PRODUCTION OF BIOMEDICAL COMPOUNDS BY ENRICHMENT CULTURES OF MICROORGANISMS

Abstract

The present invention is in the field of a method for production of biomedical compounds by enrichment cultures of microorganisms, and a product obtainable by said methods. The microorganisms are grown in a batch reactor, a continuous reactor, a semi-continuous reactor, such as a Nereda® reactor.

Claims

1. A method of producing a biomedical compound, comprising providing a microorganism culture, growing the microorganisms under aerobic and anaerobic conditions by switching at least once between aerobic and anaerobic conditions thereby favouring carbon accumulating microorganisms comprising PAOs (poly-phosphate accumulating organisms) and GAOs (glycogen accumulating organisms), forming an extracellular matrix embedding microorganisms, the matrix comprising extracellular polymeric substances, physically separating the extracellular matrix embedding the microorganisms, and extracting the biomedical compound from the extracellular matrix, wherein the biomedical compound comprises at least one of a monosaccharide and/or disaccharide and is selected from (i) at least one of a heparan like polymer, a heparin like compound, and a heparin oligomer, and from (ii) at least one of a sialic acid, a glycoprotein, and a glycolipid, or a salt thereof, or conjugate thereof, or a combination thereof.

2. The method according to claim 1, wherein the biomedical compound is selected from a 3-30 kDa glycosaminoglycan.

3. The method according to claim 1, wherein the monosaccharide or disaccharide of the biomedical compound has structural formula ##STR00002## wherein each of R1-R5 is independently selected, wherein R1 is selected from at least one of H, COOH, and OH, wherein R2 is selected from at least one of NHAc, NHSO3H, and H, wherein R3 is selected from at least one of H, and OH, wherein R4 is selected from at least one of H, NHAc, OH, and ##STR00003## wherein R6 is selected from at least one of H, OSO3H, and OH, wherein R7 is selected from at least one of OH, and H, wherein R8 is selected from at least one of H, and OH, wherein R9 is selected from at least one of H, COOH, and OH, and wherein R5 is selected from at least one of H, CH2OH, CH2OSO3, COOH, CHOHCH2OH, CH2CHOHCH2OH, and OH.

4. The method according to claim 1, wherein the microorganisms are grown in granules.

5. The method according to claim 1, wherein the microorganisms form aerobic granular sludge, or wherein the microorganisms are grown under aerobic and anaerobic conditions by switching between aerobic and anaerobic conditions in a cyclic mode.

6. The method according to claim 1, wherein the compound is a 5-15 kDa glycosaminoglycan.

7. The method according to claim 1, wherein the compound is a sialic acid.

8. The method according to claim 1, wherein the microorganisms are provided with a supplement comprising at least one of sugars, fatty acids, proteins, proteins, and minerals.

9. The method according to claim 1, wherein the microorganisms are grown in an aqueous solution, or by providing a substrate, in a reactor comprising a carbon source and linear or branched carboxylic acids, and linear or branched alkanols, and a phosphorus source, and a nitrogen source, and combinations thereof.

10. The method according to claim 1, wherein a temperature is maintained between 15-40° C., and wherein a COD is 200-500 mg/l, and wherein a N content is 40-100 mg/1, and wherein a P content is 1-20 mg/1, and wherein a S content is 1-20 mg/1, and wherein a Cl content is 1-20 mg/1, and wherein a Mg content is 1-20 mg/1, and wherein a pH is 6-8, and wherein a dissolved oxygen concentration is 10-60%, and wherein a sludge retention time is 10-50 days, and wherein an aerobic phase is 30-120 minutes/cycle, and wherein an anaerobic phase is 100-360 minutes/cycle, and wherein a settling time is 1-10 minutes per cycle, and wherein an effluent withdrawal time is 1-10 minutes per cycle.

11. The method according to claim 1, wherein the microorganisms are selected from Proteobacteria, Alphaproteobacteria, Betaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Gammaproteobacteria, Hydrogenophilalia, Oligoflexia, and fimbria comprising bacteria.

12. The method according to claim 1, wherein microorganisms are grown in a batch reactor, a continuous reactor, or a semi-continuous reactor.

13. The method according to claim 1, wherein the produced granular sludge is incubated at increased pH of 9-12, stirring the mixture, removing insoluble substances, lowering the pH to 4-6, freeze drying the precipitate, solubilizing the EPS in an alkaline aqueous solution, optionally reducing sulphide bridges, denaturating the EPS, at a temperature of 60-80° C. during 20-45 minutes, and providing enzymes for enzymatic hydrolysis of extracted EPS and separating proteins, at elevated temperature, at a temperature of 50-80° C., during 10-15 hours, at a pH of 5-8.

14. The method according to claim 1, wherein 0.1-20 wt. % biomedical compound is extracted, wherein wt. % are relative to a total mass of the extracellular matrix.

15. The method according to claim 1, wherein 0.1-10 wt. % monosaccharide is extracted, and wherein 0.1-10 wt. % disaccharide is extracted, wherein wt. % are relative to a total mass of the extracellular matrix.

