferably prepared from raw oil.

[0011] In one embodiment of the present invention, fish oil intermediates and / or algae oil intermediates are produced from raw fish oil and / or raw algae oil by means of distillation, deacidification, ethylation / esterification, saponification, mechanical separation, urea precipitation, chromatography, wintering, silver extraction and / or supercritical fluid extraction.

[0012] The fish oil intermediate and / or algae oil intermediate preferably has an ethyl ester content of at least 50% by weight, preferably at least 80% by weight.

[0013] The omega-3 fatty acids are suitably present in the intermediate product as ethyl esters, wherein preferably at least 50% by weight, preferably at least 60% by weight, particularly preferably at least 90% by weight of the omega-3 fatty acids are present as ethyl esters.

[0014] In a particularly preferred embodiment of the present invention, the separated product is a by-product of a separation process for preparing a target product, wherein the target product contains a high concentration of omega-3 fatty acids and / or omega-6 fatty acids, preferably EPA and / or DHA and / or DPA.

[0015] In one embodiment of the present invention, the target product, in particular produced from fish oil and / or algae oil, has an omega-3 fatty acid content of >50 wt.-%, preferably >80 wt.-%, particularly preferably >90 wt.-%, preferably at least one of the following components: 0-99 wt.-% EPA, 0-99 wt.-% DHA, and / or 0-20 wt.-% DPA.

[0016] The target product expediently has an EPA content of >80% by weight, preferably >90% by weight, particularly preferably >95% by weight, or a DHA content of >80% by weight, preferably >90% by weight, particularly preferably >95% by weight.

[0017] Furthermore, the target product can have a DPA content of >50% by weight, preferably >60% by weight, particularly preferably >70% by weight.

[0018] Suitably, the by-product has a saturated fatty acid content of >10% by weight, preferably >15% by weight, particularly preferably >45% by weight. In one embodiment of the invention, the by-product has a polyunsaturated hydrocarbon component, in particular omega-3 fatty acids and / or omega-6 fatty acids, preferably EPA, DHA and / or DPA, wherein said components are less than those in the target product, in particular up to 15% by weight, preferably up to 10% by weight, particularly preferably up to 5% by weight of the target product. Suitably, the polyunsaturated hydrocarbon component, in particular omega-3 fatty acids, in the by-product is <15% by weight, preferably <10% by weight, particularly preferably <5% by weight.

[0019] In one embodiment of the present invention, the by-product, in particular produced from fish oil and / or algae oil, has at least one of the following components: 0-25% by weight, preferably 0-15% by weight EPA, 0-25% by weight, preferably 0-15% by weight DHA, and / or 0-25% by weight, preferably 0-15% by weight DPA.

[0020] Suitably, the isolated product is produced by:

[0021] - distillation, in particular using a distillation apparatus, preferably HPE (High Pressure Extraction; as described, for example, in WO2017005235 Al) or short-path distillation,

[0022] - by means of SFE (supercritical fluid extraction), and / or

[0023] - processing by means of chromatography, preferably SFC (supercritical fluid chromatography), high performance liquid chromatography (HPLC), true moving bed chromatography (TMB) and / or simulated moving bed chromatography (SMB),

[0024] - by means of silver extraction and / or

[0025] -Urea precipitation method (urea complex).

[0026] In one embodiment of the present invention, the by-product produced by means of short-path distillation has at least one of the following components: 0-8% by weight, preferably 0-4% by weight of EPA, 0-5% by weight, preferably 0-3% by weight of DHA, and / or 0-5% by weight, preferably 0-3% by weight of DPA, in particular n-3-DPA.

[0027] The by-product produced by means of HPE has at least one of the following components: 0-12% by weight, preferably 0-6% by weight, EPA, 0-7% by weight, preferably 4% by weight, DHA, and / or 0-7% by weight, preferably 0-4% by weight, DPA, in particular n-3-DPA.

[0028] The by-product produced by means of the silver extraction method has at least one of the following components: 5-70% by weight, preferably 5-40% by weight EPA, 5-70% by weight, preferably 5-40% by weight DHA, and / or 0-12% by weight, preferably 0-6% by weight arachidonic acid.

[0029] The by-product produced by means of the urea precipitation method has at least one of the following components: 2-40% by weight, preferably 2-20% by weight of EPA, 2-40% by weight, preferably 2-20% by weight of DHA, 2-20% by weight, preferably 2-10% by weight of DPA, in particular n-3-DPA, and / or 5-60% by weight, preferably 5-30% by weight of monounsaturated fatty acids.

