U.S. patent application number 17/488229 was filed with the patent office on 2022-01-20 for methods for plant growth stimulation with fermentation leachates.
This patent application is currently assigned to River Road Research, Inc. The applicant listed for this patent is River Road Research, Inc. Invention is credited to Kenneth H' Nealson, Radu Popa.
Application Number | 20220017426 17/488229 |
Document ID | / |
Family ID | 1000005868952 |
Filed Date | 2022-01-20 |
United States Patent
Application |
20220017426 |
Kind Code |
A1 |
Popa; Radu ; et al. |
January 20, 2022 |
METHODS FOR PLANT GROWTH STIMULATION WITH FERMENTATION
LEACHATES
Abstract
Primary leachate is used as a plant growth stimulant. A
fermentation medium is fermented with a microbial culture in a
bioreactor to produce a primary leachate comprising microorganisms
derived from the microbial culture and/or naturally occurring
microorganisms. The primary leachate is isolated from the
bioreactor, diluted with water, and used to irrigate plants to
reduce bacterial diversity and stimulate beneficial microorganisms
in the rhizosphere around the plants. The fermentation medium may
be organic waste, preferably food waste. A secondary leachate may
also be used as a plant growth stimulant. The primary leachate is
used to culture black soldier fly larvae with a substrate in a
secondary processing bioreactor under suboptimal culture
conditions, thereby producing secondary leachate. Melanin is
extracted therefrom by acid precipitation. The secondary leachate
is then diluted with water and used to irrigate plants, reducing
bacterial diversity and stimulating beneficial microorganisms in
the rhizosphere around the plants.
Inventors: |
Popa; Radu; (Reseda, CA)
; Nealson; Kenneth H'; (South Pasadena, CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
River Road Research, Inc |
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Assignee: |
River Road Research, Inc
Tonawanda
NY
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Family ID: |
1000005868952 |
Appl. No.: |
17/488229 |
Filed: |
September 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16263899 |
Jan 31, 2019 |
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17488229 |
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14896044 |
Dec 4, 2015 |
10227626 |
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PCT/US14/41118 |
Jun 5, 2014 |
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16263899 |
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13911927 |
Jun 6, 2013 |
8815539 |
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14896044 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 1/20 20130101; C12P
1/04 20130101; C12P 17/10 20130101; C12P 17/00 20130101; C05F 17/40
20200101; Y02A 40/20 20180101; Y02P 20/145 20151101; Y02W 30/40
20150501; C05C 11/00 20130101; C05F 5/008 20130101; C05F 17/20
20200101; C12P 1/00 20130101; C07K 14/33 20130101; C07K 14/195
20130101; C05B 17/00 20130101; C05D 9/00 20130101; C05F 17/05
20200101; C12P 21/00 20130101; C05D 9/02 20130101; C12P 17/167
20130101; C07K 14/335 20130101; C12P 39/00 20130101; C05F 17/50
20200101; C12P 17/16 20130101 |
International
Class: |
C05B 17/00 20060101
C05B017/00; C12P 39/00 20060101 C12P039/00; C07K 14/195 20060101
C07K014/195; C07K 14/33 20060101 C07K014/33; C07K 14/335 20060101
C07K014/335; C12P 17/00 20060101 C12P017/00; C05F 5/00 20060101
C05F005/00; C12N 1/20 20060101 C12N001/20; C12P 1/00 20060101
C12P001/00; C12P 1/04 20060101 C12P001/04; C12P 17/10 20060101
C12P017/10; C12P 17/16 20060101 C12P017/16; C05F 17/05 20060101
C05F017/05; C05F 17/20 20060101 C05F017/20; C05F 17/40 20060101
C05F017/40; C05F 17/50 20060101 C05F017/50; C05C 11/00 20060101
C05C011/00; C05D 9/00 20060101 C05D009/00; C05D 9/02 20060101
C05D009/02; C12P 21/00 20060101 C12P021/00 |
Claims
1. A method of using a primary leachate as a plant growth stimulant
comprising the steps of: (a) providing a primary processing
bioreactor, a fermentation medium and a microbial culture
comprising microorganisms of the genera Clostridium and
Lactobacillus; (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) diluting the primary leachate
with water; and (e) irrigating plants with the diluted primary
leachate, thereby reducing bacterial diversity and stimulating
beneficial microorganisms in the rhizosphere around the plants.
2. The method of claim 2, wherein 4 ml of the primary leachate is
diluted with one liter of water.
3. The method of claim 2, wherein the fermentation medium is
organic waste.
4. The method of claim 3, wherein the organic waste is food
waste.
5. The method of claim 2, wherein the beneficial microorganisms are
Pseudomonas putida.
6. A method of using a secondary leachate as a plant growth
stimulant 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 microorganisms of the genera
Clostridium and Lactobacillus; (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; (g) extracting the melanin from the secondary leachate
by acid precipitation using an acid other than hydrochloric acid
and an alkali other than sodium hydroxide, thereby preventing
enrichment of the leachate by chloride and sodium; (h) diluting the
secondary leachate with water; and (i) irrigating plants with the
secondary primary leachate, thereby reducing bacterial diversity
and stimulating beneficial microorganisms in the rhizosphere around
the plants.
7. The method of claim 6, wherein the fermentation medium is
organic waste.
8. The method of claim 7, wherein the organic waste is food
waste.
9. The method of claim 6, wherein 4 ml of the secondary leachate is
diluted with one liter of water .
10. The method of claim 6, wherein the acid is an organic acid.
11. The method of claim 10, wherein the organic acid is selected
from the group consisting of lactic acid, ascorbic acid and citric
acid.
11. The method of claim 6, wherein the acid is sulfuric acid.
12. The method of claim 6, where in the alkali is potassium
hydroxide.
13. A method of using a secondary leachate as a plant growth
stimulant 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 microorganisms of the genera
Clostridium and Lactobacillus; (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; (g) diluting the secondary leachate with water; and (h)
irrigating plants with the secondary primary leachate, thereby
reducing bacterial diversity and stimulating beneficial
microorganisms in the rhizosphere around the plants.
14. The method of claim 13, additionally comprising the step of,
after isolating or removing the secondary leachate, extracting the
melanin from the secondary leachate by acid precipitation using an
acid other than hydrochloric acid and an alkali other than sodium
hydroxide, thereby preventing enrichment of the leachate by
chloride and sodium.
15. The method of claim 14, wherein only large melanin molecules
with a molecular weight of greater than 12,000 are extracted from
the secondary leachate and the acid precipitation occurs at a pH of
between 2 and 5.
16. The method of claim 14 wherein very small polyphenols
(<8,000 molecular weight) remain in the secondary leachate and
the acid precipitation occurs at a pH of between 1 and 2.
17. The method of claim 13, wherein the fermentation medium is
organic waste.
18. The method of claim 17, wherein the organic waste is food
waste.
19. The method of claim 13, wherein 4 ml of the secondary leachate
is diluted with one liter of water.
20. The method of claim 13, wherein the acid is an organic
acid.
21. The method of claim 20, wherein the organic acid is selected
from the group consisting of lactic acid, ascorbic acid and citric
acid.
22. The method of claim 13, wherein the acid is sulfuric acid.
23. The method of claim 13, where in the alkali is potassium
hydroxide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to and the benefit of U.S.
patent application Ser. No. 16/263,899, entitled Methods for
Producing Melanin and Inorganic Fertilizer from Fermentation
Leachates, filed Jan. 31, 2019, which in turn claims priority to
and the benefit of U.S. patent application Ser. No. 14/896,044,
filed on Dec. 4, 2015, which issued as U.S. Pat. No. 10,227,626.
U.S. patent application Ser. No. 14/896,044 is a National Stage
Entry of PCT/US14/41118, filed on Jun. 5, 2014, which is a
continuation-in-part of U.S. patent application Ser. No.
13/911,927, which issued as U.S. Pat. No. 8,815,539. These
applications are incorporated herein by reference in their
entirety.
1. TECHNICAL FIELD
[0002] The 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 INVENTION
[0003] Melanin 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.
[0004] 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.
[0005] 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 eumelanin, 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).
[0006] 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).
[0007] 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.sub.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.sup.o<2 V), the variation in the energy level associated
with electron exchanges is small relative to the strength of the
covalent bonds which hold the quinone structure together.
Henceforth, it can be predicted that as long as charging potentials
remain small (say .ltoreq.2 V) melanins can be charged:discharged
with electrons numerous times without significant alteration of the
basic structure. A set of usage conditions can be found (with
regard to redox potential and depth of charging/discharging) where
the extreme electron-load phases of melanin (i.e. quinone and
hydroquionone) are sufficiently stable to allow a melanin based
battery to be fully charged or discharged without significant
effects on the melanin' stability. Based on these properties,
melanins can be used to replace heavy metals as electron storage
substrates in the construction of long life, deep cycle
rechargeable batteries. Energy storage in melanin is ecofriendly
because melanin is composed only of elements (i.e. carbon, oxygen
and hydrogen) that are abundant and easily recycled in near earth
surface ecosystems. Henceforth, an economy using melanin-based
energy storage will not become resource limited, nor will it
compete with materials needed by other economic activities.
[0008] Relative to other types of melanin, pyomelanin has the
benefit of being produced in microbial cultures, has narrow range
molecular size, 12,000-14,000 MW (Turrick et al., 2002), and it is
easier to dissolve and purify. These properties make pyomelanin an
excellent choice for applied technologies that require predictable
product quality and predictable redox chemistry. In contrast,
eumelanin and pheomelanin are only available in low supply and
eumelanin is highly variable in size (it may have a molecular mass
as large as 10.sup.6 g/mol, making some eumelanin fractions highly
insoluble). Last but not least, the cost of producing melanin is
important in evaluating its sustainability for energy storage
devices. Commercial eumelanin is extracted from hair and squid or
polymerized from DOPA-related monomers and presently costs between
S300 and S600 per gram, which makes it unsuitable for most
economical applications. In contrast, pyomelanin extracted from
microbial cultures can be produced in large amounts and at
considerably lower costs.
[0009] Pyomelanin and pyomelanin-related molecules can be produced
in various ways. Most frequently proposed methods are: direct
extraction from microbial biomass, enrichment in culture media
spiked with tyrosine or phenylalanine, induction of genetically
modified microorganisms and chemical oxidation of homogentisate.
Microorganisms may produce between 1 and 115 fg of pyomelanin per
cell (though a 1-10 fg yield is more common) depending on strain
and physiological state (Turick et al., 2003; Turick et al., 2008;
Turick et al., 2002). Most cell cultures will produce approximately
0.03-0.3% pyomelanin relative to dry weight (DW). Obtaining higher
melanin yields requires using pure cultures, inducing agents, long
incubation times, expensive media and controlled growth conditions.
