Semi-continuous process for the production of rhamnolipids at high yield and titer
10144943 ยท 2018-12-04
Assignee
Inventors
Cpc classification
International classification
Abstract
Provided is a semi-continuous fermentation method of a rhamnolipid producing microorganism to produce rhamnolipids. The fermentation may be run as a batch process but at the end of the fermentation, at least about 70% of the fermentation medium comprising one or more rhamnolipids is drawn out and the new culture medium (feedstock) is fed in as a replacement. This process may be repeated for at least about one month without having to sacrifice RL yield and titer. It allows the fermenter to be utilized at a higher capacity with less downtime for clean-up compared to batch and fed batch fermentation strategies.
Claims
1. A semi-continuous method for producing a plurality of fermentation media comprising one or more rhamnolipids comprising: (a) culturing a rhamnolipid producing Pseudomonas aeruginosa in culture medium comprising at least one vegetable oil as a carbon source, an emulsifier, at least one nitrogen source, at least one phosphorous source, at least one magnesium source, at least one potassium source, at least one sulfur source, at least one chloride source, and at least one sodium source between for about 2 to about 5 days to obtain a first fermentation medium comprising one or more rhamnolipids; (b) removing at least about 70% of the total volume of said first fermentation medium obtained in (a), wherein said first fermentation medium removed comprises one or more rhamnolipids; (c) replacing said first fermentation medium removed in (b) with culture medium having the composition set forth in step (a); (d) repeating steps (a)-(c) at least one time to obtain a subsequent fermentation medium comprising rhamnolipids wherein at least about 45 g/L of rhamnolipids are produced.
2. The method according to claim 1, wherein said emulsifier is selected from the group consisting of Arabic gum, guar gum and rhamnolipids.
3. The method according to claim 1, wherein said emulsifier is present in the amount of about 0.1-20% by weight.
4. The method according to claim 1, wherein said vegetable oil is selected from the group consisting of olive oil, rapeseed oil, corn oil, sunflower oil, canola oil and soybean oil.
5. The method according to claim 1, wherein said carbon source is present at a concentration of about 6-12% by weight.
6. The method according to claim 1, wherein said nitrogen source is selected from the group consisting of NaNO.sub.3, Urea (CO(NH.sub.2).sub.2) and NH.sub.4Cl.
7. The method according to claim 1, wherein said nitrogen source is present in the amount of about 1-4 g/L.
8. The method according to claim 1, wherein said phosphorous source is selected from the group consisting of H.sub.3PO.sub.4 and K.sub.2HPO.sub.4.
9. The method according to claim 1, wherein said phosphorous source is present in the amount of about 1-3 g/L.
10. The method according to claim 1, wherein said magnesium source is selected form the group consisting of MgSO.sub.4.7H.sub.2O and MgCl.sub.2.
11. The method according to claim 1, wherein said magnesium source is present in the amount of about 0.001-0.2 g/L.
12. The method according to claim 1, wherein said potassium source is KCl and KOH.
13. The method according to claim 1, wherein said potassium source is present in the amount of about 0.1 to about 1 g/L.
14. The method according to claim 1, wherein said sodium source is NaCl, NaNO.sub.3, and NaOH.
15. The method according to claim 1, wherein said sodium source is present in the amount of about 1-10 g/L.
16. The method according to claim 1, wherein said chloride source is KCl and NaCl.
17. The method according to claim 1, wherein said chloride source is present in the amount of about 0.1-1 g/L.
18. The method according to claim 1, wherein said sulfur source is H.sub.2SO.sub.4.
19. The method according to claim 1, wherein said sulfur source is present in the amount of about 0.1-1 g/L.
20. The method according to claim 1, which further comprises adding a composition comprising one or more micronutrients at a concentration of no more than 20 mg/L of micronutrient solution to said culture medium in step (a) at 0.1% v/v of total fermentation volume per day.
21. The method according to claim 20, wherein said micronutrient is a Fe, Mn, Zn, Na or Cu salt.
22. The method according to claim 21, wherein said Cu salt is at least one of CuCl.sub.2.2H.sub.2O and CuSO.sub.4.5H.sub.2O.