[0030] The production of short-chain or medium-chain fatty acids (especially with a chain length <12 carbon atoms), long-chain fatty acids (especially with a chain length ≥12 carbon atoms, preferably with a chain length ≥20 carbon atoms) by means of microorganisms has proven to be particularly advantageous.

[0031] In one embodiment of the present invention, the polyunsaturated hydrocarbons produced with the aid of a microorganism are or include EPA, DHA and / or DPA.

[0032] In one embodiment of the present invention, the microorganism is selected from the group consisting of yeast, fungi, bacteria, protists, especially marine protists, preferably microalgae.

[0033] Particularly preferably, microorganisms selected from the group consisting of Schizochytrium sp., Aurantiochytrium sp., Crypthecodinium cohnii, Isochrysis galbana, Phaeodactylum tricornutum, Dunaliella salina, Nannochloropsis oceanica, Chlorella vulgaris, Mucor circinelloides, Saccharomyces cerevisiae, Marine bacteria spp., Cyanobacteria spp., Myxobacteria spp. are used.

[0034] In one embodiment of the present invention, the microorganism is or comprises a lipolytica yeast, preferably a yeast of the Yarrowia lipolytica family, particularly preferably a genetically modified variant of the Yarrowia lipolytica family, such as Yarrowia lipolytica Af4.

[0035] In the studies of the present inventors it was found that yeasts, in particular yeasts of the Yarrowia lipolytica type, produce polyunsaturated hydrocarbons, in particular DHA, from carboxylic acids, in particular the isolated products.

[0036] For use in the process according to the invention, the following yeasts can also be used, for example: Candida tropicalis, Candida albicans, Debaryomyceshansenii and / or Trichosporon.

[0037] In another embodiment of the present invention, the microorganism is or comprises a microalgae of the Schizochytrium class, preferably Schizochytrium, particularly preferably Schizochytrium SR21.

[0038] Furthermore, it has been found that microalgae of the Schizochytrium group produce polyunsaturated hydrocarbons, in particular DHA, from carboxylic acids, in particular isolated products.

[0039] For use in the method according to the invention, the following marine protists, in particular microalgae or marine bacteria, can also be used: Marinbacter, Oceanospiralles, Pseudomonas and / or Alkanivorax.

[0040] It has proven to be particularly advantageous to produce polyunsaturated hydrocarbons from carboxylic acids, especially from isolated products, by means of a combination of yeast and microalgae. In the production process using yeast and microalgae, the extracellular lipase activity of the yeast causes an emulsifying effect, which promotes the production of polyunsaturated hydrocarbons by the microalgae.

[0041] Compared to using yeast alone, the yield using the combination was about 4 times higher. Compared to using microalgae alone, the yield using the combination was almost doubled.

[0042] The production of unsaturated hydrocarbons with the aid of microalgae of the Schizochytrium group, in particular Schizochytrium SR21, together with yeast has proven to be particularly effective.

[0043] In one embodiment of the present invention, lipase is added to improve the production of polyunsaturated hydrocarbons by microorganisms. Suitably, the separated product is added to a reactor together with the microorganisms to produce polyunsaturated hydrocarbons.

[0044] In one embodiment of the invention, the biomass formed by the microorganisms is processed into a food or pharmaceutical ingredient. The food is preferably fed to aquatic organisms, preferably fish, which are raised, for example, in fish farms to produce fish oil.

[0045] In addition, biomass can be fed to an organism to enrich the nutrition of the available components or products (e.g., eggs) of the organism with polyunsaturated hydrocarbons, particularly omega-3 fatty acids and / or omega-6 fatty acids. For example, biomass can be fed to chickens to correspondingly increase the content of polyunsaturated hydrocarbons in the chicken meat and / or the eggs produced.

[0046] Suitably, the polyunsaturated hydrocarbons are extracted from biomass produced by a microorganism.

[0047] The present invention also relates to a biomass containing polyunsaturated hydrocarbons produced by microorganisms. The biomass suitably contains yeast and / or microalgae, preferably yeasts of the Yarrowia lipolytica class and / or marine protists of the Schizochytrium class, preferably Schizochytrium SR21. The content of EPA, DHA and / or DPA in the biomass is preferably at least 5 mg / g dry weight.