The biomass obtained in bacterial cultures may vary between 0.23 g
DW/L in batch media without manipulated conditions, and 37.8 g DW/L
in rich media under controlled conditions (Soini et al., 2008).
Hence, simple batch cultures may produce 0.069-0.69 mg pyomelanin
L.sup.-1, while complex controlled cultures may produce as much as
1.13 g pyomelanin L.sup.-1 (albeit obtaining such extremely high
yields is costly).
[0010] Citation or identification of any reference in Section 2, or
in any other section of this application, shall not be considered
an admission that such reference is available as prior art to the
present invention.
3. SUMMARY OF THE INVENTION
[0011] A method is provided for producing a secondary leachate for
use in the production of a product derived or isolated from the
secondary leachate comprising: [0012] a. providing a primary
processing bioreactor, a fermentation medium and a microbial
culture comprising microorganisms (e.g., bacteria, yeast and/or
fungi or combinations thereof, preferably a mixed microbial culture
dominated by Clostridium spp. and/or Lactobacillus spp.); [0013] 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; [0014] c.
isolating or removing the primary leachate from the primary
processing bioreactor; [0015] d. providing a secondary processing
bioreactor, Hermetia illucens larvae (hereinafter "black soldier
fly larvae", "BSFL", or "larvae"), and a cellulose-based and/or
mineral substrate; [0016] e. culturing the BSFL with the primary
leachate and the cellulose-based and/or mineral substrate in the
secondary processing bioreactor under suboptimal culture conditions
for culture of the BSFL, thereby producing a secondary leachate;
and [0017] f. isolating or removing the secondary leachate from the
secondary processing bioreactor.
[0018] In one embodiment of the method, the product derived or
isolated from the secondary leachate is a melanin, a
melanin-associated protein, and/or an inorganic fertilizer.
[0019] In other embodiments of the method, products derived from
the secondary leachate are products derived from BSF (i.e., BSF
eggs, larvae and adults), such as animal feed, proteins, fats,
biodiesel and chitin.
[0020] In another embodiment of the method, the product derived
from or isolated from the secondary leachate is a reagent or
intermediate product for producing melanin, proteins such as
melanin-associated proteins, phosphate, ammonia, bicarbonate,
inorganic nitrogen (e.g., ammonium, nitrite, nitrate),
micronutrients and/or microelements.
[0021] In another embodiment of the method, the method additionally
comprises the step of: [0022] g. extracting, isolating or deriving
the melanin, melanin-associated protein and/or inorganic fertilizer
from the secondary leachate.
[0023] In another embodiment of the method, the melanin is selected
from the group consisting of pyomelanin, eumelanin and
pheomelanin.
[0024] In another embodiment of the method, the melanin-associated
protein is associated with the melanin.
[0025] In another embodiment of the method, the inorganic
fertilizer comprises ammonium, phosphate, carbonate, microelements
and/or micronutrients.
[0026] In another embodiment of the method, the step of extracting,
isolating or deriving the melanin, melanin-associated protein or
inorganic fertilizer comprises the step of evaporating, titrating
for changing the pH, filtering, centrifuging, dialyzing and/or
lyophilizing the melanin, melanin-associated protein or inorganic
fertilizer.
[0027] In another embodiment of the method, the step of culturing
the BSFL with the primary leachate, the BSFL density is maintained
at 35.+-.5 kg/m.sup.2, where the depth of the culture layer is
preferably 4.+-.2 cm.
[0028] In another embodiment of the method, 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
35.+-.5 kg/m.sup.2, where the depth of the culture layer is
preferably 4.+-.2 cm.
[0029] In another embodiment of the method, the step of culturing
the BSFL with the primary leachate proceeds for 10-20 days.
[0030] In another embodiment of the method, the fermentation medium
is organic waste.
[0031] In another embodiment of the method, the organic waste is
food waste, plant waste, compost, cellulosic residues,
cellulose-rich waste, starch-rich waste, or protein-rich waste.
[0032] In another embodiment of the method, the step of fermenting
the fermentation medium with the microbial culture is conducted
under anaerobic or microaerobic conditions.
[0033] In another embodiment of the method, the microorganisms in
the microbial culture are bacteria, yeast and/or fungi.
[0034] In another embodiment of the method, the microorganisms in
the microbial culture comprise Clostridium, Lactobacillus and/or
Actetobacter bacteria.
[0035] In another embodiment of the method, the microbial culture
is a pure microbial culture or a mixed microbial culture.
[0036] In another embodiment of the method, the mixed microbial
culture is derived from inoculums produced by fermenting wheat
culture medium with fermentation microorganisms. Standard
microbiology methods known in the art can be used to purify many
strains of Clostridium, Lactobacillus and Acetobacter, Clostridium
and Lactobacillus will generally be more commonly found or frequent
in the primary leachate. Acetobacter will generally be more
commonly found or frequent in the secondary leachate. Fermenting
wheat bran with traces of molasses, until a very high microbial
density is reached, is one of the simplest methods, among many
known in the art, to produce cultures of Clostridium and
Lactobacillus. Acetobacter can be grown using standard methods
under aerobic conditions in the presence of alcohols. However, the
input materials can be highly variable and each feedstock will
favor different strains.
[0037] In another embodiment of the method, the steps of (a)
providing a primary processing bioreactor, a fermentation medium
and a microbial culture, (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.
[0038] In another embodiment of the method, the method comprises
the step of monitoring the chemical composition of the primary
leachate.
[0039] In another embodiment of the method, 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.
[0040] In another embodiment of the method, 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.
[0041] In another embodiment of the method, 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.
[0042] In another embodiment of the method, the step of culturing
the BSFL comprises the step of adding an additive or an
augmentation solution to the secondary processing bioreactor.
[0043] In another embodiment of the method, the additive or
augmentation solution comprises carbohydrate, cellulose and/or
starch.
[0044] In another embodiment of the method, the carbohydrate is a
sugar.
[0045] In another embodiment of the method, the additive or
augmentation solution comprises molasses.
[0046] In another embodiment of the method, the suboptimal culture
condition is suboptimal temperature, high density, chemical stress,
acidification, presence of toxic secondary metabolites, and/or
nutrient starvation.
[0047] In another embodiment, 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.
[0048] In another embodiment of the method, the step of culturing
the BSFL is carried out under a nitrogen-poor condition and/or a
phosphorus-poor relative to C condition.
[0049] In another embodiment of the method, the nitrogen-poor
condition or the phosphorus-poor relative to C condition is
relative to a classical Redfield ratio, wherein the ratio is
106C:16N:1P.
[0050] In another embodiment of the method, the bacteria, yeast
and/or fungi in the secondary processing bioreactor are derived
from the primary leachate or are naturally occurring.
[0051] In another embodiment of the method, after the step of
isolating or removing the secondary leachate from the secondary
processing bioreactor, new primary leachate and/or additional BSFL
are added to the secondary processing bioreactor.
[0052] In a specific embodiment, the method can be used to produce
a melanin, a melanin-associated protein or an inorganic fertilizer
and comprises the steps of: [0053] a. providing a primary
processing bioreactor, a fermentation medium and a microbial
culture comprising microorganisms; [0054] 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; [0055] c. isolating or
removing the primary leachate from the primary processing
bioreactor; [0056] d. providing a secondary processing bioreactor,
Hermetia illucens larvae (BSFL), and a cellulose-based and/or
mineral substrate; [0057] e. culturing the BSFL with the primary
leachate and the cellulose-based and/or mineral substrate in the
secondary processing bioreactor under suboptimal culture conditions
for culture of the BSFL, thereby producing a secondary leachate;
[0058] f. isolating or removing the secondary leachate from the
secondary processing bioreactor; and [0059] g. extracting,
isolating or deriving the melanin, melanin-associated protein or
inorganic fertilizer from the secondary leachate.
[0060] A primary processing bioreactor for fermenting food and
vegetal wastes and for producing primary leachate is also
provided.
[0061] A secondary processing bioreactor for converting primary
leachate to secondary leachate is also provided.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0062] The present invention is described herein with reference to
the accompanying drawings, in which similar reference characters
denote similar elements throughout the several views. It is to be
understood that in some instances, various aspects of the invention
may be shown exaggerated, enlarged, exploded, or incomplete to
facilitate an understanding of the invention.
[0063] FIG. 1. Diagram of redox changes in one unit of pyomelanin,
showing that each unit can exist in three oxidation -reduction
states: quinone, semiquinone and hydroquinone.
[0064] FIG. 2. Cross-sectional diagram of an embodiment of a black
soldier fly (BSF) nursery used to produce and harvest eggs of BSF.
Nurseries can be scaled up or down to suit a wide range of
production schemes and can have controlled environmental
conditions. The nursery is constructed with screen to hold the
flies and BSF larvae (BSFL) inside (1), and contains adult BSF (2),
larvae feeding containers (3), egg laying substrates (4), water
source (5), secondary leachate collectors (6) and larvae collectors
(7). Not shown in this diagram are additional elements that can be
employed such as temperatures controllers, moisture controllers,
air purifiers and traps for gnats and houseflies.
[0065] FIG. 3. Diagram illustrating principle of one embodiment of
the method disclosed herein, which is used to convert media rich in
sugars and polysaccharides (comprised in primary leachate) in
solutions with high concentration of pyomelanin (comprised in
secondary leachate).
[0066] FIG. 4. Flow diagram of one embodiment of the method
disclosed herein. In this embodiment, the method is used to convert
raw materials (e.g., food waste, residues of food industry;
gardening and agriculture vegetal waste) into pyomelanin.
[0067] FIG. 5. Cross-sectional diagram of an embodiment of a
primary processing bioreactor for fermenting food and vegetal
wastes and for producing primary leachate. (1) Side wall of the
primary processing bioreactor. (2) Bottom sieve. (3) Primary
leachate drainage tube. (4) Drainage system fitting. (5) Drainage
system spigot. (6) Primary leachate accumulated in the primary
processing bioreactor. (7) Primary leachate collecting container.
(8) Primary leachate in collecting container. (9) Vegetal materials
and food waste processed in the primary processing bioreactor. (10)
Gas outflow system used to monitor the production of gases produced
during fermentation (primarily carbon dioxide). (11) Main primary
processing bioreactor lid used for cleanup. (12) Secondary primary
processing lid, which can be used to add new fermentable materials
and lime during fermentation. (13) Port, which can be used for
injecting water, buffers, augmentation ingredients (e.g., molasses)
and microbial inocula as needed during fermentation and for
returning to the primary processing bioreactor any primary leachate
that is insufficiently acidic or is poor in nutrients. (14)
Electrode for monitoring the pH of the primary leachate. (15) pH
meter. (16) Side gate for the collection of solid fermented
materials.