23. The method according to claim 21, wherein said Cu salt is present in the amount of about 0.5-3 g/L of micronutrient solution.
24. The method according to claim 21, wherein said Mn is at least one of MnSO4.H.sub.2O and MnCl.sub.2.4H.sub.2O.
25. The method according to claim 21, wherein said Mn is present in the amount of about 0.1-1.5 g/L of micronutrient solution.
26. The method according to claim 21, wherein said Zn is ZnSO.sub.4.7H.sub.2O or ZnCl.sub.2.
27. The method according to claim 21, wherein said Zn is present in the amount of about 0.5.
28. The method according to claim 21, wherein said Fe is at least one of FeCI.sub.3.6H.sub.2O or FeSO.sub.4.
29. The method according to claim 21, wherein said Fe is present in the amount of about 0.1-1 g/L of micronutrient solution.
30. The method according to claim 21, wherein said sodium salt is Na.sub.3C.sub.6H.sub.5O.sub.7.2H.sub.2O, NaC.sub.6H.sub.7O.sub.7, and Na.sub.2C.sub.6H.sub.6O.sub.7.
31. The method according to claim 21, wherein said sodium salt is present in the amount of about 1-5 g/L of micronutrient solution.
32. The method according to claim 20, wherein said micronutrient composition is added continuously.
33. The method according to claim 20, wherein said micronutrient composition is added daily.
34. The method according to claim 1, wherein said method further comprises adding anti-foam.
35. The method according to claim 34, wherein said anti-foam is silicon-based or carbon-based anti-foam.
36. The method according to claim 1, wherein said Pseudomonas aeruginosa is cultured at a temperature of about 30-37 ? C. and/or at a pH of about 6-8.6.
37. The method according to claim 1, wherein step (d) is repeated for at least about 30 days.
38. The method according to claim 1, wherein said carbon source is vegetable oil.
39. The method according to claim 20, wherein said composition comprises a Fe, Mn, Zn, Na and Cu salt.
40. The method according to claim 1, wherein said method further comprises adding an emulsifier to said culture medium prior to beginning said culturing step (a).
Description
BRIEF DESCRIPTION OF THE FIGURES
(1)
(2)
(3)
DETAILED DESCRIPTION
(4) Provided herein is a semi-continuous fermentation method for producing a plurality of fermentations comprising one or more rhamnolipids. In a particular embodiment, the rhamnolipid may have the structure (I).
(5) ##STR00001##
Where m=2, 1 or 0, in particular 1 or 0, n=1 or 0, or in particular 1, R.sup.1 and R.sup.2=independently of one another identical or different organic radical with 2 to 24, preferably 5 to 13, carbon atoms, in particular optionally branched, optionally substituted, in particular hydroxyl-substituted, optionally unsaturated, in particularly optionally mono-, di- or triunsaturated, alkyl radical, preferably one selected from the group consisting of pentenyl, heptenyl, nonenyl, undeceny and tridecenyl and (CH2)o-CH.sub.3 where o=1 to 23, preferably 4 to 12.
(6) Both the main chain as well as the branches may furthermore contain heteroatoms as for instance N, O, S, Se or Si or a carbon atom may be replaced by one of these heteroatoms. An aliphatic moiety may be substituted or unsubstituted with one or more functional groups. Substituents may be any functional group, as for example, but not limited to, amino, amido, carbonyl, carboxyl, hydroxyl, nitro, thio and sulfonyl.
(7) Rhamnolipid Producing Microorganism
(8) As noted above, the method comprises culturing a rhamnolipid producing microorganism. A rhamnolipid producing microorganism may be a host cell producing rhamnolipids. A recombinant host cell producing rhamnolipids may be a host cell, such as a bacterial cell that expresses a RhlA gene or ortholog thereof and/or a RhlB gene or ortholog thereof, and/or a RhlC gene or ortholog thereof, and/or RhlR gene or ortholog thereof, and/or RhlI gene or ortholog thereof, and/or RhlG gene or ortholog thereof and others.
