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Microbial fermentation methods and compositions

A composition, fermentation product technology, applied in botanical equipment and methods, biofuels, immobilized on or in biological cells, etc.

Active Publication Date: 2015-04-08
NEWLEAF SYMBIOTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Such devices may not be constructed and operated in a commercially viable manner

Method used

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  • Microbial fermentation methods and compositions
  • Microbial fermentation methods and compositions
  • Microbial fermentation methods and compositions

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0110] The growth of embodiment 1.PPFM bacteria on solid agar plate medium.

[0111] Various standard media were tested for the growth of PPFM bacteria on solid agar plate media.

[0112] One medium used is ammonium mineral salts (AMS) medium (Whittenbury et al., 1970). AMS medium per liter contains 700 mg of anhydrous potassium dihydrogen phosphate, 540 mg of anhydrous potassium dihydrogen phosphate, 1 g of magnesium sulfate heptahydrate, 500 mg of anhydrous ammonium chloride, 200 mg of dehydrated calcium chloride, 4 mg of heptahydrate Ferric sulfate, 100 micrograms zinc sulfate heptahydrate, 30 micrograms manganese chloride tetrahydrate, 300 micrograms anhydrous boric acid, 200 micrograms cobalt chloride hexahydrate, 10 micrograms dehydrated copper chloride, 20 micrograms nickel chloride hexahydrate and 60 micrograms dehydrated Sodium molybdate.

[0113] Prepare AMS medium from the 4 stock solutions listed below.

[0114] Stock Solution I: For 1 L at 50X concentration

...

Embodiment 2

[0137] The growth of embodiment 2.PPFM bacteria in clarified single-phase liquid medium.

[0138] For those 4 solid agar plate media found in Example 1 to support the fastest and most abundant growth of the PPFM bacterium M. extorquens, corresponding liquid forms (that is, without addition of agar) were prepared and tested . These 4 liquid media prepared as described in Example 1 (with the only exception that they did not contain any agar) were all water-clear liquids with all components in solution. To a flask containing 100 ml of these 4 liquid media, add an inoculum of the PPFM bacterium M. extorquens to obtain approximately 1x10 5 Initial titer in colony forming units (CFU) / ml. The flasks were placed on a rotary shaking culture apparatus and incubated at 30°C and 250 rpm for 5 days. At the end of the 5 day incubation, determine the titer of PPFM bacteria in the flask. The result is:

[0139]

[0140] A surprising aspect of these results is that the nutrients presen...

Embodiment 3

[0141] Example 3.Growth of PPFM bacteria in a two-phase medium containing insoluble salt crystals.

[0142] To prepare biphasic media, liquid AMS+glycerol and peptone media are made turbid (ie given solid matter) by intentionally forming insoluble crystals of magnesium phosphate and / or calcium phosphate. In order to intentionally form insoluble crystals in the medium, the preparation method described in Example 1 was modified as follows. All components except trace metal stock solutions were mixed together prior to autoclaving. That is, to 940 ml of distilled water was added 20 ml each of stock solutions I, II and III, together with 10 g of glycerol and 10 g of peptone. After autoclaving, media preparation was completed by adding 1 ml of filter-sterilized trace metal stock solution. Autoclaving of the components of stock solutions I, II and III mixed together prior to autoclaving resulted in the formation of insoluble salt crystals, possibly predominantly magnesium hydrogen ...

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Abstract

The present invention provides methods for the cultivation of the Methylobacterium genus of bacteria. In particular the method provides methods for the efficient and inexpensive cultivation of these bacteria. Additionally, the invention provides methods for the utilization of these bacterial cultures to improve plant agriculture.

Description

[0001] Cross References to Related Applications [0002] This International Patent Application claims the benefit of: U.S. Patent Application No. 61 / 784,375 filed March 14, 2013 and incorporated herein by reference in its entirety; U.S. Patent Application No. 61 / 784,375 filed June 1, 2012 and incorporated herein by reference in its entirety Application No. 61 / 654,504; and US Patent Application No. 61 / 654,394, filed June 1, 2012 and incorporated herein by reference in its entirety. Background technique [0003] One-carbon organic compounds such as methane and methanol widely exist in nature, and bacteria classified as methanotrophs and methylotrophs are used as carbon sources. Methane-oxidizing bacteria include the genera Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, Methylosinus, Species of the genera Methylocystis, Methylosphaera, Methylocaldum and Methylocella (Lidstrom, 2006). Methanotrophs have the enzyme methane monooxygenase, which will be derived from ...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N11/02C12N11/14A01N63/00A01C1/06A01H3/00A01N63/20
CPCC12N1/20A01N63/00A01N63/02C12N11/02C12N11/14A01C1/06A01H3/00A01N63/20A01N25/00A01N25/08Y02E50/30C12N11/16
Inventor 格雷格·博戈西安
Owner NEWLEAF SYMBIOTICS INC
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