Process for producing a rhamnolipid produced by <i>Pseudomonas </i>or <i>Enterobacter </i>using andiroba or murumuru seed waste

Abstract

Process for producing a rhamnolipid produced by Pseudomonas or Enterobacter using andiroba or murumuru seed waste, pertaining to the sector of compounds containing monosaccharide radicals, consists of producing rhamnolipids by a biotechnological process using andiroba or murumuru seed waste, following oil extraction, as a substrate for a Pseudomonas aeruginosa, Enterobacter hormaechei or Enterobacter buriae line cultivated in a bioreactor with a non-dispersive aeration system for reducing foam, producing a rhamnolipid content of 10.5 g/L for Pseudomonas aeruginosa bacteria, in bioreactors carried out in a stirred tank with non-dispersive aeration using microporous membranes, particularly of silicone tubes, which allow oxygen to be supplied by diffusion. This type of aeration allows for various configurations, and in the embodiment of the invention, the porous membrane/tube was internally located in the liquid in the bioreactor in the form of a serpentine, under the following process conditions: pure oxygen with suitable pressure and flow rate to maintain O2 pressure in the bioreactor at 20% during the first 24 hours of the assay and stirring varying from 300 to 700 rpm, using 2 radial impellers and manual adjustment according to the decrease in the concentration of dissolved oxygen. The product produced has features that can be used primarily in the cosmetic industry due to its emulsifying, stability and non toxicity capacities.

Claims

1. A process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue, comprising the following steps: (a) reactivating the microorganism kept under refrigeration by means of growth in nutrient broth; (b) preparing an inoculum; and (c) batch bioprocessing in a stirred tank reactor under aeration conditions, wherein: step a) reactivates Pseudomonas or Enterobacter microorganisms kept under cryopreservation refrigeration at a temperature ranging from 70 to 100 C., by growing in a nutrient broth for 10 to 30 hours, at a temperature of from 25 to 40 C., on a stirring platform at a stirring speed of 170 rpm to 200 rpm, the nutrient broth comprising meat extract and peptone, which nutrients are mixed with the aid of a stirrer and are subjected to wet heat sterilization at 121 C., 1 atm, for 15 minutes; step (b) consists of preparing the inoculum by transferring the material from reactivation step (a) to a nutrient broth for 4 to 12 hours at a temperature between 20 C. to 40 C. on a stirring platform at a stirring speed of from 170 to 200 rpm; step (c) consists of batch bioprocessing the prepared inoculum of step (b) and the andiroba or murumuru seed residue in a stirred tank reactor at 300 to 700 rpm under aeration with microporous membranes through which pure oxygen at an adequate pressure and flow rate is passed to maintain the O.sub.2 pressure in the bioreactor at 20% over the first 24 hours, wherein stirring settings should be varied as the microorganism grows, in order to maintain 20% O.sub.2 saturation at all times by using radial impellers and manual or automatic settings as the oxygen concentration decreases, hence keeping the process constant at a temperature of 28 C. to 37 C., and pH 6.5 to 7.2, the mineral medium of this step comprising salts and trace elements.

2. The process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue according to claim 1, wherein: step (a) consists of reactivating the microorganisms in nutrient broth for 15 to 25 hours at a temperature between 28 C. and 35 C. on a stirring platform at a stirring speed of 180 rpm and wherein the nutrient broth comprises 3 g/L meat extract and 5 g/L peptone; step (b) consists of preparing the inoculum by transferring 1 mL of the material of step (a) into 50 mL of nutrient broth for 6 and 10 hours at a temperature between 28 C. to 35 C. on a stirring platform at a stirring speed of 180 rpm; step (c) consists of batch bioprocessing the prepared inoculum of step (b) and the andiroba or murumuru seed residue in a stirred tank reactor under aeration with microporous membranes of silicone tubes located internally into the bioreactor liquor in the shape of a coil, the process being maintained at a temperature of 30 C. and pH 6.8 and being controllable by the addition of 4 mol/L NaOH or addition of 2 mol/L H.sub.2SO.sub.4.

3. The process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue according to claim 1, wherein: step (a) consists in reactivating the microorganism in nutrient broth for 21 hours at a temperature of 30 C.; and step (b) consists of preparing the inoculum by transferring the material from step (a) into the nutrient broth for 8 hours at 30 C.

4. The process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue according to claim 1, wherein it uses Pseudomonas aeruginosa ARS-NRRL B-59183 or ARS-NRRL B-59184 or ARS-NRRL B-59188 or ARS-NRRL B-59193; or Enterobacter hormaechei ARS-NRRL B-59185 or Enterobacter buriae ARS-NRRL B-59189 bacteria.

