Methods for producing melanin and inorganic fertilizer from fermentation leachates-gctid245013

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Publication number	US8815539 B1
Publication type	Grant
Application number	US 13/911,927
Publication date	Aug 26, 2014
Filing date	Jun 6, 2013
Priority date	Jun 6, 2013
Also published as	CA2914363A1, 4 More »
Inventors	Radu Popa, Kenneth H. Nealson
Original Assignee	River Road Research, Inc.
Export Citation	BiBTeX, EndNote, RefMan
Patent Citations (2), Non-Patent Citations (12), Referenced by (2),Classifications (11), Legal Events (2)
External Links: USPTO, USPTO Assignment, Espacenet

Methods for producing melanin and inorganic fertilizer from fermentation leachates
US 8815539 B1
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.

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CLAIMS(23)
What is claimed is:1. A method for producing microbial melanin and/or a microbial melanin-associated protein 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;
(f) isolating or removing the secondary leachate from the secondary processing bioreactor, wherein the secondary leachate from the secondary processing bioreactor comprises melanin and/or a melanin-associated protein; and
(g) extracting or isolating the melanin or melanin-associated protein from the secondary leachate.

2. The method of claim 1 wherein the melanin is selected from the group consisting of pyomelanin, eumelanin and pheomelanin.

3. The method of claim 1 wherein the melanin-associated protein is associated with the melanin.

4. The method of claim 1 wherein the step of extracting or isolating the melanin or melanin-associated protein comprises the step of evaporating, titrating for changing the pH, filtering, centrifuging, dialyzing and/or lyophilizing the melanin or melanin-associated protein.

5. 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.

6. 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.

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

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

9. The method of claim 8 wherein the organic waste is food waste, plant waste, compost, cellulosic residues, cellulose-rich waste, starch-rich waste, or protein-rich waste.

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

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

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

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

14. 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.

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

16. The method of claim 15 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.

17. 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.

18. 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.

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

20. The method of claim 19 wherein the additive comprises carbohydrate, cellulose and/or starch.

21. The method of claim 20 wherein the carbohydrate is a sugar.

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

23. 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.

DESCRIPTION
1. TECHNICAL FIELDThe present invention relates to methods for producing melanin, melanin-associated proteins and inorganic fertilizer from fermentation leachates or from nutrient rich solutions spiked with low cost, sugar-rich sources.
2. BACKGROUND OF THE INVENTIONMelanin is one of a very few examples of natural organic semiconductors and was demonstrated to be such in the early 1970s. Melanin is thus a desirable natural, environmentally friendly material with many known applications for the electronics industry. Melanin can be used to produce a wide variety of biologically friendly electronic devices and batteries used in applications such as medical sensors and tissue stimulation treatments.
Many metazoans and microorganisms form melanin naturally. Because the concentration of melanin in these organisms is generally low and melanin is very insoluble, melanin extraction is inefficient and natural melanin is expensive. It is known in the art that the yield of melanin in a microbial culture can be increased by using chemically defined culture media, targeted extraction from natural populations, culturing pure strains, mutation and selection, genetic modification, and by spiking culture media with melanin precursors such as tyrosine and phenylalanine.
Melanins (such as eumelanin, pheomelanin and pyomelanin) are natural polyphenols produced by living cells. Pyomelanin is a negatively charged hydrophobic polymer of imprecise structure and size (Turick et al., 2003, 2009). It is present in fungi (Nosanchuk and Casadevall, 1997; Carreira et al., 2001; Schmaler-Ripcke et al., 2009), but also in many bacteria such as species of Pseudomonas (Yabuuchi and Ohyama, 1972; Arai, 1980), Legionella (Chatfield and Cianciotto, 2007) and Shewanella (Turick et al., 2008). Unlike the well-known eumalanin, which is produced from dihydroxyphenylalanine (DOPA), pyomelanin is metabolically derived from homogentisic acid (HGA), which upon elimination from cells autooxidizes and polymerizes as pyomelanin (David et al. 1996; Chatfield and Cianciotto 2007; Schmaler-Ripcke, 2009; Yabuuchi and Ohyama, 1972; Ruzafa et al., 1994; Kotob et al., 1995). In cells, pyomelanin is often associated with proteins (albeit melanin associated proteins are relatively little studied), and it is more concentrated in the outer cell envelopes such as the lipopolysaccharide layer and cell capsule (Turick et al., 2003).
The primary role of pyomelanin in living cells remains debated as melanins were proposed to play various roles in different species. Melanins can alter the electrical charge of a cell, especially when the polysaccharide capsule is small or absent (Nosanchuk and Casadevall, 1997). In Cryptococcus spp. the expression of pyomelanin is correlated with virulence (Kwon-Chung, 1982). In Legionella, pyomelanin increases resistance to light (Steinert et al., 1995). An antioxidant role for pyomelanin has been often proposed and has been demonstrated in Burkholderia sp. (Boles et al., 2004; Boles and Singh, 2008) and Methylococcus thermophilus (Sokolov et al., 1992). Pyomelanin confers Legionella ferric reductase capabilities (Chatfield and Cianciotto, 2007) and may help cells reduce, immobilize or chelate metals (Chatfield and Cianciotto, 2007; Turick et al., 2008; Nyhus et al., 1997). Pyomelanin may also bind and help recycle soluble electron shuttles such as riboflavin or may be used to transfer electrons toward solid phases (Marsili et al., 2008; Turick et al., 2009). The capacity of melanins to transfer electrons is derived from their ability to change the state of their monomers between quinone, semiquinone and hydroquinone (FIG. 1) (McGinness et al., 1974; Turrick et al., 2010). Because melanins have broad energy absorbing properties, their capacity to exchange electrons are influenced by many types of energy sources, including ionizing radiation, UV light, visible light, IR light and heat (Dadachova et al., 2007). It was proposed that in nature pyomelanin may also serve as a terminal electron acceptor (Turick et al., 2008), electron shuttle (Arai et al., 1980; Keith et al., 2007), or conduit for electrons (Turick et al., 2010).
Due to the complex architecture and broad size range of melanin the chemical entourage of the various quinone centers vary within a melanin polymer. Hence, their redox properties also vary, albeit they can exchange electrons within and between melanin polymers. For this reason, rather than having a narrow redox potential (E_o) as most simple redox chemicals do, melanin shows a broad redox potential profile. The discharge or recharge of electrons from some quinone centers is likely followed by re-partition of electrons and protons within the polymer until equilibrium is reached. Because most redox transformations involving melanin in nature occur at low redox potential (dE^o