(9) Alternatively, a rhamnolipid-producing microorganism may be any microorganism, such as bacteria, which has the capacity to synthesize/produce rhamnolipids under suitable conditions which includes but is not limited to bacterium of the phyla Actinobacteria, Fimicutes and Proteobacteria. In a particular embodiment, the rhamnolipid-producing microorganism is a bacterium of the Gammaproteobacteria class. In a further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonadales order. In yet another further embodiment, the rhamnolipid producing microorganism is a bacterium of the Pseudomonadacae family. In an even further embodiment, the rhamnolipid-producing microorganism is a bacterium of the Pseudomonas genus, such as P. alcaligenes, P. aeruginosa, P. chlororaphis, P. clemancea, P. collierea, P. fluorescens, P. luteola, P. putida, P. stutzeri and P. teessidea. In a further embodiment, the rhamnolipid-producing microorganism is P. aeruginosa.
(10) Culture Medium
(11) The rhamnolipid containing microorganism is cultured in culture medium. Said culture medium comprises at least one carbon source, at least one nitrogen source, at least one phosphorous source, at least one sulfur source, at least one sodium source, at least one magnesium source, at least one potassium source, at least one sulfur source and at least one chloride source.
(12) The carbon source may be a monosaccharide, e.g. glucose, a disaccharide, e.g. sucrose, a sugar alcohol, e.g. glycerol, a long chain alkane, e.g., n-hexadecane, a fatty acid such as caprylic acid (also termed octanoic acid), vegetable oils (fresh or waste; e.g., soybean oil) or mixtures thereof, organic acids (e.g. lactic acid, acetic acid, citric acid, propionic acid), alcohols (e.g. ethanol), and mixtures of these. In a particular embodiment, the carbon source is a vegetable oil selected from the group consisting of olive oil, rapeseed oil, olive oil, corn oil, sunflower oil, canola oil and soybean oil. The carbon source may be present in the amount of about 6% to about 12% w/w.
(13) The nitrogen source may be ammonium sulfate, ammonium phosphate, urea, yeast extract, meat extract, peptone, and corn steep liquor. In a particular embodiment, the nitrogen source is NaNO.sub.3. In yet another embodiment, the nitrogen may be present in the amount of about 1-10 g/L.
(14) The phosphorous source may in a particular embodiment be H.sub.3PO.sub.4 or K.sub.2HPO.sub.4. In yet another particular embodiment, said phosphorous is present in the amount of about 1-10 g/L.
(15) The magnesium ion in a particular embodiment may be MgSO.sub.4*7H2O and/or MgCl.sub.2. In a particular embodiment, the magnesium is present in the amount of about 0.01-1 g/L.
(16) The potassium may be KCl and/or KOH. In a particular embodiment, the potassium is present in the amount of about 0.1 to about 2 g/L.
(17) The sodium may be NaCl, NaNO.sub.3, and NaOH. In a particular embodiment, said sodium ion is present in the amount of about 1-15 g/L.
(18) The chloride may be KCl and NaCl. In a particular embodiment, said chloride ion is present in the amount of about 0.1-1 g/L.
(19) The sulfur may be H.sub.2SO.sub.4. In a particular embodiment, said sulfur ion is present in the amount of about 0.1-1 g/L.
(20) The sulfur, chloride and nitrogen sources may be derived from the aqueous layer, or also referred to as the aqueous liquid phase or aqueous phase of an acid treated and aged microorganism containing fermentation medium obtainable using procedures described in application Ser. No. 14/992,995. In a specific embodiment, the fermentation or culture medium comprising one or more rhamnolipids may be aged by incubating for at least about 1 day and between about 24-72 hrs at between about 0-30 C. In a particular embodiment, the aged aqueous medium may be treated with acid so the culture medium is adjusted to a pH of about 1.5 to 2.5, preferentially, about 2.05 to about 2.15. The acid can be an organic acid such as acetic acid, or a mineral acid. In a preferred embodiment, the acid is a mineral acid, e.g. HCl, H.sub.2SO.sub.4, HNO.sub.3, or H.sub.3ClO.sub.4. As a result, an aqueous liquid phase, oily phase and solid phase is generated. The aqueous liquid phase is removed using procedures known in the art and in a specific embodiment using methods set forth above (e.g., filtration, or centrifugation or settling combined with decanting).