[0068] FIG. 6. Cross-sectional diagram of an embodiment of a
secondary processing bioreactor comprising wood shavings,
organotrophic bacteria, yeasts and Black Soldier Fly larvae (also
referred to herein as "BSFL," "BSF larvae" or "larvae"). The
secondary processing bioreactor can be used to convert primary
leachate to secondary leachate rich in melanin, melanin-associated
proteins and inorganic fertilizer. (1) Rear or back side of the
secondary processing bioreactor. (2) Exit ramp for mature
(wandering) BSFL. This embodiment has an approximately
30-60.degree. slope angle. (3) Larvae exit path. (4) Lid for adding
more materials as needed (e.g., additional primary leachate, young
larvae and/or an augmentation ingredient or additive such as
molasses). (5) Secondary leachate drainage tube. (6) Drainage
system fitting. (7) Drainage system spigot. (8) Secondary leachate
in the secondary processing bioreactor. (9) Collection container
with secondary leachate enriched in melanin. (10) Light, solid,
slowly decaying, non-toxic substrate material such as wood
shavings. (11) BSFL crawling on top. (12) BSFL processing the
primary leachate into secondary leachate. (13) BSFL crawling out of
the processor (in the wandering stage) when they have reached
maturity. (14) Air pump for aerating the bottom portion of the
primary leachate being converted into secondary leachate and to
circulate the leachate inside the secondary processing bioreactor.
(15) Air pump for providing air to the BSFL. (16) Electrode for
monitoring the pH of the leachate (i.e., primary leachate being
converted into secondary leachate). (17) pH meter.
[0069] FIGS. 7A-C. The effect of the fermentate composition and
leachate treatment on the pH of the primary leachate (modified
after Alattar et al., 2012). The graphs show the evolution of pH in
primary processing bioreactors with three treatments: A) Primary
leachate continuously eliminated; B) Primary leachate returned
un-aerated to the fermentors; and C) Primary leachate aerated for
three days then returned to the primary processing bioreactor. A1,
A2 and A3 are incubations of 100% lignocellulose materials and 0%
produce. B, B2 and B3 are incubations of 90% lignocellulose
materials and 10% produce. C1, C2 and C3 are incubations of 50%
lignocellulose materials and 50% produce.
[0070] FIGS. 8a-b. (a) The effect of BSFL on the evolution of pH
and Chemical Oxygen Demand (COD) while feeding on compost primary
leachate: .DELTA.=pH without BSFL; .tangle-solidup.=pH with BSFL;
.quadrature.=COD without BSFL; and .box-solid.=COD with BSFL.
Errors bars are .+-.1 SD of duplicates using 20 BSFL in 10 ml
primary leachate. (b) The turnover of the dominant VOAs and
alcohols from primary leachate.+-.BSFL, using ten larvae per 2 ml
primary leachate: Et=ethanol; nP=n-propanol; Ac=acetic acid;
Pr=propanoic acid; Bu=butyric acid; and Is=isovaleric acid (Popa
and Green, 2012).
5. DETAILED DESCRIPTION OF THE INVENTION
[0071] For clarity of disclosure, and not by way of limitation, the
detailed description of the invention is divided into the
subsections set forth below.
5.1. Black Soldier Flies (BSF)
[0072] Black soldier flies (BSF), Hermetia illucens, are insects in
the order Diptera. BSF are ubiquitous throughout much of the world
extending between roughly the equator and 45th degree latitude
(Newton et al., J. Anim Sci., 44:395-400, 1977; Bondari and
Sheppard, Aquaculture and Fisheries Management, 18:209-220, 1987;
Sheppard et al., Bioresource Technology, 50:275-279, 1994;
Tomberlin et al., Ann. Entomol. Soc. Am., 95:379-386, 2002;
St-Hilaire et al., J. World Aquaculture Society, 38:59-67, 2007;
St-Hilaire et al., J. World Aquaculture Society, 38:309-313, 2007).
They are holometabolous insects that undergo a life cycle of
complete metamorphosis. The life cycle progresses from the egg to
hatching of the larva, typically 5-6 larval instars or stages,
pre-pupa, pupation into the pupal stage, then ecdysis (emergence)
into the adult fly. During the pre-pupa stage, between the larva
and pupal stages, the larva enters the wandering stage, moves away
from or out of the nutrient source to find a pupation site on a dry
surface. The skilled artisan is familiar with larvae generally, and
with methods of breeding and propagating larvae. For example,
methods of breeding and propagating dipterans including Hermetia
illucens larvae can be found, e.g., in Fatchurochim et al., J.
Entomol. Sci., 24:224-231, 1989).
[0073] Methods of breeding and propagating black soldier fly larvae
(BSFL), including methods of breeding BSFL in captivity, as well as
methods for using BSFL to process solid wastes and organic
leachates, are familiar to the skilled artisan (see for example,
Tomberlin et al., Environ Entomol., 38 (3):930-4, 2009; Sheppard et
al., J. Med. Entomol., 39 (4):695-8, 2002; Tomberlin, J. Econ.
Entomol., 95:598-602, 2002; U.S. Pat. No. 6,780,637; US
2012/0187041; Popa et al., J. Econ. Entomol., 105(2): 374-378,
2012).
[0074] Additionally, BSFL can be purchased commercially, for
example BioGrubs.TM. BSFL (Prota Culture, LLC, Dallas, Tex.) and
Phoenix Worms.TM. BSFL (Insect Science Resource, LLC, Tifton, Ga.).
Alternatively, BSFL, and eggs laid by adults, can be harvested in
the wild by gathering eggs and larvae present in animal manure,
particularly chicken and pig manure, on farms and at commercial
animal facilities open to the elements, especially in warmer
climates where the insects are known to lay eggs throughout the
year in the wild.
[0075] BSF eggs take approximately 4 days to hatch and are
typically deposited in crevices or on surfaces above or adjacent to
the food source. BSFL approaching the pupae stage reach a size in
excess of 2 cm in length and 0.4 cm in diameter relative to
immature larvae which start out on hatching from eggs at less than
0.2 cm in length and less than 0.1 cm in diameter. Although they
can be stored at room temperature for several weeks, their longest
shelf life is achieved at 50-60.degree. F. (10-16.degree. C.).
[0076] BSFL feed on a variety of vegetal and manure wastes of
varying extreme pH ranges and O.sub.2 tensions, self-harvest on
entering the pupae stage from the organic matter they are feeding
on, and are ubiquitous throughout much of the world extending
between roughly the equator and 45th degree latitude (Newton et
al., J. Anim Sci., 44:395-400, 1977; Bondari and Sheppard,
Aquaculture and Fisheries Management, 18:209-220, 1987; Sheppard et
al., Bioresource Technology, 50:275-279, 1994; Tomberlin et al.,
Ann. Entomol. Soc. Am., 95:379-386, 2002; St-Hilaire et al., J.
World Aquaculture Society, 38:59-67, 2007; St-Hilaire et al., J.
World Aquaculture Society, 38:309-313, 2007). BSF larva consume
organic matter, including kitchen waste, spoiled feed, and manure,
and assimilates organic compounds in the organic matter into larva
biomass. BSFL, like most dipteran insects, wander out of, or
"self-harvest" from, the organic matter they are feeding on upon
entering the pupal stage. Adult BSF do not need to eat; they
survive on the fat stored from the larva stage.
[0077] Prior to their introduction into the step of the method of
culturing the BSFL with primary leachate, as described hereinbelow,
BSFL can be hatched from egg clutches laid by mating adult flies in
an insect nursery (FIG. 2) set up for rearing flies on decomposing
vegetal and food scrap residues (banana peelings, leftover
fragments of lettuce, stale bread, rotting tomatoes, apples, and
other discarded produce, yard debris including mowed grass
clippings, etc.) under conditions known in the art (Tomberlin et
al., Environ Entomol., 38 (3):930-4, 2009). The eggs can be hatched
in wheat bran moistened with tap water. At approximately 1 week of
age, larvae can be freed of wheat bran by washing them in stainless
steel mesh colander (mesh size 1 mm) with several liters of tap
water.
[0078] FIG. 2 is a diagram of an embodiment of a black soldier fly
(BSF) nursery used to produce and harvest eggs of BSF. Nurseries
can be scaled up or down to suit a wide range of production
schemes.
5.2. Method for Producing Melanin, Melanin-Associated Protein
and/or Inorganic Fertilizers from Fermentation Leachates
[0079] A method is provided for producing melanin,
melanin-associated protein and/or inorganic fertilizers from
fermentation leachates or from low-cost nutrient-rich solutions.
The method for producing the melanin or inorganic fertilizer
comprises repetitive trophic cycling, under the controlled
conditions of bioreactors, of nutrients between organisms with
complementary physiology: natural microorganisms such as bacteria,
yeast and fungi and larvae of Hermetia illucens, the Black Soldier
Fly (BSF). According to the method, 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 and sugars-rich liquid waste of the food industry.
These leachates can be used raw or they 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.
[0080] BSFL can be used for the processing and disposal of food
waste products. BSFL are grown first on a low-cost feed, as
disclosed herein. Food waste is the cheapest source of nutrients
and fermented food waste is excellent for producing both leachate,
which can be used produce melanin, melanin-associated proteins
and/or fertilizer, and fermented solid residue, which can be used
to grow the BSFL from the first instar hatchling larval stage to
the last instar, largest size, white larval stage. The large white
larvae are grown to a sufficient size to withstand, for at least
two weeks (and up to 6 months), the harshness of conditions in
secondary processing bioreactors. Using the methods disclosed
herein, approximately 300-400 kg dry weight food waste produces
about 100-130 kg of last instar larvae (about a 3:1 ratio of food
waste to larvae produced).
[0081] As disclosed herein, BSFL are grown in large quantities and
used to digest microbes (e.g., bacteria) present in the primary
leachate and convert it to secondary leachate. As a result of this
fermentation process, the concentration of melanin goes from a very
low percentage in each microbe or bacterium to a highly
concentrated product in the secondary leachate as the microbes are
digested by the BSFL. This large-scale, bio-concentration method
allows production of useful products including, but not limited to,
melanin, melanin-associated proteins, fertilizers and products
derived from insect bodies such as animal feed, protein, fats,
biodiesel and chitin. For example, chitin can be extracted, using
methods well known in the art, from both the pupal shells that are
left behind after eclosion of BSF pupae into the adult fly stage,
and the fly carcasses left behind after the adult flies mate and
die.
[0082] BSFL are grown to the large, late instar (e.g., last instar)
white larval stage because in the late or last instars, they are of
sufficient size to withstand for at least two weeks (up to 6
months) the harshness of conditions in melanin producing
incubators. It takes 1 kg of larvae approximately 2 weeks to
convert 1 L of primary leachate and molasses into 1 L of secondary
leachate containing 2.5-10 g melanin.
[0083] Diagrams of one embodiment of the method are shown in FIGS.