5. The process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue according to claim 1, wherein Pseudomonas aeruginosa ARS-NRRL B-59183 or ARS-NRRL B-59184 or ARS-NRRL B-59188 or ARS-NRRL B-59193 bacteria are used.

6. The process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue, according to claim 1, wherein the bioreactor culture of step (c) is carried out in mineral medium comprising 3.5 g/L dibasic sodium phosphate (Na.sub.2HPO.sub.4), 1.5 g/L potassium phosphate (KH.sub.2PO.sub.4), 1.0 to 3.0 g/L ammonium sulfide (NH.sub.4).sub.2SO.sub.4, 0.2 g/L magnesium sulfide (MgSO.sub.4.7H.sub.2O), 0.01 g/L calcium chloride (CaCl.sub.2.2H.sub.2O); 0.06 g/L ammonium iron(III) citrate, 0 a 2.0 g/L glucose, 100 g/L solid Andiroba residue, 1.0 ml/g/L trace element solution, q.s.p. 1 L distilled H.sub.2O; and the trace element solution comprising 0.3 g/L boric Acid (H.sub.3BO.sub.3), 0.2 cobalt chloride (CoCl.sub.2.6H.sub.2O); 0.1 zinc sulfate (ZnSO.sub.4.7H.sub.2O), 0.03 g/L manganese chloride (MnCl.sub.2.4H.sub.2O), 0.03 g/L sodium molybdate (NaMoO.sub.4.2H.sub.2O), 0.02 g/L nickel chloride (NiCl.sub.2.6H.sub.2O), 0.01 g/L copper sulfate (CuSO.sub.4.5H.sub.2O), q.s.p. 1 L Distilled H.sub.2O.

7. The process for obtaining rhamnolipid from Pseudomonas or Enterobacter using andiroba or murumuru seed residue, according to claim 1, wherein the used andiroba and murumuru residues are pre-ground and, after grinding, when necessary, the residues are sifted through a set of sieves ranging from 1.0 to 0.25 mm.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Plot showing the surface tension of strains Pseudomonas aeruginosa ARS-NRRL B-59183, Pseudomonas aeruginosa ARS-NRRL B-59184, Enterobacter hormaechei ARS-NRRL B-59185, Pseudomonas aeruginosa ARS-NRRL B-59188, Enterobacter buriae ARS-NRRL B-59189 and Pseudomonas aeruginosa ARS-NRRL B-59193 using glucose, murumuru and andiroba.

(2) FIG. 2: Plot showing the rhamnolipid production kinetics after daily withdrawals of samples over 240 hours of culture of Pseudomonas aeruginosa in a mineral medium with 10% of androba, murumuru residues or a mixture thereof as growth substrates in a rotary incubator at 180 rpm, 30 C.

(3) FIG. 3: Plot of the surface tension profile measured in samples withdrawn daily over 240 hours of culture of Pseudomonas aeruginosa in mineral medium with 10% of andiroba, murumuru residues or the mixture thereof as growth substrates in a rotary incubator at 180 rpm, 30 C.

(4) FIG. 4: Plot of rhamnolipid production kinetics in a bioreactor in an oxygen pressure- and pH-controlled bubble-free system in mineral medium with 10% andiroba seed residues and 2% glucose and 3 g/L ammonium sulfate.

(5) FIG. 5: Plot of the surface tension kinetics in a bioreactor in an oxygen pressure- and pH-controlled bubble-free system in mineral medium with 10% andiroba seed residue and 2% glucose and 3 g/L ammonium sulfate.

(6) FIG. 6: Plot showing the foaming test comparing the rhamnolipid produced with andiroba seed residue and with commercially available rhamnolipid (Sigma Aldrich).

(7) FIG. 7: Plot showing a comparative assessment of foam stability using the rhamnolipid produced with andiroba seed residue and commercially available rhamnolipid (Sigma Aldrich).

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS

(8) Rhamnolipid production is carried out according to the following steps:

(9) step (a) of the process consists of reactivating the microorganism maintained under refrigeration at a temperature of from 70 to 100 C. by means of growth in nutrient broth for 10 to 30 hours, preferably 15 to 25 hours, preferably 21 hours, between 25 and 40 C., preferably at 28 C. and 35 C., more specifically 30 C., on a stirring platform at a stirring speed of 170 rpm to 200 rpm, preferably 180 rpm.