(21) The culture medium may further comprise an emulsifier. In a particular embodiment, the emulsifier is selected from the group consisting of Arabic gum, guar gum and rhamnolipids. In yet another particular embodiment, the ratio of emulsifier to carbon source in said culture medium is between about 0.1% to about 20% w/w. In yet another particular embodiment, wherein said emulsifier may be present in the amount of about 5-10% by weight.
(22) In a particular embodiment, the culture or fermentation medium is sterilized using methods known in the art. These methods may be filtration based, heat based, chemical based or ultraviolet light radiation based. In a particular embodiment, the heat based treatment may be via moist heat sterilization, particularly autoclaving.
(23) In one embodiment, the aqueous medium (e.g., fermentation medium) may be sterilized by one of the above procedures. In another embodiment, the fermentation media may be sterilized by more than one of the procedures set forth above and these sterilizations could be in any order. It may be sterilized in the fermentation during the first cycle of fermentation, but should be sterilized in another vessel in subsequent cycles.
(24) Micronutrient Composition
(25) As noted above, said method may further comprise adding a micronutrient solution or composition. Said micronutrient may be a trace of Fe, Mn, Zn, Cu, Na. In a particular embodiment, said micronutrient is a Fe, Mn, Zn, Na or Cu salt. In a more particular embodiment said micronutrient composition comprises Fe, Mn, Zn, Na and Cu salts. The composition may be sterilized by filtration.
(26) In particular embodiments, said Cu salt is at least one of CuCl.sub.2*2H.sub.2O and CuSO.sub.4*5H.sub.2O and may be present in the amount of about 0.5-3 g/L of micronutrient solution; said Mn salt is at least one of MnSO4*H.sub.2O and MnCl.sub.2.4H.sub.2O and may be present in the amount of about 0.1-1.5 g/L of micronutrient solution; said Zn salt is ZnSO.sub.4*7H.sub.2O or ZnCl.sub.2 and may be present in the amount of about 0.5-3 g/L of micronutrient solution; said Fe salt is at least one of FeCl.sub.3*6H.sub.2O or FeSO.sub.4 and may be present in the amount of about 0.1-1 g/L of micronutrient solution; said sodium salt is Na.sub.3C.sub.6H.sub.5O.sub.7*2H.sub.2O and may be present in the amount of about 1-5 g/L of micronutrient solution.
DESCRIPTION OF SPECIFIC EMBODIMENTS
EXAMPLE 1
Culture Medium Preparation
(27) A composition of the 8% soybean oil in the culture medium for the fermentation of Pseudomonas aeruginosa for rhamnolipids production is shown in Table 2, infra. Gum Arabic is used at 10% w/w of soybean oil.
(28) TABLE-US-00002 TABLE 2 Culture Medium Composition Component Concentration Gum Arabic 8.00 g/L Soybean oil 80.00 g/L 85% H.sub.3PO.sub.4 9.69 g/L 99% NaOH 5.21 g/L 99% MgSO.sub.4*7H.sub.2O 0.50 g/L 99% KCl 1.00 g/L 99% NaNO.sub.3 15.00 g/L 98% H.sub.2SO.sub.4 0.92 g/L Deionized water The rest
(29) To make the culture medium, gum Arabic is first dissolved in deionized water to obtain 5% w/w gum Arabic solution under agitation at 150-250 rpm and at 40-45 C in a separate container. This step will take at least 30 min to allow all gum Arabic to dissolve in water. Second, mix soybean oil and 5% gum Arabic solution with deionized water using a blender such as kitchen blender. Make sure it is well mixed to obtain an emulsion (i.e. the solution becomes white like milk). After the emulsion of soybean oil is obtained, H.sub.3PO.sub.4 is added into the emulsion under stirring and the next chemical NaOH is added in after H.sub.3PO.sub.4 is well dissolved, The next chemical which are MgSO.sub.4 then KCl then NaNO.sub.3 and the last H.sub.2SO.sub.4 are added into the emulsion under stirring. Before adding the next chemical, make sure that it is well dissolved in the emulsion. The culture medium then can be stream sterilized (autoclave).