16. A product obtained by claim 1, comprising 0.1-30 wt. % heparan like polymer, a heparin like compound, and a heparin oligomer is/are present, and 0.1-30 wt. % a neuraminic acid glycosaminoglycan, a sialic acid, a glycoprotein, and a glycolipid, and optionally comprising trace compounds of the microorganism culture.

17. (canceled)

18. (canceled)

Description

FIGURES

[0070] FIG. 1a-f shows disaccharide units of heparin.

[0071] FIG. 2a-d show sialic acids.

[0072] FIG. 3 shows images of SDS-PAGE gels.

[0073] FIGS. 4a-b show structural formulas of the present monosaccharide and disaccharide.

DETAILED DESCRIPTION OF THE FIGURES

[0074] FIG. 1a-f show GlcA=β-D-glucuronic acid, IdoA=α-L-iduronic acid, IdoA(2S)=2-O-sulfo-α-L-iduronic acid, GlcNAc=2-deoxy-2-acetamido-α-D-glucopyranosyl, GlcNS=2-deoxy-2-sulfamido-α-D-glucopyranosyl, GlcNS(6S)=2-deoxy-2-sulfamido-α-D-glucopyranosyl-6-O-sulfate.

[0075] FIG. 2a-d show N-acetylneuraminic acid (Neu5Ac), 2-keto-3-deoxynonic acid Kdn, an α-anomer, and a β-anomer.

[0076] FIG. 3 shows Images of SDS-PAGE gels, showing 12 kDa and 8 kDa bands. On the left (A) stained with Alcian Blue pH 2.5. On the right (B) stained with PAS. The same numbering applies as to FIG. 1; Lane 1: Dye-sGAG complex from Blyscan assay of untreated Anammox, lane 2: Dye-sGAG complex from blyscan assay of denatured Anammox hydrolysed with Papain, lane 3: untreated Anammox, lane 4: Denatured Anammox, lane 5: Anammox hydrolysed with Trypsin, lane 6: Denatured Anammox, hydrolysed with Trypsin. FIGS. 4a-b have been detailed above.

[0077] The invention is further detailed by the accompanying examples, which is exemplary and explanatory of nature and are not limiting the scope of the invention. To the person skilled in the art it may be clear that many variants, being obvious or not, may be conceivable falling within the scope of protection, defined by the present claims.

EXAMPLE

Reactor Operation and Dominant Microorganisms

[0078] Reactor Operation

[0079] Seawater-adapted aerobic granular sludge was cultivated in a 2.7 L bubble column (5.6 cm diameter), operated as a sequencing batch reactor (SBR). The reactor was inoculated with Nereda® sludge. The temperature was controlled at 20° C., the pH at 7.0±0.1, dissolved oxygen (DO) concentration at 50% saturation. The average sludge retention time (SRT) was 20 days, and reactor cycles related to 60 minutes of anaerobic feeding, 170 minutes aeration, 5 minutes settling and 5 minutes effluent withdrawal. A feed of 1.5 L per cycle consisted of 1200 mL of artificial seawater (Instant Ocean®, final concentration 35 g/L), 150 mL of medium A, and 150 mL of medium B. Medium A contained 7.785 g/L sodium acetate trihydrate (3.66 g/L COD), 0.88 g/L MgSO.sub.4.7H.sub.2O, and 0.35 g/L KCl. Medium B contained 2.289 g/L NH.sub.4Cl (600 mg/L NH.sub.4.sup.+—N), 349 mg/L K.sub.2HPO.sub.4, and 136 mg/L KH.sub.2PO.sub.4. The combination of these feed streams led to influent concentrations of 366 mg/L COD, 60 mg/L NH.sub.4.sup.+—N and 9.3 mg/L PO.sub.4.sup.3−—P. Acetate was completely consumed anaerobically within the first 60 minutes of the cycle, while phosphate was released up to 75 mg P043-P/L (5.9 net P-mol release). This corresponds to 0.34 P-mol/C-mol of anaerobic phosphate release per carbon uptake. Fluorescence in situ hybridization (FISH) analysis was performed for microbial community analysis. Probes for polyphosphate accumulating organisms (PAOmix), glycogen accumulating organisms (GAOmix), and a general probe for all bacteria (EUB338) were used. The results indicate dominance of PAO over GAO in the present system.

[0080] Commercially available lectins (FITC or Alexa488) were applied as an individual probe to one granule. After this glycoconjugates screening, granules were stained specifically for proteins and sialic acids. The result of lectin staining showed that sialic acid is abundantly distributed in granular sludge. To quantify the amount of sialic acids, neuraminidase was applied that cleave α(2.fwdarw.3,6,8,9) N-acetylneuraminic acid linkages, as well as branched N-acetylneuraminic acid. Subsequent quantification yields an amount of 11.33±3.80 mg N-acetylneuraminic acid per gram of volatile solids (VS).

