# US 2014/0360237 A1 · Methods for producing melanin and inorganic fertilizer from fermentation leachates

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- Number: US 2014/0360237 A1
- Document ID: US20140360237A1
- Status: Published application
- Kind: A1
- Application number: 14/330,790
- Filed: 2014-07-14
- Published: 2014-12-11
- Earliest claimed: 2013-06-06
- Assignees: River Road Research, Inc.
- Applicants: River Road Research, Inc.
- Inventors: Radu Popa; Kenneth H. Nealson
- Web page: https://patentgrep.com/patent/US20140360237A1

## CPC

- C05B 17/00: Other phosphatic fertilisers, e.g. soft rock phosphates, bone meal
- C05C 11/00: Other nitrogenous fertilisers
- C05D 9/00: Other inorganic fertilisers
- C05D 9/02: containing trace elements
- C05F 5/008: {Waste from biochemical processing of material, e.g. fermentation, breweries}
- C05F 17/05: Treatments involving invertebrates, e.g. worms, flies or maggots
- C05F 17/20: using specific microorganisms or substances, e.g. enzymes, for activating or stimulating the treatment (use of invertebrates C05F17/05)
- C05F 17/40: Treatment of liquids or slurries
- C05F 17/50: Treatments combining two or more different biological or biochemical treatments, e.g. anaerobic and aerobic treatment or vermicomposting and aerobic treatment
- C07K 14/195: from bacteria
- C07K 14/33: from Clostridium (G)
- C07K 14/335: from Lactobacillus (G)
- C12N 1/20: Bacteria; Culture media therefor
- C12P 1/00: Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P 1/04: by using bacteria
- C12P 17/00: Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms (C12P13/04 - C12P13/24 take precedence)
- C12P 17/10: Nitrogen as only ring hetero atom
- C12P 17/16: containing two or more hetero rings {(thiamine open chain analogs C12P17/167, i.e. not condensed among themselves or through a common carbocyclic ring system)}
- C12P 17/167: {Heterorings having sulfur atoms as ring heteroatoms, e.g. vitamin B1, thiamine nucleus and open chain analogs}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 39/00: Processes involving microorganisms of different genera in the same process, simultaneously
- Y02A 40/20: Fertilizers of biological origin, e.g. guano or fertilizers made from animal corpses
- Y02P 20/145: the feedstock being materials of biological origin
- Y02W 30/40: Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
- C05B: PHOSPHATIC FERTILISERS
- C05C: NITROGENOUS FERTILISERS
- C05D: INORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C; FERTILISERS PRODUCING CARBON DIOXIDE
- C05F: ORGANIC FERTILISERS NOT COVERED BY SUBCLASSES C05B, C05C, e.g. FERTILISERS FROM WASTE OR REFUSE
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02P: CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02W: CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT

## Abstract

Melanin or inorganic fertilizers are produced from fermentation leachates or from low-cost nutrient-rich solutions. The method for producing the melanin or inorganic fertilizer comprises repetitive trophic cycling in the controlled conditions of primary and secondary bioreactors. Nutrients are cycled between microorganisms such as bacteria, yeast and fungi and black soldier fly larvae, Hermetia illucens. Polysaccharides are partly converted into natural melanins or inorganic fertilizer, which are difficult to biodegrade and hence accumulate in the bioreactors. The method can employ, as a source of nutrients, leachates produced from food waste or from sugar-rich liquid waste of the food industry. These leachates can be used raw or can be augmented with low-cost sugar-rich solutions such as molasses, hydrolyzed cellulose or starch. The method is inexpensive and does not require the use of expensive chemically-defined culture media.

## Claims

### Claim 1

A method for producing a secondary leachate for use in producing inorganic fertilizer from the secondary leachate comprising the steps of:
(a) providing a primary processing bioreactor, a fermentation medium and a microbial culture comprising microorganisms, wherein the microorganisms in the microbial culture comprise Lactobacillus bacteria;
(b) fermenting the fermentation medium with the microbial culture in the primary processing bioreactor, thereby producing a primary leachate, wherein the primary leachate comprises microorganisms derived from the microbial culture and/or naturally occurring microorganisms acquired during the fermentation step;
(c) isolating or removing the primary leachate from the primary processing bioreactor;
(d) providing the primary leachate isolated or removed from the primary processing bioreactor, a secondary processing bioreactor, Hermetia illucens (black soldier fly) larvae (BSFL), and a cellulose-based substrate;
(e) culturing the BSFL in an aerated culture with the primary leachate isolated or removed from the primary processing bioreactor and the cellulose-based substrate in the secondary processing bioreactor under suboptimal culture conditions for culture of the BSFL, thereby producing a secondary leachate, wherein the suboptimal culture condition is suboptimal temperature, high density, chemical stress, acidification, presence of toxic secondary metabolites, and/or nutrient starvation; and
(f) isolating or removing the secondary leachate from the secondary processing bioreactor, wherein the secondary leachate from the secondary processing bioreactor comprises inorganic fertilizer; and
(g) extracting or isolating the inorganic fertilizer from the secondary leachate.