EXAMPLE 2
Micronutrient Composition Preparation
(30) The composition of micronutrient is shown in Table 3. Please note that the concentration of each salt is in g/L of micronutrient solution.
(31) TABLE-US-00003 TABLE 3 Composition of Micronutrient Component Concentration Na.sub.3C.sub.6H.sub.5O.sub.7*2H.sub.2O 2.0 g/L FeCl.sub.3*6H.sub.2O 0.3 g/L ZnSO.sub.4*7H.sub.2O 1.4 g/L CuCl.sub.2*2H.sub.2O 1.2 g/L CuSO.sub.4*5H.sub.2O 1.2 g/L MnSO.sub.4*H.sub.2O 0.8 g/L
(32) All chemicals are ACS grades (highest purity available). Na.sub.3C.sub.6H.sub.5O.sub.7*2H.sub.2O is first added into deionized water using stirring. After it is all dissolved, the next chemical which is FeCl.sub.3*6H.sub.2O can be added in. This step is repeated until all the chemicals listed in the table are added into the solution in the order of FeCl.sub.3*6H.sub.2O then ZnSO.sub.4*7H.sub.2O then CuCl.sub.2*2H.sub.2O then CuSO.sub.4*5H.sub.2O and MnSO.sub.4*H.sub.2O the last. The micronutrient can be sterilized using a 0.2 micron sterilized filtration. Do not steam autoclave the micronutrient.
EXAMPLE 3
Seed Culture of P. aeruginosa (Schroeter) Migula (R4 Strain)
(33) R4 strain obtained from ATCC #55734 is first inoculated in agar plates containing 40 g/L Tryptic Soy Agar at 32 C for 18-24 hours. After R4 colonies are formed, a single colony is then cultured in 5 ml of 20 g/L LB Broth (Lennox) in a shake tube at 37 C for 20-24 hours. The final OD.sub.600 is about 3-5 and the LB Broth (Lennox) solution in the shake tube will change from yellow to green. Then, the R4 culture obtained from a shake tube is inoculated in a shake flask containing 20 g/L LB Broth (Lennox) at 1% inoculation and incubated at 37 C for 20-24 hours. This process is repeated as necessary in order to generate sufficient inoculum of R4 required for the fermentation. The LB Broth (Lennox) is obtained from Sigma Aldrich #3022 which contains 10 g/L Tryptone, 5 g/L yeast extract and 5 g/L NaCl.
EXAMPLE 4
Semi-Continuous Fermentation of Rhamnolipids with Deionized Water and 10% w/w Gum Arabic (as Emulsifier) to Soybean Oil
(34) The fermentation of P. aeruginosa (Schroeter) Migula obtained from ATCC #55734 (R4) is performed in 10 L bioreactor and is schematically set forth in
(35) The fermentation is run at a temperature of 37 C with the initial pH of the culture medium of 6.2 with no pH control over the course of fermentation. The stirring rate automatically increases as necessary in order to keep % dissolved oxygen (% DO) at 15%-20%. The stirring rate goes up 500 rpm before the air flow rate increases from 1.5 to 3.5 L/min at 40-48 hours post inoculation in order to keep up with the oxygen demand of the microbes during growth rate. After 60 hours post inoculation, the pH is increasing after it slightly dips down (or remains stable). Additionally, the % DO increases while the agitation and air flow are at the lowest values (250 rpm and 1.5 L/min, respectively) indicating that the fermentation is completed at 72 hours. This can be confirmed with a residual soybean oil in fermentation medium comprising one or more rhamnolipids to be less than 0.8%.