[0048] The invention is explained in more detail below with reference to exemplary embodiments and tables and figures relating to these exemplary embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The graph shows the dry cell weight content and the DHA content as a function of the culture duration when carrying out the method according to the invention. Specific implementation plan

[0050] A. Materials used:

[0051] The pretreated fish oil forming the intermediate product is processed by means of a separation process hereinafter referred to as “HPE (High Pressure Extraction)” and described in document WO2017005235A1.

[0052] The intermediate product has the composition shown in Table 1:

[0053]

[0054]

[0055] Table 1 After HPE, the target product has the following composition shown in Table 2:

[0056]

[0057]

[0058] Table 2

[0059] From the hydroxy acids of one of the fractions, which do not form the target product of the separation process but are usually discarded as waste, polyunsaturated hydrocarbons are produced in the manner according to the invention with the aid of microorganisms, as will be explained in more detail below.

[0060] The fatty acid composition of the fractions used relative to the total amount of fatty acids contained is shown in Table 3:

[0061] fatty acid Content in fractions [%] C16:0 16.5 C16:1 0 C18:0 9.1 C18:1 54.7 C18:2 4.9 C18:3 0.6 C18:4 8.5 C20:5 3.8 C22:5 0 C22:6 0 other 1.8

[0062] Table 3

[0063] 1. Microorganisms

[0064] For cultivation on the fractions shown in Table 3, the oleaginous yeast Yarrowia lipolytica and the marine protist Schizochytrium, a microalgae, were used.

[0065] a) The following strain of Yarrowia lipolytica was used: Yarrowia lipolytica, Po1h::SynPfaPptAf4 (Yarrowia lipolytica Af4) [Gemperlein et al., 2019].

[0066] b) The following strain of the seafood protist Schizochytrium was used: Schizochytrium SR21 (ATCC MYA-1381) (renamed Aurantiochytrium limacinum in 2007).

[0067] 2. Cultivation

[0068] Use the following media to grow Yarrowia lipolytica and Schizochytrium:

[0069] a) YNB medium without carbon source

[0070] For the cultivation of Yarrowia lipolytica, YNB medium with a carbon source was used. All components were prepared as corresponding stock solutions in MilliQ water and either autoclaved at 120°C for 20 minutes (ammonium sulfate) or sterilized with a PES membrane filter with a pore size of 0.2 μm (all other components). The solutions were stored at room temperature (ammonium sulfate) or at 4°C. Concentrations are shown in Table 4.

[0071]

[0072] Table 4

[0073] b) Artificial seawater culture medium

[0074] For the cultivation of Schizochytrium, an artificial seawater medium was used, which contained yeast extract as a nitrogen source and a carbon source according to the composition shown in Table 5. For the mixed cultivation of Schizochytrium and Yarrowia lipolytica, half the amount of salt was provided for the cultivation (see the values ​​in parentheses).

[0075]

[0076] Table 5

[0077] c) Carbon source

[0078] For pre-culture, 10 g / L glycerol (200 g / L strain concentration; autoclaved at 120°C for 20 minutes) was used as a carbon source. For the main culture, the above fractions were used at an initial concentration of 25 to 100 g / L. The fractions were freshly sterilized using a PES membrane filter with a pore size of 0.2 μm for each culture. After replacing the air in the bottle with nitrogen, the fractions were stored at 4°C to reduce the risk of oxidation.

[0079] d) Culture conditions

[0080] The cells were cultivated in 250 or 500 mL shake flasks with 10% fill capacity. The flasks were shaken in an orbital shaker (Multitron-Shaker, Infors HT, Bottmingen, Switzerland) at 200 or 230 rpm at 28°C for Schizochytrium / mixed culture and Yarrowia lipolytica.

[0081] 3. Methods

[0082] a) Sampling

[0083] Transfer 1.5 mL of culture broth from each three-necked flask to a pre-weighed glass vial in duplicate. Measure the exact volume gravimetrically. Centrifuge the glass vial at 10,000 x g and 4 °C for 3 minutes. Collect an additional 100 μL of culture broth in a reaction vessel for pH measurement, microscopy, and lipase assay.

[0084] b) Supernatant

[0085] The supernatant still containing residual oil was removed from the cytospin pellet using a syringe with a needle and filtered through a PES membrane filter (0.2 μm pore size; HPLC quality) into a new reactor. The oil-free supernatant was stored at -20°C until further analysis by means of HPLC.