3 and 4. FIG. 3 shows an overview of the method used to convert
media rich in sugars and polysaccharides (comprised in primary
leachate) in solutions with high concentration of melanin,
including but not limited to pyomelanin (comprised in secondary
leachate). FIG. 4 is a flow diagram showing an embodiment of the
method that can be used to convert raw materials (e.g., food waste,
residues of food industry; gardening and agriculture vegetal waste)
into melanin, including but not limited to pyomelanin. Bacteria
from the mixed culture produce extracellular cellulases which
hydrolyze cellulose and hemicellulose to sugars (hexoses and
pentoses). Along with other sugars present in the primary leachate,
these added sugars are used by microorganisms (e.g., bacteria and
fungi) as a source of energy and carbon. During growth,
microorganisms uptake nutrients and produce biomass and secondary
metabolites (mostly organic acids, alcohols and amines). These
microorganisms also produce melanin. BSFL grind partly digested
polysaccharides in order to uptake digestible organics and
microbial biomass and also consume microbial biomass and
metabolites directly from liquid. BSFL do not digest cellulosic
polysaccharides, but they grind larger particles into finer
particles making them more prone to enzymatic hydrolysis. BSFL
eliminate unused food in the form of a finely ground suspensions
called frass (i.e., 20-200 .mu.m diameter colloids), thus returning
nutrients (C, N, P, S, etc.) and bioavailable energy to the medium.
This recycling of nutrients and the removal of toxic metabolites
make microorganisms divide indefinitely as long as the primary
carbon and energy sources are available. As they grow,
microorganisms continuously produce polyphenols (such as melanin),
but neither BSFL nor microorganisms digest polyphenols efficiently.
In this system melanins are not recycled, but they accumulate.
[0084] In an exemplary, non-limiting embodiment of the method, a
method is provided for producing a secondary leachate for use in
the production of a product derived or isolated from the secondary
leachate comprising: [0085] a. providing a primary processing
bioreactor, a fermentation medium and a microbial culture
comprising microorganisms (e.g., bacteria, yeast and/or fungi or
combinations thereof, preferably a mixed microbial culture
dominated by Clostridium spp. (also referred to herein as
"Clostridium") and/or Lactobacillus spp. (also referred to herein
as "Lactobacillus"), [0086] b. fermenting the fermentation medium
with the microbial culture in the primary processing bioreactor,
thereby producing a primary leachate, wherein the primary leachate
comprises naturally occurring microorganisms acquired during the
fermentation step; [0087] c. isolating or removing the primary
leachate from the primary processing bioreactor; [0088] d.
providing a secondary processing bioreactor, Hermetia illucens
larvae (hereinafter "black soldier fly larvae" or "BSFL"), and a
cellulose-based and/or mineral substrate; [0089] e. culturing the
BSFL with the primary leachate and the cellulose-based and/or
mineral substrate in the secondary processing bioreactor under
suboptimal culture conditions for culture of the BSFL, thereby
producing a secondary leachate; and [0090] f. isolating or removing
the secondary leachate from the secondary processing
bioreactor.
[0091] In one embodiment of the method, the product derived or
isolated from the secondary leachate is melanin, a
melanin-associated protein, or an inorganic fertilizer.
[0092] In another embodiment of the method, the product derived or
isolated from the secondary is a reagent or intermediate product
for producing melanin, proteins, phosphate, ammonia, bicarbonate,
inorganic nitrogen (e.g., ammonium, nitrite, nitrate),
micronutrients and/or microelements.
[0093] In another embodiment of the method, the method additionally
comprises the step of: [0094] g. extracting, isolating or deriving
the melanin, melanin-associated protein and/or inorganic fertilizer
from the secondary leachate.
[0095] In another embodiment, a method is provided for producing a
melanin, a melanin-associated protein and/or an inorganic
fertilizer, comprising the steps of: [0096] a. providing a primary
processing bioreactor, a fermentation medium and a microbial
culture comprising microorganisms (e.g., bacteria, yeast and/or
fungi or combinations thereof, preferably a mixed microbial culture
dominated by Clostridium and/or Lactobacillus); [0097] 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; [0098] c.
isolating or removing the primary leachate from the primary
processing bioreactor; [0099] d. providing a secondary processing
bioreactor, Hermetia illucens larvae (hereinafter "black soldier
fly larvae", "BSFL", or "larvae"), and a cellulose-based and/or
mineral substrate; [0100] e. culturing the BSFL with the primary
leachate and the cellulose-based and/or mineral substrate in the
secondary processing bioreactor under suboptimal culture conditions
for culture of the BSFL, thereby producing a secondary leachate;
[0101] f. isolating or removing the secondary leachate from the
secondary processing bioreactor; and p1 g. extracting, isolating or
deriving the melanin, melanin-associated protein or inorganic
fertilizer from the secondary leachate.
5.2.1. Providing a Primary Processing Bioreactor, a Fermentation
Medium and a Microbial Culture Comprising Microorganisms
[0102] In the first step of the method, a primary processing
bioreactor, a fermentation medium and a microbial culture
comprising microorganisms (e.g., bacteria, yeast and/or fungi or
combinations thereof, preferably a mixed microbial culture
dominated by Clostridium spp. and/or Lactobacillus spp.), are
provided.
[0103] The primary processing bioreactor is an incubator for
converting food waste and vegetal residues into primary leachate
via fermentation. An embodiment of a primary processing bioreactor
is shown in FIG. 5. Microorganisms (MOs) used in the primary
processing bioreactor can be naturally occurring bacteria,
preferably from the genera Clostridium and Lactobacillus. The role
of Clostridium is to produce exoenzymes digesting polysaccharides
such as cellulose and starch. The role of Lactobacillus is to
ferment sugars and to lower the pH of the primary leachate making
it shelf stable in anaerobiosis. This process is akin to pickling
or silage.
[0104] In one embodiment, the fermentation medium is organic waste,
such as food waste, plant waste, compost, cellulosic residues,
cellulose-rich waste, starch-rich waste, or protein-rich waste.
Carbohydrates (particularly polysaccharides) are most important in
this process because they can be used by microorganisms as a source
of energy, yet have little value as feedstock for the BSFL. Vegetal
wastes that are rich in sugars, starch and cellulosic polymers are
particularly preferred. Vegetal wastes that are very rich in
cellulose but poor in sugars and starch (such as hay, straw or corn
stover), can be used, but are of lesser value because they do not
decompose fast enough in the primary processing bioreactor (FIG. 3,
also referred to herein as "primary bioreactor"). Woody biomass
(which is very rich in lignin) cannot be processed by this method,
though it can be used as filler or for increasing permeability in
primary bioreactors (FIG. 5). Wood shavings are also used in the
secondary bioreactors (FIGS. 3 and 6, also referred to herein as
"secondary processing bioreactors") as a crawling substrate for the
larvae.
[0105] Animal waste, dairy products and animal and vegetal fats are
preferably present in low proportion (<5%) in the primary
bioreactors, should be fed directly to the larvae from the nursery
(FIGS. 2 and 5) and should not be used in the secondary
bioreactors. BSFL can eat animal waste, but prefer a mixed diet,
which includes vegetal waste as well. Animal carcasses, bones,
skins, entrails, oils, grease and dairy products aggravate
management, odor and pest problems, and greatly increase health
risks due to food-borne pathogens such as Salmonella, Listeria, E.
coli and Campylobacter (to name only a few). Sewage liquid and
sludge are not recommended as feedstock because they are also a
health hazard. They are also inefficient in the production of
melanin because they are eaten by both microorganisms and BSFL, and
because they do not stunt the growth of the larvae. Stunting the
growth of the larvae in the secondary bioreactors is very
important, because it helps limit the removal of nutrients and
energy in the form of mature larvae biomass and lowers the amount
of newborn larvae that have to be constantly produced. Manure, used
in excess, increases the risk of spreading coliforms and other
pathogens (especially if the larvae produced in this process are to
be later used as feedstock for animals). Excessive use of manure
will also aggravate odor problems (due primarily to ammonia and
organic amines).
[0106] The types of raw materials preferred for this process
include, but are not limited to, non-animal derived food waste
(from groceries, bakeries, restaurants, domestic sources and food
industry) and vegetal waste (green biomass from vegetable farms and
greenhouses, aquaponics and hydroponic stations, leaves and grass
clippings). Products such as manure, hay, straw and soiled animal
bedding can also be used, but only to a lesser extent and should be
mixed with the above recommended raw materials.
[0107] The microbial culture comprises bacteria, yeast and/or
fungi. The source of the microbes can vary widely, e.g., from
naturally occurring wild microbes, to laboratory grown or
commercially grown microbes. Likewise, the growth media on which
they are cultivated can vary widely among different kinds of food
waste and other types of organic waste.
[0108] In a preferred embodiment, the microorganisms in the
microbial culture are species of Clostridium and Lactobacillus
bacteria. In another embodiment, the microbial culture is a pure
microbial culture or a mixed microbial culture. Numerous methods
are known in the art for growing Clostridium and Acetobacter.
Fermenting wheat bran with traces of molasses, until very high
microbial density is reached, is one of the simplest. Thus, in a
specific embodiment, the mixed microbial culture is derived from
inoculums produced by fermenting wheat culture medium with
fermentation microorganisms.
5.2.2. Fermenting the Fermentation Medium with the Microbial
Culture in the Primary Processing Bioreactor
[0109] In the next step of the method, the fermentation medium is
fermented 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. The primary leachate is thus
a liquid byproduct of anaerobic fermentation of the organic
waste.
[0110] The step of fermenting the fermentation medium with the
microbial culture can be conducted under anaerobic or microaerobic
conditions.
[0111] The method can additionally comprise the step of monitoring
the chemical composition of the primary leachate. Preferably, 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.
5.2.3. Isolating or Removing the Primary Leachate from the Primary
Processing Bioreactor
[0112] In the next step of the method, the primary leachate is
isolated or removed from the primary processing bioreactor (FIG.
5). This can be accomplished by many methods known in the art, such
as by draining, pumping, etc. In one embodiment, 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, which can be determined using
methods known in the art. For example, the pH can be measured with
a pH meter. Acidification, i.e., accumulation of organic acids
(mainly acetic acid), changes the pH and can stop the fermentation.
The concentration of organic acids and alcohols can also be
measured to determine inefficient fermentation, using methods known
in the art. Sugars (mono and disaccharides) can be measured by
standard methods of liquid chromatography, gas chromatography,
colorimetric methods and spectroscopy.
[0113] In another embodiment, the step of isolating or removing the
primary leachate from the primary processing bioreactor is
conducted when the pH of the primary leachate is
3.4-4.0.+-.0.4.
[0114] In a preferred embodiment, the steps of (a) providing a
primary processing bioreactor, a fermentation medium and a
microbial culture, (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. After this many repetitions,
the food source can become exhausted and can be removed from the
primary incubator and replaced.