(36) The peristatic pump is then started to remove 6 L (75% of the total) of the fermentation medium comprising one or more rhamnolipids thru line #5. Once 6 L of broth is removed which so called Draw#1, the recently sterilized 6 L of 8% soybean oil culture medium prepared in sterilized tank 20 is fed into the fermenter 10 using a gravity feed. The initial fermentation pH is 6.5. The fermentation parameters described in the above paragraph are used. In this case, after 48 hours, the fermentation is completed and 75% of the fermentation medium comprising one or more rhamnolipids is being ready to be drawn out (Draw#2). Subsequently, the next batch of sterilized soybean oil culture medium is fed in from the sterilized tank 20. The process repeats for 6 weeks with no sign of loss in rhamnolipids yields and titers before the fermenter is shut down for cleaning. A change in pH over the course of fermentation between the drawing and for each drawing is shown in the
(37) TABLE-US-00004 TABLE 4 Rhamnolipid Conc and Titer During Semi- Continuous Fermentation With Soybean Oil Fermenta- % Initial tion time Total RL Soybean (between draw) RL titer Micro- Draw# oil (h) (g/L) (g/L/h) nutrient 1 8.0% 72 66 0.92 Continuously 2 6.0% 48 62 1.29 added 3 6.0% 46 71 1.54 4 7.5% 69 77 1.12 5 6.0% 46 74 1.61 Added 6 6.0% 48 74 1.54 daily 7 7.5% 71 79 1.11 8 6.0% 47 70 1.49 9 6.0% 46 66 1.43 10 7.5% 69 65 0.94 Continuously 11 6.0% 48 68 1.42 added 12 6.0% 48 69 1.44
EXAMPLE 5
Semi-Continuous Fermentation of Rhamnolipids with Deionized Water, 5% w/w Gum Arabic to Soybean Oil with Continuously Added Micronutrient
(38) The composition of the culture medium used in Table 5. 7.3 L of 8% soybean oil with 5% w/w gum Arabic to soybean oil is prepared as described in Example 1. All chemicals used in this example are industry grade containing impurities.
(39) TABLE-US-00005 TABLE 5 Composition of Culture Medium w/5% Gum Arabic, 8% Soybean Oil and 5% R4 Inoculum Component Concentration Gum Arabic 4.00 g/L Soybean oil 80.00 g/L 85% H.sub.3PO.sub.4 9.69 g/L 99% NaOH 5.21 g/L 99% MgSO.sub.4*7H.sub.2O 0.50 g/L 99% KCl 1.00 g/L 99% NaNO.sub.3 15.00 g/L 98% H.sub.2SO.sub.4 0.92 g/L Deionized water The rest
(40) The fermentation is run as in Example 4 except that 5% (360 ml) R4 inoculum is used to inoculate the fermenter and 77% of the fermentation medium comprising one or more rhamnolipids (5.6 L) is drawn out and the new culture medium contains 5% w/w gum Arabic to soybean oil. Micronutrient is prepared as described in Example 2. By diluting 7.3 ml of micronutrient in 32.7 ml deionized water (per day), it is added continuously at the flow rate of 40 ml/day using a peristatic pump. Silicon based antifoam (DOW AFE-1510) is automatically added to knock down the foam during fermentation. The stirring rate automatically increases as necessary in order to keep % dissolved oxygen (% DO) at 15%-20% with air flow rate of 1.5 L/min. Pure oxygen is additionally added to the fermenter at 0.005-0.1 L/min in order to keep the agitation down and thus, less foaming issue. A typical change in pH over the course of fermentation post inoculation or after filling with the new culture medium is shown in
(41) TABLE-US-00006 TABLE 6 Rhamnolipid Conc and Titer Using Semi- Continuous Method Using 5% R4 Inoculum Fermenta- % Initial tion time Total RL Soybean (between draw) RL titer Draw# oil (h) (g/L) (g/L/h) 1 8.0% 59 56 0.90 2 7.7% 70 79 1.13 3 6.1% 65 76 1.17 4 8.0% 96 70 0.73 5 7.7% 91 76 0.84 6 8.0% 82 70 0.85 7 7.7% 70 78 1.11
EXAMPLE 6
Semi-Continuous Fermentation of Rhamnolipids with Deionized Water, 5% w/w Gum Arabic to Soybean Oil with Continuously Added 2? of Micronutrient
(42) In this example, the culture medium and fermentation parameters are the same as shown in Example 5 except that the amount of micronutrient used is double. By diluting 14.6 ml of micronutrient (prepared in Example 2) in 26.4 ml deionized water (per day), it is added continuously at the flow rate of 40 ml/day using a peristatic pump. The stirring rate automatically increases as necessary in order to keep % dissolved oxygen (% DO) at 15%-20% with air flow rate of 1.5 L/min. Pure oxygen is additionally added to the fermenter at 10-30% of air flow to keep the total flow rate constant at 1.5 L/min in order to keep the agitation down and thus, less foaming issue. The rhamnolipid (RL) concentration and titer are shown in Table 7 infra.