[0086] c) Centrifugal plaque

[0087] The remaining centrifugal pellet in the glass vial was resuspended in 600 μL hexane: water (1: 6, v / v) and then centrifuged as described above to remove the adherent oil on the cell surface and the glass wall of the vial. The water-hexane-oil mixture was discarded, or the hexane-oil phase was collected in a separate glass vial for non-quantitative residual fatty acid determination and only the lower aqueous phase was discarded. Both the hexane-oil phase and the wet cell centrifugal pellet were stored at -20°C until further processing.

[0088] d) Determination of dry cell weight

[0089] The wet cell pellet was dried in a rotary vacuum concentrator (RVC 2-33CDplus, Christ, Germany, with infrared heating) at 30 mbar, 240 rpm and 40° C. for 30 min or until dry. The cell dry weight (CDW) was determined gravimetrically.

[0090] e) Analysis of fatty acids

[0091] The intracellular fatty acids in each centrifugal pellet sample (after determination of CDW) and the fatty acids in the remaining substrate HPE-oil (hexane-oil phase) were determined. For the next step, the hexane-oil phase had to be dried in a rotary vacuum concentrator (RVC 2-33CDplus, Christ, Germany, with infrared heating) at 30 mbar, 240 rpm and 40° C. for 30 minutes or until dry.

[0092] f) Preparation of fatty acid methyl esters (FAME)

[0093] The preparation process of FAME is carried out as follows.15 μ g of henicosapentaenoic acid methyl ester (HPA) is added in each sample as the internal standard of gas chromatography-mass spectrometry (GCMS).Add 300 μ L 50:50:2 methanol-toluene-sulfuric acid mixture (v / v / v), for carrying out 24 hours acid catalytic transesterification reaction at 80 ℃.Reaction mixture is cooled to ice, by adding 250 μ L 0.5MNH4HCO3 and 2M KCl solution and vortexing 30 seconds to stop transesterification reaction subsequently.Phase separation is by at room temperature with 10,000xg centrifugal 3 minutes to realize.75 μ L upper phase containing FAME is transferred in the glass vial with GCMS measurement inlet.

[0094] g) GCMS measurement

[0095] Gas chromatography analysis was performed using an HP-6890 gas chromatography system (Hewlett-Packard, CA, USA) with an HP-88 column (30 m x 250 μm x 0.2 μm, Agilent Technologies) and helium (He 5.0) as carrier gas. The system was equipped with an injection system (7683B series, Agilent Technologies, CA, USA) and an autosampler (7683 series, Agilent Technologies, CA, USA). The injection volume was set to 0.2 or 1.0 μL, and the split mode was selected to be 10:1 or 5:1, depending on the expected fatty acid concentration of the sample.

[0096] Other parameters are shown in Table 6.

[0097] Entry data flow 8.4mL / min pressure 0.76bar temperature 250℃ Analyte / Sample flow 1mL / min pressure 0.76bar Initial furnace temperature 110℃ for 1 minute Temperature gradient in the oven 4℃ / min Final furnace temperature 240℃

[0098] Table 6

[0099] FAMEs were detected using a mass selective detector (mass spectrometer, MS; 5973 Network Series, Agilent Technologies, CA, USA). The solvent delay time of the mass spectrometer was set to 5 min. The mass selective detector was set to SCAN mode and the total ion current (TIC) from m / z 25 to m / z 500 was measured. To identify individual fatty acids, TIC and mass spectra were adjusted using the mass spectral database NIST08 (NIST / EPA / NIH Mass Spectral Library, 2008, version 2.0). In addition, the retention times (RTs) of individual fatty acid standard solutions (Sigma) were measured and compared with the retention times of fatty acids with unclear identities for clarification. Data analysis and system control were performed using MSD ChemStation G1701 EA software.

[0100] h) Quantification of fatty acids

[0101] The area under the curve of each peak is calculated using the integration function of MSD Check G1701 EA software. The result is paired with the retention time and exported to a spreadsheet file. The corresponding fatty acid is matched to each measured retention time. The amount of each detected FAME is then calculated according to the AUC signal of the HPA-ME standard. The fatty acid concentration is calculated based on volume and CDW.

[0102] 4. Results

[0103] The fraction having the fatty acid composition shown in Table 3 was cultured in a reactor for 108 hours with Yarrowia lipolytica (see paragraph 1.a), the marine protist Schizochytrium (see paragraph 1.b), and a mixture of yeast and marine protists. Prior to the culture, the fraction did not contain any DHA, did not contain any DPA, and contained 3.83% EPA.