5.2.4. Providing a Secondary Processing Bioreactor, Hermetia
illucens Larvae (BSFL) and a Carbohydrate-Based Substrate
[0115] In the next step of the method, a secondary processing
bioreactor, Hermetia illucens larvae (BSFL) and a
carbohydrate-based substrate are provided. The secondary processing
bioreactor (FIG. 6) can contain wood shavings, organotrophic
bacteria, yeasts and BSFL. The secondary processing bioreactor
(also referred to herein as "secondary bioreactor") can be used to
convert primary leachate into melanin-rich secondary leachate. (1)
Rear or back side of the secondary processing bioreactor. (2) Exit
ramp for mature (wandering) BSFL. This embodiment has an
approximately 30-60.degree. slope angle. (3) Larvae exit path. (4)
Lid for adding more materials as needed (e.g., additional primary
leachate, young larvae and/or molasses). (5) Secondary leachate
drainage tube. (6) Drainage system fitting. (7) Drainage system
spigot. (8) Secondary leachate in the secondary bioreactor. (9)
Collection container with secondary leachate enriched in melanin.
(10) Light, solid, slowly decaying, non-toxic substrate material
such as wood shavings. (11) BSFL crawling on top. (12) BSFL
processing the primary leachate into secondary leachate. (13) BSFL
crawling out of the processor (in the wandering stage) when they
have reached maturity. (14) Air pump for aerating the bottom
portion of the leachate and to circulate the primary leachate
inside the secondary processing bioreactor. In the beginning of
secondary processing the leachate will be primary leachate, which
in time will be converted into secondary leachate. (15) Air pump
for providing air to the BSFL. (16) Electrode for monitoring the pH
of the leachate (i.e., primary leachate being converted into
secondary leachate). (17) pH meter.
[0116] The carbohydrate-based substrate is preferably rich in
sugars, and can be, for example, molasses, starch, cellulose (e.g.,
hydrolyzed cellulose), wood-chips or wood shavings and/or
combinations thereof. Wood chips or shavings are particularly
preferred as a substrate.
[0117] Mineral substrates suitable for use in the methods of the
invention can be, for example, glass or rock substrates.
5.2.5. Culturing the BSFL with the Primary Leachate and the
Carbohydrate-Based Substrate in the Secondary Processing Bioreactor
Under Suboptimal Culture Conditions
[0118] In the next step of this embodiment of the method, the BSFL
are cultured with the primary leachate and the carbohydrate-based
substrate in the secondary processing bioreactor under suboptimal
culture conditions for culture of the BSFL, thereby producing a
secondary leachate.
[0119] In one embodiment, BSFL are preferably cultured in layers
that are approximately 4.+-.2 cm deep. In layers deeper than this,
the BSFL may potentially risk suffocation or drowning. Thus, in
another embodiment, in the step of culturing the BSFL with the
primary leachate and the carbohydrate-based substrate, the volume
of food/larvae mix is approximately 40.+-.20 L per square meter
where the depth of the culture layer is approximately 4.+-.2
cm.
[0120] In another embodiment, in the step of culturing the BSFL
with the primary leachate and the carbohydrate-based substrate, the
BSFL density is maintained, e.g., at approximately 35.+-.5
kg/m.sup.2 where the depth of the culture layer is preferably
4.+-.2 cm.
[0121] In another embodiment, the step of culturing the BSFL with
primary leachate and the carbohydrate-based substrate further
comprises adding BSFL to the secondary processing bioreactor to
maintain the BSFL density at a desired density, e.g., 35.+-.5
kg/m.sup.2 where the depth of the culture layer is preferably
4.+-.2 cm.
[0122] As mentioned above, wood chips or shavings are particularly
preferred as the carbohydrate-based substrate, as very high
densities of BSFL can be maintained in cultures containing wood
chips/shavings. In a preferred embodiment, a density is maintained
of at least 500,000 larvae/m.sup.2 in culture layers that are
4.+-.2 cm deep. In other embodiments, larval density is 2-4
kg/ft.sup.2 in culture layers that are 4.+-.2 cm (1.6.+-.0.8
inches) deep.
[0123] In another embodiment, the step of culturing the BSFL with
the primary leachate proceeds for 10-20 days.
[0124] In another embodiment, the step of culturing the BSFL with
the primary leachate comprises the step of adding an additive to
the secondary processing bioreactor. The additive, which can be a
solid, liquid, or a gas, can be used to augment the components
being cultured in the secondary processing bioreactor. When the
additive is a liquid or is added as a solution to the culture, it
is also referred to herein as an "augmentation solution." The
additive can comprise carbohydrate (e.g., sugar), cellulose and/or
starch. In another embodiment, the carbohydrate is a sugar, e.g., a
monosaccharide or a disaccharide sugar. In specific embodiments,
the sugar is glucose, fructose, galactose, sucrose, maltose and/or
lactose. In another specific embodiment, the additive is molasses.
In other embodiment, the additive can be any organic molecule,
polymer or small metabolite that is known in the art to promote or
assist the growth of bacteria, yeast and/or fungi can be amino
acids, pentoses, proteins, nucleic acids, carboxylic acids,
etc.
[0125] An additive or augmentation solution could be added, for
example, upon a decrease in biological oxygen demand, when no
change in pH of the leachate being processed by the BSFL is
observed or when very little change in melanin yield or
concentration is seen over time compared with earlier rounds of
culturing.
[0126] Air can also be an additive that is pumped into the
secondary processing bioreactor to provide air to the BSFL. Due to
high density, the BSFL may run out of oxygen. White BSFL crawling
out of the secondary bioreactor can be evidence of lack of food
provided by the primary leachate, or more likely, excess carbon
dioxide and lack of oxygen.
[0127] The bacteria, yeast or fungi in the secondary processing
bioreactor can be comprised in the primary leachate or can be
naturally occurring and introduced naturally from the environment
into the culture in the secondary processing bioreactor.
[0128] The step of culturing the BSFL with the primary leachate is
preferably carried out under suboptimal culture conditions for the
growth of the BSFL. Suboptimal culture conditions for BSFL are
known in the art and can be, for example, suboptimal temperature,
high density, chemical stress, acidification, presence of toxic
secondary metabolites, and/or nutrient starvation. All of these
conditions delay metamorphosis and force the BSFL to remain as
larvae for a prolonged period of time. Whereas BSFL would normally
pupate after approximately 14-21 days as larvae, under suboptimal
culture conditions they can remain as larvae as long as 2-6
months.
[0129] In another embodiment, the suboptimal condition is nutrient
starvation, 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.
[0130] In one embodiment, the step of culturing the BSFL is carried
out under nitrogen-poor or phosphorus-poor relative to C conditions
as compared to the classical Redfield ratio of approximately
C:N:P=106:16:1. Such nitrogen-poor or phosphorus-poor relative to C
conditions as compared to the classical Redfield ratio can be
determined by one of skill in the art.
5.2.6. Isolating or Removing the Secondary Leachate from the
Secondary Processing Bioreactor
[0131] In the next step of this embodiment of the method, the
secondary leachate is isolated or removed from the secondary
processing bioreactor. In one embodiment, the secondary leachate is
isolated or removed by draining using methods known in the art. For
example, a preferred method is to drain the liquid through a spigot
placed near the bottom of the secondary processing bioreactor. In
certain embodiments, e.g., embodiments using a large secondary
processing bioreactor, a pump can be added to facilitate
drainage.
[0132] In another embodiment, after the step of isolating or
removing the secondary leachate from the secondary processing
bioreactor, new primary leachate and/or additional BSFL are added
to the secondary processing bioreactor.
5.2.7. Extracting, Isolating or Deriving the Melanin,
Melanin-Associated Protein or Inorganic Fertilizer from the
Secondary Leachate
[0133] In certain embodiments of the method, melanin,
melanin-associated protein or inorganic fertilizer is extracted,
isolated or derived from the secondary leachate. In another
embodiment, the step of extracting, isolating or deriving the
melanin, melanin-associated protein or inorganic fertilizer
comprises the step of evaporating, titrating for changing the pH,
filtering, centrifuging, dialyzing and/or lyophilizing the melanin,
melanin-associated protein or inorganic fertilizer. Such methods
are commonly known in the art.
[0134] In certain embodiments, melanin yield can be increased by
using selected or genetically modified microorganisms, exposure of
the liquid to UV or oxidizing conditions to increase melanin
expression, using chemicals for increasing melanin expression.
5.2.8. Melanins and Melanin-Associated Proteins
[0135] The methods disclosed herein can be used to produce
melanins. Melanins are a class of natural polyphenols composed
mainly of carbon, oxygen, hydrogen, nitrogen and sulfur and made of
monomers derived from aromatic amino acids (tyrosine and
phenylalanine). At the macromolecular level (primary, secondary and
tertiary structure level) melanins are a family of molecules and do
not have a single chemical structure and molecular size. The method
disclosed herein produces a mixture of melanins comprising
pyomelanin, eumelanin and pheomelanin Melanins comprise melanin
monomers, which are chemical derivatives of tyrosine. Pyomelanin
comprises homogentisate monomers. Eumelanin comprises DOPA
monomers.
[0136] Thus, in one embodiment of the method, the melanin is
selected from the group consisting of pyomelanin, eumelanin and
pheomelanin.
[0137] Melanins are known in the art to be associated with
melanin-associated proteins. Melanin-associated proteins form
complexes with melanins and are commonly present in melanin
extracts. In one embodiment, the melanin produced by the method is
isolated with or without its melanin-associated protein(s)
associated with the melanin. The melanin-associated proteins can be
isolated in association with the melanin or separately from the
melanin using methods known in the art.
[0138] Since melanin-associated proteins have a selective affinity
for melanin, they can be used as "molecular handles", using methods
known in the art, to tether or immobilize melanin to a substrate,
such as nanoparticles (e.g., magnetic or fluorescently-labeled
nanoparticles, microbeads, microvesicles, electrode tips, membranes
or any other substrate known in the art to be suitable for binding,
or being associated with, a tethering protein.
[0139] Methods for isolating melanin are well known in the art.
Melanins can be isolated with or without their associated proteins.
The following references are a sample of methods known in the art
for isolating melanin and melanin-associated proteins from
organisms. The skilled practitioner can easily adapt these methods
for isolating melanin from secondary leachate: [0140] Araujo et
al., 2012, Marine sponge melanin: a new source of an old
biopolymer, Structural Chemistry, 23:115-122. [0141] Chen et al.,
2008, 2008, Isolation and characterization of natural melanin
derived from silky fowl (Gallus gallus domesticus Brisson), Food
Chemistry, 111:745-749. [0142] Crippa et al., 1989, Chemistry of
melanins, The Alkaloids 36:253-322, Academic Press, N.Y., N.Y.
