# US 2019/0169564 A1 · Methods of culturing microorganisms in non-axenic mixotrophic conditions

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- Number: US 2019/0169564 A1
- Document ID: US20190169564A1
- Status: Published application
- Kind: A1
- Application number: 16/267,912
- Filed: 2019-02-05
- Published: 2019-06-06
- Earliest claimed: 2012-11-09
- Assignees: Heliae Development, LLC
- Applicants: Heliae Development, LLC
- Inventors: ENEKO GANUZA TABERNA; ANNA LEE TONKOVICH; JASON D. LICAMELE
- Web page: https://patentgrep.com/patent/US20190169564A1

## CPC

- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- A01G 33/00: Cultivation of seaweed {or algae}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01G: HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING (picking of fruits, vegetables, hops or the like A01D46/00; propagating unicellular algae C12N1/12)

## Abstract

Methods of culturing microorganisms in non-axenic mixotrophic conditions are disclosed. A method of culturing microalgae mixotrophically and controlling bacterial contamination with an acetic acid/pH auxostat system is specifically described. Methods of culturing microalgae mixotrophically with an increased productivity through an increase in oxygen transfer to the culture are also described.

## Claims

### Claim 1

A method of culturing Chlorella in non-axenic mixotrophic culture conditions, comprising:
(a) inoculating an aqueous culture medium with a culture comprising Chlorella in an open culturing vessel;
(b) supplying light comprising photosynthetically active radiation (PAR) in a photoperiod in the range of 10-16 hours of light for each 24 hour day;
(c) activating a pH auxostat system with an initial concentration of sodium acetate in the range of 0.05-10 g/L in the aqueous culture medium to start supplying acetic acid, at a concentration in the range of 15-50% (v/v) to the culture comprising Chlorella, wherein the pH auxostat system operates with in the range of 6-9, and the temperature ranges from 10° C. to 30° C.; and
(d) maintaining a dissolved oxygen (DO) concentration in the range of 1-6 mg O2/Liter.

### Claim 2 (depends on 1)

The method of claim 1, the method further comprising controlling temperature of the culture with heating and cooling to maintain the temperature within the range of 10-30° C.

### Claim 3 (depends on 1)

The method of claim 1, wherein the supplied acetic acid is mixed with a second organic carbon component comprising propionic acid.

### Claim 4 (depends on 3)

The method of claim 3, wherein the supplied acetic acid and propionic acid are mixed in an acetic acid:propionic acid ratio in the range of 10:0.01 to 10:2.

### Claim 5 (depends on 1)

The method of claim 1, wherein the supplied acetic acid is mixed with at least one additional nutrient comprising at least one selected from the group consisting of nitrates and phosphates.

### Claim 6 (depends on 5)

The method of claim 5, wherein the at least one additional nutrient comprises NO3 wherein the acetic acid and NO3 are mixed in an acetic acid: NO3 ratio of 10:0.5 to 10:2.

### Claim 7 (depends on 1)

The method of claim 1, the method further comprising harvesting Chlorella from the culture on a periodic basis once the density of Chlorella in the culture reaches a threshold density of 5 g/L.

### Claim 8 (depends on 1)

The method of claim 1, wherein the initial concentration of sodium acetate is in the range of 0.1-6 g/L.

### Claim 9 (depends on 1)

The method of claim 1, wherein the initial concentration of sodium acetate is supplied for the first 1-5 days after the culture is inoculated.