### Claim 2 (depends on 1)

The method of claim 1 wherein the inorganic fertilizer comprises ammonium, phosphate, carbonate and/or micronutrients.

### Claim 3 (depends on 1)

The method of claim 1 wherein the step of extracting, isolating or deriving the inorganic fertilizer comprises the step of evaporating, titrating for changing the pH, filtering, centrifuging, dialyzing and/or lyophilizing the inorganic fertilizer.

### Claim 4 (depends on 1)

The method of claim 1 wherein, in the step of culturing the BSFL with the primary leachate, the BSFL density is maintained at a larvae:liquid ratio, wherein the larvae:liquid ratio is 1 kg of larvae:1 kg of liquid to 1 kg of larvae:5 kg of liquid.

### Claim 5 (depends on 1)

The method of claim 1 wherein the step of culturing the BSFL with the primary leachate further comprises adding BSFL to the secondary processing bioreactor to maintain the BSFL density at a larvae:liquid ratio, wherein the larvae:liquid ratio is 1 kg of larvae:1 kg of liquid to 1 kg of larvae:5 kg of liquid.

### Claim 6 (depends on 1)

The method of claim 1 wherein the step of culturing the BSFL with the primary leachate proceeds for 10-20 days.

### Claim 7 (depends on 1)

The method of claim 1 wherein the fermentation medium is organic waste.

### Claim 8 (depends on 7)

The method of claim 7 wherein the organic waste is food waste, plant waste, compost, cellulosic residues, cellulose-rich waste, starch-rich waste, or protein-rich waste.

### Claim 9 (depends on 1)

The method of claim 1 wherein the step of fermenting the fermentation medium with the microbial culture is conducted under anaerobic or microaerobic conditions.

### Claim 10 (depends on 1)

The method of claim 1 wherein the microorganisms in the microbial culture are bacteria, yeast and/or fungi.

### Claim 11 (depends on 1)

The method of claim 1 wherein the microorganisms in the microbial culture further comprise Clostridium and/or Acetobacter bacteria.

### Claim 12 (depends on 1)

The method of claim 1 wherein the providing step (a) comprises providing a microbial culture that is a mixed microbial culture.

### Claim 13 (depends on 1)

The method of claim 1 wherein the steps of (a) providing a primary processing bioreactor, a fermentation medium and a microbial culture comprising microorganisms, (b) fermenting the fermentation medium with the microbial culture in the primary processing bioreactor, thereby producing a primary leachate, and (c) isolating or removing the primary leachate from the fermentation medium, are repeated in sequence (a)-(c) at least 1-5 times.

### Claim 14 (depends on 1)

The method of claim 1 comprising the step of monitoring the chemical composition of the primary leachate.

### Claim 15 (depends on 14)

The method of claim 14 wherein the step of monitoring the chemical composition of the primary leachate is conducted prior to the step of isolating or removing the primary leachate from the primary processing bioreactor.

### Claim 16 (depends on 1)

The method of claim 1 wherein the step of isolating or removing the primary leachate from the primary processing bioreactor is conducted at a point at which the fermentation becomes inefficient.

### Claim 17 (depends on 1)

The method of claim 1 wherein the step of isolating or removing the primary leachate from the primary processing bioreactor is conducted when the pH of the leachate is 3.4−4.0±0.4.

### Claim 18 (depends on 1)

The method of claim 1 wherein the step of culturing the BSFL comprises the step of adding an additive to the secondary processing bioreactor.

### Claim 19 (depends on 18)

The method of claim 18 wherein the additive comprises carbohydrate, cellulose and/or starch.

### Claim 20 (depends on 19)

The method of claim 19 wherein the carbohydrate is a sugar.

### Claim 21 (depends on 1)

The method of claim 1 wherein the nutrient starvation is a nitrogen-poor and/or phosphorus-poor condition as defined by the classical Redfield ratio of approximately C:N:P=106:16:1.

### Claim 22 (depends on 1)

The method of claim 1 wherein the culture in the secondary bioreactor comprises bacteria, yeast and/or fungi, and wherein the bacteria, yeast and/or fungi are derived from the primary leachate or are naturally occurring and introduced naturally from the environment into the culture in the secondary processing bioreactor.