[0143] Dong and Yao., 2012, Isolation, characterization of melanin
derived from Ophiocordyceps sinensis, an entomogenous fungus
endemic to the Tibetan Plateau, Journal of Bioscience and
Bioengineering, 113:474-479. [0144] Harki et al., 1997,
Purification, characterisation and analysis of melanin extracted
from Tuber melanosporum Vitt., Food Chemistry, 58:69-73. [0145] Liu
et al., 2003, Comparison of the structural and physical properties
of human hair eumelanin following enzymatic or acid/base
extraction, Pigment Cell Research, 16:355-365. [0146] Loganathan
and Kalyanasundaram, 1999, The melanin of the myxomycete Stemonitis
herbatica, Acta Protozoologica, 38:97-103. [0147] Medes et al.,
1928, Proceedings of the Society for Experimental Biology and
Medicine, 25:635-637. [0148] Prota, 1988, Progress in the chemistry
of melanins and related metabolites, Medical Research Reviews,
8:525-556. [0149] Ravishankar et al., 1995, Isolation and
characterization of melanin from a marine fungus, Botanica Marina,
38:413-416. [0150] Rosas et al., 2000, Isolation and serological
analyses of fungal melanins, Journal of Immunological Methods,
244:69-80. [0151] Zeise et al., 1992, Melanin standard
method--particle description, Pigment Cell Research, 5:132-142.
[0152] Zou et al., 2010, Optimization of ultrasound-assisted
extraction of melanin from Auricularia auricula fruit bodies,
Innovative Food Science & Emerging Technologies, 11:611-615.
[0153] Young, 1921, The extraction of melanin from skin with dilute
alkali, Biochemical Journal., 15:118-122. [0154] Wakamatsu and Ito,
2002, Advanced chemical methods in melanin determination, Pigment
Cell Research, 15:174-183. [0155] Wang et al., 2006, Isolation and
characterization of melanin from Osmanthus fragrans seeds, LWT-Food
Science and Technology, 39:496-502. [0156] Wheeler et al., 1978,
Ultrastructural and chemical distinction of melanins formed by
Verticillium dahlia, from (+)-scytalone, 1,8-dihydroxynaphthalene,
catechol, and .sub.L-3,4-dihydroxyphenylalanine, Can. J.
Microbiol., 24:289-297.
5.2.9. Methods for Isolating Inorganic Fertilizers
[0157] The methods disclosed herein can also be used to produce
inorganic fertilizers. Inorganic fertilizers can be extracted,
using methods well known in the art, from solids and liquids left
in the solid growth media and liquid incubated with the BSFL, or
can be obtained by amending the products with specific nutrients
according to various fertilizing needs. In one embodiment,
secondary leachate is mineralized and used to obtain concentrated
fertilizers.
[0158] Inorganic fertilizers are inorganic molecules rich in
carbon, nitrogen, phosphorus, potassium and microelements and/or
micronutrients. The inorganic fertilizer produced by the method
disclosed herein can comprise ammonium, phosphate, carbonate and
/or micronutrients. In certain embodiments, the inorganic
fertilizer obtained using the methods disclosed herein can include,
but are not limited to:
[0159] Phosphate rich minerals, for example, a mixture dominated by
struvite (NH.sub.4MgPO.sub.4.6H.sub.2O), but can comprise, in other
embodiments, other precipitates with phosphorus such as calcium
phosphates (Ca(H.sub.2PO.sub.4).sub.2; CaHPO.sub.4 and
Ca.sub.3(PO.sub.4).sub.2) and bobbierite
((Mg.sub.3(PO.sub.4).sub.2x 8H.sub.2O).
[0160] Nutrient rich mineral solution containing: bicarbonate
(HCO.sub.3.sup.-), nitrate (NO.sub.3.sup.-), sulfate
(SO.sub.4.sup.2-), potassium (K.sup.+) and microelements such as
iron, manganese, calcium and/or boron (commonly released during the
mineralization of organic matter).
[0161] Inorganic fertilizers can be isolated from the secondary
leachate using methods well known in the art. According to the
method disclosed herein, after melanin has been extracted from the
secondary leachate, what remains is a liquid rich in organic and
inorganic molecules. These organic molecules can include, but are
not limited to, a wide variety of small metabolites, partly
hydrolyzed polymers and non-hydrolyzed polymers.
[0162] The most abundant inorganic molecules or ions that can be
obtained from the secondary leachate are bicarbonate, ammonium and
phosphate. Based on the composition of biological materials
introduced in the primary bioreactor, the actual percentage of
these inorganic molecules or ions will vary. Mg, K, Na, Cl and
other microelements common in biological materials can also be
obtained or isolated from the secondary leachate using methods
known in the art. The liquid that remains after melanin has been
extracted from the secondary leachate can be used as fertilizer as
is or it can be dried first and the resulting mixture reconstituted
later with liquids such as water. The chemical composition of the
liquid or the dried mixture will vary depending on the source
materials introduced in the primary bioreactor and the degree of
biomineralization.
[0163] In certain embodiments, to use the liquid or dried mixture
in standard fertilizers may involve performing chemical analyses
and amending the mixture with various fertilizing chemicals to
reach desired concentrations and proportions.
[0164] Exemplary methods for extracting inorganic fertilizers from
these complex mixtures are described hereinbelow.
5.2.10. Struvite Extraction
[0165] Struvite is a mineral with the formula NH.sub.4MgPO.sub.4.
6H.sub.2O and a solubility of 0.2 g/L or 0.815 mM (Barak and
Stafford, 2006; Bhuiyan et al., 2009). In agriculture, struvite is
used as a component in various fertilizer mixtures. In one
embodiment, precipitating struvite can be used to extract phosphate
from the secondary leachate. Part of the ammonium and most of the
magnesium can be extracted as well.
[0166] In a preferred embodiment, struvite is extracted from the
liquid residue of secondary leachate using the methods of Golubev
et al., 2001; Matynia et al., 2006; and Kurtulus and Tas, 2011.
This method comprises the steps of analyzing the concentration of
ammonium, phosphate and magnesium in water and amending the
solution in such a way so that desired stoichiometric proportions
will exist between these three chemicals in accordance to the
formula of struvite.
[0167] Because BSF leachates are byproducts of decayed organic
materials, BSF leachates will often contain nitrogen (and thus
ammonium) in excess relative to phosphate. Hence the limiting
factor in the precipitation of struvite is expected to be in most
cases Mg. Prior to extracting struvite, the liquid is preferably:
filtered; analyzed for pH, ammonium, phosphate and magnesium;
adjusted as needed with magnesium chloride or ammonium chloride; pH
adjusted with NaOH or CO.sub.2 to approximately 6.5-6.8; and heated
to 90.degree. C. to evaporate while stirring. Struvite precipitates
when supersaturation is reached. Other phases containing phosphate
may also precipitate, albeit in lower amounts (these are calcium
phosphate and bobbierite) (Golubev et al., 2001). Struvite is
difficult to separate by gravity alone because it has a density of
1.6 and part of the precipitate is amorphous. The precipitate is
thus preferably filtered or centrifuged. An ammonium/potassium
magnesium phosphate fertilizer well known in the art and marketed
as MagAmp.RTM. (WR Grace & Co.) is a slow-release fertilizer,
made by adding magnesium oxide or magnesium hydroxide to
monoammonium phosphate (Peng et al., 1979). The cost of production
of MagAmp.RTM. has restricted the usefulness of this product to
high value-added applications, such as floriculture (Barak and
Stafford, 2006). This is not the case with the method disclosed
herein, because ammonium and phosphate are already present in the
secondary leachate and the sole amendment needed is magnesium. Thus
by using the secondary leachate that remains after melanin is
extracted, the added expense of amendment with ammonium and
phosphate can be avoided.
5.2.11. Ammonium Extraction
[0168] Ammonium is a toxic chemical, especially at high pH and in
the secondary leachates produced by BSFL, ammonium may reach
concentrations as high as 100 mM (Green and Popa, 2012). A
preferred method for removing ammonium is conversion of ammonium by
nitrification to nitrate, which is the most important form of
inorganic nitrogen absorbed by plants. In a preferred embodiment,
this can be achieved by aeration of the secondary leachate for 2-7
days at room temperature after struvite has been extracted, in the
presence of nitrifying microorganisms, while monitoring the
evolution of ammonia, nitrite and nitrate. The process can be
considered completed when nitrification is complete and most
ammonium has been converted to nitrate. The evolution of nitrite is
preferably monitored because nitrite (an intermediary product in
nitrification) is also toxic. Aeration has the benefit of better
mineralization of small metabolites and removal of toxic volatiles
such as alcohols, organic amines and hydrogen sulfide. If the
extraction of ammonium is desired, the solution can be amended with
phosphate and magnesium, followed by precipitation to obtain
struvite (as described above).
5.2.12. Microelement Extraction
[0169] Microelements remain in the solution after the ammonium,
phosphate and magnesium have been removed. Because of complex and
variable chemistry it is not practical to attempt to extract and
separate the remaining chemicals from solution. This mixture
contains organic carbon, carbonate, K, Na, Cl and numerous
microelements. The composition of this mixture can be amended as
needed using methods known in the art and/or used directly as
fertilizer in liquid or dried form.
[0170] The following references disclose exemplary methods known in
the art that can be used to isolate inorganic fertilizer from
secondary leachate: [0171] Barak P and Stafford A., 2006, Struvite:
A recovered and recycled Phosphorus Fertiliser. URL [Accessed:
24.03.2011],
http://www.soils.wisc.edu/extension/wcmc/2006/pap/Barak.pdf. [0172]
Bhuiyan M. I. H., Mavinic D. S. and Beckie R. D. 2009, Dissolution
kinetics of struvite pellets grown in a pilot-scale crystallizer.
(Report), Canadian Journal of Civil Engineering, March 1. [0173]
Green T. R. and Popa R., 2012, Enhanced ammonia content in compost
leachate processed by Black Soldier Fly larvae. J. Appl. Biochem.
Biotechnol. 166:1381-1387. [0174] Golubev S V, Pokrovsky O. S,
Savenko V. S. 2001, Homogeneous precipitation of magnesium
phosphates from seawater solutions, J Cryst Growth, 223:550-556.
[0175] Kurtulus G. and Tas A. C., 2011, Transformations of neat and
heated struvite (MgNH4PO4.6H2O), Materials Letters 65:2883-2886.
[0176] Matynia A., Koralewska J., Wierzbowska B. and Piotrowski K.,
2006, The Influence of Process Parameters on Struvite continuous
crystallization kinetics, Chemical Engineering Communications,
193:160-176. [0177] Peng P. H., Ernst W. R., Bridger G. L. and
Hartley E. M., 1979. Slow release fertilizer materials based on
magnesium ammonium phosphate. Pilot-plant granulation studies. Ind.