(43) TABLE-US-00007 TABLE 7 RL Conc and Titer During Semi-Continuous Fermentation With 2X Micronutrient Fermenta- % Initial tion time Total RL Soybean (between draw) RL titer Draw# oil (h) (g/L) (g/L/h) 1 7.7% 73 75 1.03 2 6.1% 48 64 1.33 3 7.7% 68 69 1.01 4 6.1% 69 66 0.96
EXAMPLE 7
Semi-Continuous Fermentation of Rhamnolipids with 8% Soybean Oil and 85% Cold Tap Water/15% Aqueous Top Layer Waste Stream from Fermentation Medium Comprising One or More Rhamnolipids
(44) The composition of the culture medium used in this Example is shown in Table 8.
(45) TABLE-US-00008 TABLE 8 Culture Medium with Aqueous Layer Component Concentration Soybean oil 80.00 g/L 85% H.sub.3PO.sub.4 9.69 g/L 99% NaOH 5.21 g/L 99% MgSO.sub.4*7H.sub.2O 0.50 g/L 99% KCl 1.00 g/L 99% NaNO.sub.3 15.00 g/L Aqueous layer waste water 150.00 g/L Cold tap water The rest
(46) The aqueous top layer waste stream may be obtained using the procedures described in U.S. application Ser. No. 14/992,995, filed Jan. 11, 2016 (See Example 3 of said application). Briefly, aqueous top layer waste stream is obtained from clarified fermentation broth. Clarified broth is made by allowing fermentation medium containing P. aeroginosa that ends at a pH of 6.0 to 6.5 to age under ambient conditions for about 2 days. The biomass settles to the bottom of the vessel used for this aging process and the clear supernatant, after removal, is clarified broth. The next step in the process is to add acid, such as concentrated sulfuric acid, until the pH is about 2.1. The rhamnolipids precipitate out of solution and form a solid phase and an oily liquid phase at the bottom of the vessel used for this step. The separation of the solid and oily liquid phases can be sped up by centrifugation. The solid and oily liquid phases are separated from the aqueous top phase or layer, which can be discarded or recycled. The above-referenced aqueous layer is a source of H.sub.2SO.sub.4 and micronutrients of which 15% w/w is used in the culture medium with 8% soybean oil in the balance of cold tap water. The waste stream aqueous top layer is first filtered at 1 micron to remove large particles prior to its use.
(47) 7.3 L of the culture medium containing 8% soybean oil with 15% waste stream aqueous top layer and cold tap water is prepared by first mixing soybean oil, aqueous top layer and cold tap water using a kitchen blender. After they all are well mixed, H.sub.3PO.sub.4, NaOH, MgSO.sub.4, KCl and NaNO.sub.3 are added into the solution in that order under stirring. The culture medium then can be stream sterilized (autoclave). All chemicals used in this example are industry grade containing impurities.
(48) The fermentation is carried out at the same parameters as Example 5. Micronutrient composition is added continuously. Pure oxygen is additionally added to the fermenter at 10-30% of air flow to keep the total flow rate constant at 1.5 L/min in order to keep the agitation down and thus, less foaming issue. The rhamnolipid (RL) concentration and titer are shown in Table 9 infra.