[0104] The fraction containing DHA was produced by culturing yeast as shown in Table 7. After culturing, the content of DHA in the total fatty acids was 0.29%.

[0105] The content of DHA produced by using the marine protist culture fraction was 0.38%, and the content of DPA produced was 0.07%.

[0106] The fraction, produced by culturing it with a mixed culture of yeast and marine protists, contained 6.34% DHA, 1.96% DPA and 5.80% EPA.

[0107]

[0108]

[0109] Table 7

[0110] Table 8 shows the biomass cell dry weight (CDW) produced, as well as the content of total fatty acids in the cell dry weight and the content of DHA under the cell dry weight. It can be seen that the DHA content when cultivated with marine protists is greater than that cultivated with yeast. When cultivated with mixed cultures, significantly higher contents are achieved.

[0111]

[0112] Table 8

[0113] The higher EPA and DHA contents could be explained by a synergistic effect, namely, the lipase produced by the yeast, which promoted the production of EPA and DHA by the marine protists.

[0114] Figure 1 The graphs show biomass formation as a function of cell dry weight (CDW) versus culture time for addition of 25 g / L and 75 g / L fraction ("HPE") to yeast, protists or mixed cultures, respectively.

[0115] In addition, the graph shows changes in DHA content at two added amounts over culture time.

[0116] It can be seen that the cell dry weight continued to increase until the culture time reached 108 hours. For the addition amount of 25g / L, the DHA content increased to 10mg / mL in the culture time up to 160 hours. For the addition amount of 75g / L, the DHA content increased to 10mg / mL in the culture time up to 1250 hours.

[0117] In another embodiment, the oil obtained from algae is processed by means of the above-mentioned separation process. The hydroxy acids of one of the fractions do not form the target product of the separation process, and polyunsaturated hydrocarbons are produced by means of microorganisms in a manner according to the present invention. Table 9 lists the measurement results of DHA output when processing algae oil fractions and fish oil fractions. It can be seen that yeast is more suitable for producing DHA from fish oil, and DHA output is higher when utilizing microalgae for the production of algae oil fractions.

[0118]

[0119] Table 9

Claims

1. A method for processing carboxylic acid-containing separation products of separation processes, in particular processes for separating fish oil and / or algae oil, wherein polyunsaturated hydrocarbons are produced from at least part of the carboxylic acids with the aid of microorganisms.

2. The method according to claim 1, characterized in that: The polyunsaturated hydrocarbons are or include omega-3 fatty acids and / or omega-6 fatty acids, preferably EPA, DHA and / or DPA.

3. The method according to claim 1 or 2, characterized in that: The microorganism is or comprises yeast, fungi, bacteria and / or in particular marine protists, preferably microalgae.

4. The method according to any one of claims 1 to 3, characterized in that The yeast is a yeast of the Yarrowia lipolytica family, preferably a genetically modified variant of Yarrowia lipolytica.

5. The method according to any one of claims 1 to 4, characterized in that The marine protist is a microalgae of the Schizochytrium genus, preferably Schizochytrium Limacinum SR21.

6. The method according to any one of claims 1 to 5, characterized in that The products are waste products of industrial separation processes, in particular distillation or chromatography.

7. The method according to any one of claims 1 to 6, characterized in that Lipase is added to improve production with microorganisms.

8. The method according to any one of claims 1 to 7, characterized in that The separated product is added to a reactor together with microorganisms and optionally lipase to produce polyunsaturated hydrocarbons.

9. The method according to any one of claims 1 to 8, characterized in that Biomass is formed with the aid of microorganisms and is preferably processed into a food or pharmaceutical ingredient.

10. The method according to claim 9, characterized in that The diet is fed to aquatic organisms, preferably fish, for the production of fish oil.

11. The method according to any one of claims 1 to 9, characterized in that The polyunsaturated hydrocarbons are extracted from biomass formed by microorganisms.

12. A biomass produced by a microorganism, characterized in that The biomass contains polyunsaturated hydrocarbons.

13. The biomass according to claim 12, characterized in that The biomass contains yeasts, fungi, bacteria and / or in particular marine protists, preferably microalgae.

14. The biomass according to claim 12 or 13, characterized in that The biomass contains yeasts of the Yarrowia lipolytica group and / or marine protists of the Schizochytrium group, preferably Schizochytrium SR21.

15. The biomass according to any one of claims 12 to 14, characterized in that The content of EPA and / or DHA is at least 5 mg / g dry cell weight.

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