Engr., Chem. Process Des. Dev. 18:453-458.
[0178] The following examples are offered by way of illustration
and not by way of limitation.
6. EXAMPLES
6.1. Example 1: First Embodiment of Method for Producing Natural
Melanin and/or Melanin-Associated Proteins from Fermentation
Leachates
[0179] This example describes one non-limiting embodiment of the
method.
[0180] BSFL are propagated in a nursery (such as shown in FIG. 2).
In the nursery, the BSFL are grown to a larval stage where they are
tough enough to withstand high density, chemical stress and
nutrient starvation, i.e., about 5-7 days old and approximately 0.5
cm long. BSFL are then separated from their initial food source in
the nursery and mixed in an incubator, referred to herein as a
secondary processing bioreactor, with fermentation (primary)
leachates derived from primary processing in a primary processing
bioreactor. Air is continuously provided, e.g., with a pump to
provide O.sub.2 to the larvae, to remove toxic volatile chemicals
(such as ammonia, organic amines and sulfide) and to help microbial
respiration.
[0181] In a preferred aspect, the temperature at which the BSFL are
maintained in the secondary processing bioreactor (the incubation
temperature) is kept suboptimal for larval growth and
metamorphosis. In one embodiment, the BSFL are maintained in the
secondary processing bioreactor at about 22-27.degree. C., which is
in contrast to the optimal temperature for larval growth and
metamorphosis, which is 30-35.degree. C.
[0182] The primary leachate added to the secondary processing
bioreactor is preferably poor in nutrients that can be used
directly by the larvae. In one embodiment, the primary leachate is
rich in sugars and micronutrients, yet poorer in nitrogen and
phosphorus relative to C (compared to the classical Redfield
ratio). This nutrient deficiency forces the larvae to digest
microorganisms in the primary leachate and their metabolic
byproducts. The microorganisms are only partially digested,
however, as BSFL cannot degrade polyphenols such as melanin, which
is released in the solution in the secondary processing
bioreactor.
[0183] In another embodiment, the nitrogen-poor and/or
phosphorus-poor condition is defined by the classical Redfield
ratio of approximately C:N:P=106:16:1.
[0184] In a preferred embodiment, BSFL are maintained in the
secondary processing bioreactor at a super high density of 35.+-.5
kg/m.sup.2, where the depth of the culture layer is approximately
4.+-.2 cm and with a high larvae: liquid ratio that can preferably
range from 1:1-1:5 (kg of larvae : kg of liquid) and is more
preferably 1:2-1:4 (kg of larvae:kg of liquid). Such ranges are
preferred because if the BSFL cannot process the liquid quickly
enough, the liquid exposed to air and to airborne microorganisms,
e.g., airborne fungi, will start decaying or form mold. In certain
embodiments, less liquid may be used and consequently, the
secondary leachate can be drained out of the secondary processing
bioreactor more often.
[0185] The stress of this super high density, along with nutrient
deficiency and possibly the suboptimal temperature conditions in
the secondary processing bioreactor, stunt the growth of the
larvae. The maturation of the larvae toward the pupal stage is
delayed from two weeks to more than a month. Owing to their
advanced age when the BSFL are removed from the nursery (about
10-12 days) and to good prior feeding and nutritional conditions in
the nursery, the BSFL do not die in secondary processing
bioreactor. Some larvae do mature and self-harvest from the
secondary processing bioreactor as they near pupation age. In
certain embodiments, a tilted ramp or other suitable exit can be
provided in the secondary processing bioreactor for the larvae
crawl out of the liquid in the secondary processing bioreactor so
that they can pupate on a dry surface.
[0186] As larvae mature and exit the secondary processing
bioreactor, more larvae propagated in the nursery are preferably
added to the secondary processing bioreactor to maintain high
density. One embodiment of a secondary processing bioreactor is
shown in FIG. 6. Like BSF nurseries, secondary processing
bioreactors can be scaled up or down to suit a wide range of
production schemes.
[0187] In one embodiment, the secondary processing bioreactor has
dimensions of approximately 30 cm.times.30 cm.times.30 cm and can
contain about 7 liters of liquid leachate and 3.5 kg of BSFL. Wood
shavings can be added to the liquid in the secondary processing
bioreactor to provide an additional physical substrate for the
larvae to crawl through and also as a substrate that can release
more sugars due to the extracellular cellulases produced by
microorganisms
[0188] In the secondary processing bioreactor, microorganisms grow
up to a density at which their growth becomes inhibited due to
acidification, toxic secondary metabolites (acetate, propionate,
butyrate, etc.) and depletion of nutrients. Yet, in this stage, the
remaining solution is still rich in undigested energy-rich sugars.
These sugars have little bioavailability to the BSFL. The BSFL feed
on naturally occurring microorganisms in the primary leachate and
their metabolic byproducts (e.g., alcohols, organic acids, amines,
etc.). In doing this, the density of microorganisms decreases,
nutrients (e.g., ammonium, phosphate, microelements) are released
in solution, chemicals that are toxic to the microorganisms (e.g.,
acetate, butyrate, propionate, alcohols) are lowered.
[0189] In certain embodiments, the concentrations of some of these
nutrients (ammonia, volatile alcohols, volatile organic acids,
volatile amines and H2S) are decreased by purging the secondary
processing bioreactor with air periodically (preferably
continuously). The improved environmental conditions allow the
microorganisms to multiply again. The cycle of nutrients between
microorganisms and BSFL can repeat many times until most sugars
from the solution are exhausted. For example, the biological oxygen
demand (BOD) can be approximately 8,000 ppm or below. Biological
Oxygen Demand (BOD) is a measure indicating how much bioavailable
materials are present that can be decomposed by aerobic
microorganisms in 5 days.
[0190] In each step, undigested melanin and inorganic fertilizer
accumulates in solution. Multiple such cycles are possible because
the large size (2 cm in length and 0.4 cm in diameter) of the BSFL
makes them highly resistant to toxic chemicals and starvation,
because microorganisms and BSFL have complementary physiology and
because the microorganism feed the BSFL with the nutrients
otherwise unavailable to the larvae.
[0191] In one embodiment, after about 2 weeks of incubation the
primary leachate decreases to about 50-60% of the initial volume
and the concentration of melanin is about 0.75%. In another
embodiment, the sum of all salts (containing nitrogen, phosphorus
and micronutrients) can be approximately equal to 10 g/L (.+-.2
g/L). The exact proportions between the various components will
vary considerably with the source material. The concentration will
also depend on how much water is lost by evaporation.
[0192] The melanin- and inorganic fertilizer-enriched secondary
leachate is drained from the secondary processing bioreactor, new
primary leachate is added and more BSFL are supplied as needed. The
secondary processing bioreactors do not need to be paused or
stopped, but can be continuously replenished with primary leachate,
BSFL and wood shavings. Melanin, with or without melanin-associated
proteins and inorganic fertilizer can be extracted from the
processed secondary leachate by using conventional chemical
methods. This extraction can be carried out preferably starting 1
week after secondary processing begins in the secondary processing
bioreactor, and more preferably 10-14 days after the after
secondary processing begins in the secondary processing
bioreactor.
6.2. Example 2: Second Embodiment of Method for Producing Natural
Melanin and/or Melanin-Associated Proteins from Fermentation
Leachates
[0193] This example describes another non-limiting embodiment of
the method.
[0194] 1. Producing the primary leachate [0195] a)
Anaerobic/microaerobic fermentation of food waste and cellulosic
residues is carried out in the presence of mixed microbial cultures
dominated by species of Clostridium and/or Lactobacillus. The mixed
cultures come from inoculums produced in the lab by fermenting a
wheat culture medium with microorganisms obtained from earlier
fermentations. Fermentation is conducted in primary processing
bioreactor. [0196] b) The chemical composition of the primary
leachate is continuously monitored. The primary leachate is drained
from the primary processing bioreactor when the pH is about 3.4-4.0
and stored at room temperature in closed containers in the absence
of air. [0197] c) More water is added to the primary processing
bioreactor and the primary leachate-producing steps of the method
above are repeated 2-5 times until most metabolites and easily
fermentable organic molecules are degraded. Each new fermentation
step takes more time to occur than its predecessor. A fermentation
results in a primary leachate that is poorer in nutrients than the
preceding fermentation. The number of repeated fermentations varies
depending on the source materials, the duration of time to reach
chemical stability and the composition of the primary leachate.
Cellulose-rich wastes are fermented less times and release
nutrients more slowly, while starch and protein rich wastes ferment
faster and are fermented more times. [0198] d) When fermentation
becomes inefficient, the primary leachate is drained, the residue
is squeeze-pressed to remove all remaining liquid and the leftover
solid residue is composted.
[0199] 2. Propagating the black soldier fly larvae (BSFL) [0200] a)
BSFL derived from BSF adults are propagated in a nursery (FIG. 2)
under controlled conditions of temperature, illumination, humidity
and food availability. [0201] b) Eggs laid by BSF adults are
hatched in a 30.degree. C. incubator. [0202] c) Newly hatched BSFL
are grown at 30.degree. C. to an age of 10-12 days or an individual
size of about 100-150 mg (wet weight). The BSFL are fed with a
puree of food ground to <0.1 mm particle size. [0203] d) When
the desired size is reached, the BSFL are separated from their food
source and washed free of the solid feed.
[0204] 3. Producing the secondary leachate [0205] a) Larvae,
primary leachate and wood shavings (or another cellulose-based
substrate known in the art) are mixed in a secondary processing
bioreactor in a proportion of about 35 kg BSFL per m.sup.2 and
about 70 L primary leachate per m.sup.2. Wood shavings are added to
the incubator to a final height of about 15-25 cm. [0206] b)
Incubate at 22-27.degree. C. During incubation air is continuously
pumped into the bottom of the secondary processing bioreactor.
[0207] c) The design of the secondary processing bioreactor
includes a side that is tilted at an angle of 30-60.degree., in a
specific embodiment about 45.degree. C. (FIG. 6) which allows
larvae to exit the secondary processing bioreactor when they reach
the final instar. [0208] d) The chemical composition (nutrients, pH
and melanin concentration) and the amount of BSFL self-harvested
are monitored. More BSFL are added to maintain high density of
larvae to about 35 kg/m.sup.2. Maintaining BSFL to such high
density is very important for forcing the larvae to feed on
micoorganisms and on their metabolic byproducts. Lower density of
BSFL (similar to density in natural environments) decreases the
efficiency of producing melanin (per volume of primary leachate,
per time and per amount of biomass of larvae). [0209] e) After
about 14 days of incubation (which depends on the quality of the
primary leachate, the amount of larvae and the melanin
concentration in the secondary leachate) the secondary leachate is
drained from the secondary processing bioreactor and can be stored
at room temperature. [0210] f) The density of larvae is determined
and more larvae are added as needed. New primary leachate is added
and incubation is repeated multiple times. The secondary processing
bioreactor does not need to be stopped and cleaned; it can be used
multiple times.