(10) The microorganism of step (a) consists of one of the bacteria listed in Table 1, preferably Pseudomonas aeruginosa, which is preferably kept refrigerated under cryopreservation in ultrafreezer at a temperature of from 70 to 100 C., the nutrient broth preferably comprising 3 g/L meat extract and 5 g/L peptone. These nutrients are mixed with the aid of a magnetic stirrer and subjected to wet heat sterilization at 121 C., 1 atm, for 15 minutes.

(11) Step (b) consists of the preparation of the inoculum, wherein each 1 mL from reactivation step (a) is transferred, preferably in 50 mL of nutrient broth, over 4 to 12 hours, preferably between 6 and 10 hours, more specifically about 8 hours, at a temperature of from 20 C. to 40 C., preferably about 28 C. to 35 C., more specifically at 30 C., on a stirring platform at a stirring speed of from 170-200 rpm, preferably 180 rpm.

(12) Step (c) consists of batch bioprocessing in a stirred tank reactor with aeration, preferably using microporous membranes, more specifically made of silicone tubes that supply oxygen by bubble-free diffusion avoiding foaming.

(13) Such aeration type provides different configurations, preferably the membrane/porous tube being located internally into the bioreactor liquor in the shape of a coil. Pure oxygen is passed through this hose at a suitable pressure and flow rate to maintain the O.sub.2 pressure in the bioreactor, preferably at 20% over the first 24 hours. Stirring should be carried out in a range of from 300 to 700 rpm, adjusted as the microorganism grows in order to maintain a 20% O.sub.2 saturation at all times using radial impellers and manual or automatic settings being adjusted as the dissolved oxygen concentration decreases, this being a differential of the process over those previously disclosed in the state of the art.

(14) The bioreactor culture of step (c) is carried out in mineral medium consisting of salts and trace elements as per RAMSAY et al. (RAMSAY, B. A.; LOMALIZA, K.; CHAVARIE, C.; DUBE, B.; BATAILLE, P.; RAMSAY, J. A. Production of Poly-(P-Hydroxybutyric-Co-3-Hydroxyvaleric) Acids. Applied and Environmental Microbiology, p. 2093-2098 v. 56 (7), 1990) as described in Tables 2A and 2B. The process should be kept constant at a temperature of 28 C. to 37 C., preferably 30 C. and a pH of 6.5 to 7.2, preferably pH 6.8, and may be automatically controlled by addition of NaOH, preferably at 4 mol/L, or by the manual addition of H.sub.2SO.sub.4, preferably at 2 mol/L.

(15) TABLE-US-00001 TABLE 1 List of bacterial strains used in the steps for producing rhamnolipid. Bank of Origin and IPT Strain name registration number number Pseudomonas aeruginosa (Schroeter ARS-NRRL B-59183 998 1872) Migula 1900 E03-31 Pseudomonas aeruginosa (Schroeter ARS-NRRL B-59184 999 1872) Migula 1900 E03-36 Enterobacter hormaechei O'Hara et al. ARS-NRRL B-59185 1000 1990E03-50 Pseudomonas aeruginosa (Schroeter ARS-NRRL B-59188 1001 1872) Migula 1900H05-11 Enterobacter buriae Brenner et al. 1988 ARS-NRRL B-59189 1002 H05-14 Pseudomonas aeruginosa (Schroeter ARS-NRRL B-59193 1005 1872) Migula 1900 H05-45

(16) TABLE-US-00002 TABLE 2A Composition of the mineral medium used for growth and rhamnolipid production. Mineral medium components Concentration (g/L) Dibasic sodium phosphate (Na.sub.2HPO.sub.4) 3.5 Potassium phosphate (KH.sub.2PO.sub.4) 1.5 Ammonium sulfide (NH.sub.4).sub.2SO.sub.4 1.0 to 3.0 Magnesium sulfide (MgSO.sub.47H.sub.2O) 0.2 Calcium chloride (CaCl.sub.22H.sub.20) 0.01 Ammonium iron(III) citrate 0.06 Solution of trace elements (Table 3) 1.0 mL Glucose 0 to 2.0 Andiroba solid residue 100 Distilled H.sub.2O q.s.p. 1 L

(17) TABLE-US-00003 TABLE 2B Composition of the trace element solution Components of the trace element solution Concentration (g/L) Boric Acid (H.sub.3BO.sub.3) 0.3 Cobalt chloride (CoCl.sub.26H.sub.2O) 0.2 Zinc sulfate (ZnSO.sub.47H.sub.2O) 0.1 Manganese chloride (MnCl.sub.24H.sub.2O) 0.03 Sodium molybdate (NaMoO.sub.42H.sub.2O) 0.03 Nickel chloride (NiCl.sub.26H.sub.2O) 0.02 Copper sulfide (CuSO.sub.45H.sub.2O) 0.01 Distilled H.sub.2O q.s.p. 1 L

(18) All strains listed in Table 1 exhibited rhamnolipid production using murumuru and andiroba seed residues, evidencing that they can be used to obtain surfactant.