(49) TABLE-US-00009 TABLE 9 RL Conc and Titer During Semi-Continuous Fermentation (Aqueous Layer) Fermenta- % Initial tion time Total RL Soybean (between draw) RL titer Draw# oil (h) (g/L) (g/L/h) 1 8.0% 95 74 0.78 2 6.1% 75 64 0.85 3 7.7% 92 73 0.79 4 6.1% 60 62 1.03 5 7.7% 83 71 0.85 6 6.1% 71 68 0.96 7 7.7% 89 77 0.86
EXAMPLE 8
Semi-Continuous Fermentation of Rhamnolipids in 100 L Scale
(50) 100 L of the culture medium (composition as in Example 5) is prepared as in Example 1 and sterilized in 120 L bioreactor. Its composition is shown in Table 10. Seed culture of R4 strain is prepared according to Example 2. The 100 L fermenter is inoculated with 2.9 L R4 strain incubated in a shake flask at 37 C for 24 hours in 20 g/L LB Broth Lennox.
(51) The fermentation is run at a temperature of 37 C with the initial pH of the culture medium of 6.2 with no pH control over the course of fermentation. Silicon based antifoam (DOW AFE-1510) is diluted with 50% deionized water which is then autoclaved prior to use. The antifoam is automatically added to knock down the foam during fermentation. The agitation is at 150 rpm with 17 L/min of air. The stirring rate automatically increases as necessary in order to keep % dissolved oxygen (% DO) at 15%-20%. 800 ml of micronutrient prepared and composition as Example 2 is diluted with 2.2 L deionized water and thus, it is being fed in continuously at 375 ml/day for 8 days.
(52) 70 L of culture medium containing 9% soybean oil (composition in Table 10 infra) is prepared as in Example 1 and is sterilized in a different 100 L bioreactor a day prior to the draw. After 105 hours post inoculation, the pH starts to increase after it remains constant around 7. The fermentation is completed at 115 hours with a pH at 7.3.
(53) TABLE-US-00010 TABLE 10 Composition of 9% Soybean Oil Culture Medium (70 L) Component Concentration Gum Arabic 4.50 g/L Soybean oil 90.00 g/L 85% H.sub.3PO.sub.4 9.69 g/L 99% NaOH 5.21 g/L 99% MgSO.sub.4*7H.sub.2O 0.50 g/L 99% KCl 1.00 g/L 99% NaNO.sub.3 15.00 g/L 98% H.sub.2SO.sub.4 0.92 g/L Deionized water The rest
(54) After 115 hours post inoculation, the peristatic pump is then started to remove 70 L (70% of the total) of the fermentation medium comprising one or more rhamnolipids. Once 70 L of broth is removed which so called Draw#1, the recently sterilized 70 L of 9% soybean oil culture medium is fed into the fermenter using a peristatic pump. The initial fermentation pH is 6.5. The fermentation parameters described in the above paragraph are used. In this case, after 70 hours, the fermentation is completed with a pH at 7.14. The rhamnolipid (RL) concentration and titer are shown in Table 11 infra.
(55) TABLE-US-00011 TABLE 11 RL Concentration and Tier (100 L Scale) Fermenta- % Initial tion time Total RL Soybean (between draw) RL titer Draw# oil (h) (g/L) (g/L/h) 1 .sup.8% 115 70 0.61 2 6.3% 70 75 1.07
EXAMPLE 9
Shake Flask Experiment without Micronutrient
(56) 50 ml of culture medium (composition as below) is prepared as Example 1 in a 250 ml shake flask. Its composition is shown in Table 12. After being autoclaved and cooled down to room temperature, the shake flasks are inoculated with 5% frozen stock of R4 strain. The frozen stock is obtained from mixing 70% R4 tube culture having OD.sub.600 of 3-4 with 30% glycerol and stored at ?80 C. The incubation is carried out at 37 C in a shaker for 92 hours without the addition of micronutrient. After 92 hours post inoculation, the rhamnolipid concentrations are average 47?3 g/L for 5 shake flasks.
(57) TABLE-US-00012 TABLE 12 Culture Medium Concentration For Use In Fermentation Without Micronutrient Component Concentration Gum Arabic 6.00 g/L Soybean oil 60.00 g/L 85% H.sub.3PO.sub.4 9.69 g/L 99% NaOH 5.21 g/L 99% MgSO.sub.4*7H.sub.2O 0.50 g/L 99% KCl 1.00 g/L 99% NaNO.sub.3 15.00 g/L 98% H.sub.2SO.sub.4 0.92 g/L Deionized water The rest
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