Enhanced production of rhamnolipids using at least two carbon sources

1. A semi-continuous fermentation method for producing rhamnolipids (RL), said method comprising:

(a) culturing a rhamnolipid-producing microorganism in a culture medium comprising:

i. at least two carbon sources, wherein a first carbon source is a sweetener and a second carbon source is an oil containing medium or long chain triglycerides;

ii. at least one nitrogen source;

iii. at least one phosphorous source;

iv. at least one magnesium source;

V. at least one potassium source;

vi. at least one sulfur source;

vii. at least one chloride source; and

viii. at least one sodium source

wherein said culture medium comprises between about 0.1% to about 2.0% by weight of said sweetener (w/v) in said culture medium and between about 3-15% by weight of said oil (w/v) in said culture medium;

wherein said rhamnolipid-producing microorganism is cultured in said culture medium for at least about 1 day to obtain a first fermentation medium comprising the rhamnolipids and the rhamnolipid-producing microorganism;

(b) removing at least about 70% of said first fermentation medium obtained in step (a);

(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 the rhamnolipids and the rhamnolipid-producing microorganism;

wherein said culturing step (a) yields rhamnolipids at a rate of at least about 1.7 g/L/h; and

wherein said method yields a rhamnolipid titer of at least about 40 g/L.

2. The method of claim 1 , wherein said rhamnolipid-producing microorganism is cultured in step (a) for about 1 to about 4 days.

3. The method of claim 1 , further comprising adding a micronutrient composition comprising one or more micronutrients at a concentration of no more than 20 mg/L of micronutrient composition to said culture medium in step (a) at 0.1% v/v of total fermentation volume per day.

4. The method of claim 1 , wherein said first fermentation medium or said subsequent fermentation medium is removed in step (b) during agitation and while maintaining air flow.

5. The method of claim 4 , wherein said air flow is oxygen enriched air.

6. The method of claim 1 , wherein there is no sedimentation step between steps (a) and (b).

7. The method of claim 1 , wherein said semi-continuous fermentation is sustained for at least about 20 days.

8. The method of claim 1 , wherein said rhamnolipid-producing microorganism is a Pseudomonas microorganism.

9. The method of claim 8 , wherein said Pseudomonas microorganism is Pseudomonas aeruginosa.

10. The method of claim 1 , wherein said sweetener is an unrefined sweetener and/or said oil is a vegetable oil.

11. The method of claim 10 , wherein said unrefined sweetener is derived from sap, one or more roots, fruit, one or more seeds, one or more trees, or one or more animals.

12. The method of claim 1 , wherein said sweetener is at least one of molasses, rice or barley malt syrup, nectar, yacon syrup, sugar beet syrup, sorghum syrup and/or said oil is at least one of soybean oil, safflower oil, peanut oil, hempseed oil, jatropha oil, coconut fat, calabash oil, linseed oil, corn oil, poppy seed oil, evening primrose oil, olive oil, palm kernel oil, palm oil, rapeseed oil, sesame oil, sunflower oil, grapeseed oil, walnut oil, wheat germ oil, coconut oil, or medium chain triglyceride oil.