(19) From among the assessed strains all of them exhibited similar surface tension results using both andiroba and murumuru seed residues.

(20) Rhamnolipid production is carried out according to the following steps:

(21) Step (a) of the process consisted of reactivating Pseudomonas aeruginosa, or Enterobacter hormaechei or Enterobacter buriae over 21 hours at a temperature of 30 C. on a stirring platform at a stirring speed of 180 rpm, the nutrient broth containing meat extract and peptone at 3 g/L and 5 g/L, respectively.

(22) Step (b) consisted of preparing the inoculum, wherein each 1 mL from reactivation step (a) was inoculated into 50 mL of the nutrient broth for about 8 hours at a temperature of 30 C. on a stirring platform at a stirring speed of 180 rpm.

(23) Step (c) consisted of a batch bioprocessing in a stirred tank reactor under aeration by means of silicone tubes that allowed the supply of pure oxygen by bubble-free diffusion, at suitable pressure and flow rate to maintain the O.sub.2 pressure at 20% over the first 24 hours, hence avoiding foaming. Stirring at 300 rpm to 700 rpm in the reactor was carried out by radial impellers and the dissolved oxygen concentration decrease was manually set.

(24) The process was kept constant at a temperature of 30 C. and a pH of 6.8, being controlled by the addition of 4 mol/L NaOH, or by 2 mol/L H.sub.2SO.sub.4.

(25) Andiroba and murumuru residues used in the tests were pre-ground using a domestic food processor at 10-second pulses or an industrial blender equipped with a high rotation stainless steel cup (22,000 rpm) and a power of 1200 w. After grinding, when necessary, the residues were sifted through a set of sieves ranging from 1.0 to 0.25 mm.

(26) Results

(27) The Pseudomonas aeruginosa strain was able to synthesize about 10 g/L rhamnolipid from ground andiroba residues used as an alternative substrate at a concentration of 100 g/L.

(28) Rhamnolipid production using andiroba seed residue was about 4 times higher than the production using murumuru seed residue. Surface tension of the culture supernatant with andiroba seed residue (<35 dynes/cm) was also better than the tension obtained with murumuru seed residue (>35 dynes/cm).

(29) Andiroba and murumuru seed residues were characterized with respect to their composition, showing the presence of carbohydrates, lipids and proteins in both residues. The murumuru seed residue exhibited 53.4% carbohydrates, 29.0% lipids and 6.8% proteins. The andiroba seed residue exhibited 63.4% carbohydrates, 14.8% lipids and 10.4% proteins. Other components such as ash and moisture were also quantified, wherein 1.4% ash was found in murumuru seed residue and 4.3% ash in andiroba seed residue.

(30) The rhamnolipid biosurfactant molecule produced using andiroba seed residue had a surface tension of from 30 to 40 dynes/cm and an emulsification index greater than 60%. With murumuru residue, the surface tension obtained was 40 to 50 dynes/cm and the emulsification index was also greater than 60%.

(31) The produced rhamnolipid did not present any cytotoxicity in in vitro assays and exhibited good performance in terms of surfactant and foaming properties. Said performance was measured in an apparatus that evaluates the foaming ability of surfactant-containing solutions via stirring and assessing stability of the foam obtained after stirring. The average foaming values are obtained after four foam decay readings for five minutes. In this evaluation a corrected concentration of surfactants was used considering a sample content to 0.1% of active. Tests were made using the rhamnolipid produced in a bioreactor and dried in a freeze-drying equipment versus Sigma Aldrich standard rhamnolipid.

(32) The average foaming results for rhamnolipids produced with andiroba seed residue were shown to be better than those obtained using Sigma Aldrich standard rhamnolipid.

(33) Rhamnolipid foam stability was shown to very good when compared to the rhamnolipid standard, which makes the product interesting to be applied in cosmetics as it maintains the foam stable over the course of the analysis.

(34) The rhamnolipid molecule was also shown to be quite stable, which means that the produced biosurfactant can be applied in the cosmetic industry due to its in vitro emulsifying ability, stability and non-toxicity.