[0081] Quantification of sialic acid (neuraminic acid, Neu5Ac) in the seawater-adapted AGS was performed with a Sialic Acid Quantitation Kit (Sigma-Aldrich, USA). The protocol was followed as described in the manual that was supplied with the quantitation kit for a whole cell assay. 80 μL of homogenized cells were taken, added to 20 μL sialidase buffer and 1 μL of α(2.fwdarw.3,6,8,9)-neuraminidase, and incubated overnight at 37° C. Afterwards, 20 μL 0.01M β-NADH solution, 1 μL of N-Acetylneuraminic Acid Aldolase and 1 μL of Lactic Dehydrogenase were added, and incubated at 37° C. for 1 hour. Absorbance at 340 nm was measured prior and after addition of the last enzymes and used for calculation of the Neu5Ac concentration.

[0082] Typically the sialic acid, and likewise the heparin-like compound can be obtained in relatively pure form. Typically purities found are >50%, such as >60%.

Genome Transferase

[0083] The species from which the reference protein sequences were taken were a range of pathogenic bacteria (Neisseria meningitides, Campylobacter jejuni, Helicobacter cetorum, Photobacterium damselae), extremophiles (Chitinivibrio alkaliphilus, Psychrobacter arcticus, Salinibacter ruber, Halanaerobium praevalens), and the common fruit fly (Drosophila melanogaster). The criteria for low E-value, and thereby high probability for presence in its genome, are set at <1E-40.

[0084] Glycosaminoglycan Extraction

[0085] Anammox granules and AGS were obtained from commercially operated reactors in Sluisjesdijk and Dinxperlo respectively. The AGS reactor was operated as described above. The anammox reactor was operated as follows. A full-scale anammox reactor of 70 m.sup.3 was used. The reactor combines a high loading rate with efficient biomass retention, characteristics which the anammox process has in common with anaerobic wastewater treatment. The lower compartment (ca 40 m.sup.3) is mixed by influent and down-corner flow as well as by gas recycled from the top of the reactor. On top of the lower compartment, gas is collected for the riser of the gas lift. The liquid moves from the lower compartment to the less mixed and thus stratified upper compartment, serving mainly for biomass retention and effluent polishing. The feed is introduced from the bottom of the reactor and is (during loads lower than ca 8 m.sup.3/h or 150 kg-N/d) mixed with an additional recirculation flow from the effluent of the reactor to maintain adequate up flow velocity and shear stress to favour granule formation.

[0086] The design load was 500 kg-N/d (7.1 kg-N/m.sup.3/d) but the practical maximum loading is determined by the amount of nitrogen in the sludge digestate (on average ca 700 kg-N/d). At the sludge treatment site sludge is thickened and digested (residence time ca 30 days, temperature 32-33° C.). The start-up involved two phases. The start-up regime was characterized by a relatively high influent flow rate (on average 3.6 m.sup.3/h, HRT ¼ 19.4 h) with a low concentration of nitrite (on average 120 mg-N/1). During the start-up of the anammox reactor, the aim was to produce an effluent containing nitrite at non-toxic levels. An additional economical advantage of this mode of operation was that the nitrogen removal of the sludge treatment as a whole remained high during this phase in the start-up of the anammox reactor. In the second part of the start-up, methanol dosing to the nitritation reactor was completely stopped and the reactor was running as a nitritation reactor with nitrite effluent concentrations close to 600 mg-N/l. The nitrite:ammonium ratio of circa 1:1—which is required for the anammox process was obtained automatically. A flow-adjustable recycle stream from the top of the reactor was mixed with the influent to maintain a sufficiently high up flow rate (2-3 m/h) during the phases in the start-up when the influent rate. After the reactor was converting at its design capacity of 500 kg-N/d, sludge was removed periodically from the bottom of the reactor. A total amount of 36 m.sup.3 of sludge was removed in amounts varying from 0.5 to 2 m.sup.3.

[0087] The extracted EPS's were pre-treated in line with a general method to prepare proteins before they are used in mass-spectrometry (MS) (ThermoFischer). Enzyme hydrolysis (trypsin, papain, proteinase K), dimethylmethylene blue assay (DMMB), and sodium dodecyl sulphate polyacrylamide gel electrophoresis we sequentially used to prepare samples. Glycoproteins were stained using the periodic acid-Schiff (PAS) method, Pierce™ glycoprotein staining kit. For negatively charged glycans Alcian Blue 8GX (Sigma) was used at pH 2.5. Sulphated glycans were stained using Alcian Blue at pH 1.0. Initial results showed that AGS contains around 4.5 μg sGAG per mg dry weight and Anammox around 0.33 μg sGAG per mg dry weight for the DMMB assay. A first optimization led to increased measured sGAG content (1.2% for Anammox EPS and 6.4% for AGS EPS). The annamox sGAG content was found to be between 1.4% and 2.0% (w/w) after denaturation. It was found that protein digestion and denaturation led to an increased amount of sGAG content. For annamox a concentration between 1.2% and 2.6% was found, whereas for AGS a concentration between 5.5% and 6.7% was found (w/w).

[0088] Obtained sGAG's had typical weights of 10-15 kDa, such as about 12 kDa. Also 8 kDa and 5 kDa sGAG's were found.