[0211] 4. Extracting melanin and inorganic fertilizers
[0212] Melanin and inorganic fertilizers (ammonium, phosphate,
carbonate and micronutrients) are extracted from the secondary
leachate using a combination of chemical methods. They can include,
but are not limited to, evaporation, titration for changing the pH,
filtration, centrifugation, dialysis and lyophilization. Methods
for extracting melanin or inorganic fertilizer are well known in
the art and references providing guidance in these methods are
disclosed herein.
6.3. Example 3: Method for Producing Pyomelanin-Enriched Melanin
from Organic Waste Using Microorganisms and Black Soldier Fly
Larvae
Introduction
[0213] This example demonstrates another non-limiting embodiment of
the method. In this example, the method is used to obtain high
yield, low cost pyomelanin-enriched melanin mixtures. The method
uses low cost nutrients and energy from organic waste, maintains
cultures in batch conditions, and has a pyomelanin yield of
.gtoreq.7.5 g L.sup.-1. The stationary phase of batch cultures is
initiated owing to the depletion of energy and nutrients and/or the
accumulation of inhibitory metabolic byproducts. In this stage,
i.e., the end of the exponential growth phase and the onset of the
stationary phase, the biomass is largest, yet the melanin
concentration is still low.
[0214] In the method described in this example, bacteria and yeasts
are grown in secondary processing bioreactors that also contain
larvae of the Black Soldier Fly (B SF) (Hermetia illucens). The
overall principle is described in FIG. 3. In these cultures, small
sugars and polysaccharides are the principal source of energy and
carbon. Because BSFL feed on microorganisms and their toxic
secondary metabolites, but less on sugars and polysaccharides, the
larvae recycle nutrients and decrease the toxicity of secondary
metabolites. Henceforth, inhibition by nutrient deprivation, energy
deprivation and accumulated metabolic byproducts have little
influence on cell division, and microbes continue to divide
actively as long as the primary energy resource is available. The
larvae do not significantly diminish the availability of nutrients
to the microbial community because the overall biomass of BSFL in
these cultures changes little as the BSFL growth and metamorphosis
are stunted. Undigested materials released by the larvae are used
as nutrients for new generations of microorganisms.
[0215] The exception is polyphenols (such as melanins), which
cannot be digested by the larvae or easily decomposed by microbial
activity, and consequently accumulate in the medium. The enrichment
in melanin continues and can exceed 7.5 g L.sup.-1 provided that
the primary carbon and energy sources (i.e., small sugars and
polysaccharides such as starch, cellulose and hemicellulose) are
present. In these cultures, active aeration can be used to promote
the digestion of small metabolites and the removal of toxic
volatiles such as carbon dioxide, organic amines, ammonia and
hydrogen sulfide. After approximately 10-14 days of incubation, the
resultant liquid, called secondary leachate, is harvested and used
to extract pyomelanin using methods known in the art. Fresh
nutrient media and more young larvae are then added and the cycle
is repeated for producing more melanin. With good management, there
is no need to ever stop a melanin-producing bioreactor once it is
started.
[0216] The method is preferably carried out so that the main source
of energy and nutrients is available to the microorganisms, but not
to the BSFL. The BSFL preferably feed mostly on microorganisms and
their metabolic byproducts. The BSFL can lower the toxicity of the
primary leachate and recycle nutrients. The BSFL remain alive but
their growth and metamorphosis are preferably stunted. Polyphenols
are preferably produced continuously, but should not be digested by
microorganisms or larvae. Based on these principles, this example
demonstrates a process for producing low cost pyomelanin-enriched
melanin mixtures (FIG. 4).
Materials and Method
[0217] Raw feedstock materials (FIG. 4). The microorganisms and
BSFL used in this process are capable of feeding on raw materials
from many sources. Not all sources, however, are appropriate for
high melanin yield. The worth of various types of feedstock was
therefore assessed based on balancing costs, availability, health
risks and nutritional value. Carbohydrates (particularly
polysaccharides) are most important in this process because they
can be used by microorganisms as a source of energy, yet have
little value as feedstock for the BSFL (Popa and Green, 2012).
Vegetal wastes that are rich in sugars, starch and cellulosic
polymers are particularly preferred. Vegetal wastes that are very
rich in cellulose but poor in sugars and starch (such as hay, straw
or corn stover), can be used, but are of lesser value because they
do not decompose fast enough in the primary bioreactor (FIG. 3).
Woody biomass (which is very rich in lignin) cannot be processed by
this method, though it can be used as filler or for increasing
permeability in primary bioreactors (FIGS. 3 and 5). Wood shavings
are also used in the secondary bioreactors (FIG. 6) as a crawling
substrate for the larvae.
[0218] Animal waste, dairy products and animal and vegetal fats are
preferably present in low proportion (<5%) in the primary
bioreactors, should be fed directly to the larvae from the nursery
(FIGS. 2 and 5) and should not be used in the secondary
bioreactors. BSFL can eat animal waste, but prefer a mixed diet,
which includes vegetal waste as well. Animal carcasses, bones,
skins, entrails, oils, grease and dairy products aggravate
management, odor and pest problems, and greatly increase health
risks due to food-borne pathogens such as Salmonella, Listeria, E.
coli and Campylobacter (to name only a few). Sewage liquid and
sludge are not recommended as feedstock because they are also a
health hazard. They are also inefficient in the production of
melanin because they are eaten by both microorganisms and BSFL, and
because they do not stunt the growth of the larvae. Stunting the
growth of the larvae in the secondary bioreactors is very
important, because it helps limit the removal of nutrients and
energy in the form of mature larvae biomass and lowers the amount
of newborn larvae that have to be constantly produced. Manure, used
in excess, increases the risk of spreading coliforms and other
pathogens (especially if the larvae produced in this process are to
be later used as feedstock for animals). Excessive use of manure
will also aggravate odor problems (due primarily to ammonia and
organic amines).
[0219] The types of raw materials recommended for this process
include: non-animal derived food waste (from groceries, bakeries,
restaurants, domestic sources and food industry), vegetal waste
(green biomass from vegetable farms and greenhouses, aquaponics and
hydroponic stations, leaves and grass clippings). Products such as
manure, hay, straw and soiled animal bedding can also be used, but
only to a lesser extent and should be mixed with the above
recommended raw materials.
Fragmentation
[0220] Before being added to the primary processing bioreactors,
large size raw materials are preferably chopped or fragmented into
smaller particles (FIG. 4). A preferred size range is 1-10 mm
Particles of this size are sufficiently small to allow easy
penetration by enzymes, microorganisms and secondary metabolites,
yet sufficiently large to be produced at low cost and to maintain
permeability (an important consideration in the functioning of the
primary bioreactors). The raw materials are preferably not
fragmented too extensively and too finely (<1 mm in size),
because when softened by the "fermentation and pickling" process
from the primary bioreactors, such mixtures will form a compact
low-permeability paste and clog the drainage system of the primary
bioreactor.
Inoculum
[0221] Enriched cultures are produced in dedicated fermentors using
wet mixtures of approximately 95% wheat bran and 5% molasses (by
weight), moistened with tap water and incubated for 7-10 days, at
20.degree. C. and 0% O.sub.2, with occasional mixing and exposure
to oxygen. These mixtures are inoculated with .about.100-500 fold
diluted solutions of mature leachates, harvested at pH 4.0-4.5 from
former primary bioreactors (Green and Popa, 2010; 2011; Alattar et
al., 2012). The microbial enrichments produced this way can be
dominated by strains of microorganisms such as Clostridium,
Lactobacillus, yeasts and/or Acetobacter. This inoculum (FIG. 4) is
mixed with the raw materials during the fragmentation step (FIG.
3). Clostridium species are dominant in the inoculum
(.about.60-70%) based on cell counts and are preferred in this
process because they grow in anaerobic conditions. Clostridium
species produce extracellular cellulases and metabolize hexoses and
pentoses into organic acids such as formic, acetic, propionic and
butyric (Montville et al., 1985; Green and Popa, 2010). Yeasts such
as Saccharomyces cerevisiae convert sugars to ethanol and
Acetobacter spp. convert ethanol to acetic acid, an efficient
metabolic inhibitor that extends the storage lifetime of the
fermented products, but is also metabolized by the larvae (Popa and
Green, 2012). High yield of organic acids is preferred for
establishing the mutualistic relationship between microorganisms
and BSFL in the secondary bioreactors, which in turn helps increase
the pyomelanin yield.
Primary Processing Bioreactor
[0222] A primary processing bioreactor (also referred to herein as
primary bioreactor, FIG. 5) is also provided herein. A primary
processing bioreactor is an anaerobic fermentor constructed in a
way that allows monitoring the evolution of the fermentation
process and also continuous adding of raw materials and harvesting
of products during fermentation. The functioning of such fermentors
of various sizes 140 mL, 1 L, 20 L and 200 L was verified.
Fermentation in these bioreactors occurred at a temperature of
approximately 20-30.degree. C. and can result in partial hydrolysis
of cellulosic polysaccharides and release of sugars, organic acids
(Alattar et al., 2012) but also many other metabolites, biological
polymers and pigments. This type of fermentation is akin to silage
and it is sometimes called Bokashi fermentation (Green and Popa,
2011). Woody biomass (even finely grounded) cannot be digested by
this method and its only justification in primary fermentors it as
filling materials for increasing permeability.
Primary Leachate
[0223] Primary leachate is liquid produced and drained from primary
bioreactors. Primary leachate is considered to be mature when its
pH has stabilized for at least 24 hours at an acidic value less
than pH 5, with a variation of +/-0.1 pH units and its Chemical
Oxygen Demand (COD) is .gtoreq.10,000 ppm. The COD yield can be
increased by adding .about.400 g of powdered limestone (CaCO.sub.3)
to each 10 kg of raw materials. The primary leachate is rich in
sugars, partly hydrolyzed polysaccharides, organic acids, organic
amines, ammonia, bicarbonate and plant pigments (Green and Popa,
2011; Green and Popa, 2012; Alattar et al., 2012). In a preferred
embodiment, mature primary leachate can be drained 2-4 times during
the residence of a feedstock material in the primary processing
bioreactor. The number of times mature leachate is extracted
depends on the material's composition. Mixtures that are rich in
easily fermentable molecules such as sugars and starch may produce
mature primary leachate up to 3-4 times, while mixtures that are
rich in lignocellulose will only generate mature primary leachate
1-2 times (FIGS. 7A-C). After each extraction of primary leachate,
fresh water is added to the fermentors to dilute metabolic
inhibitors and reinitiate fermentation. The acid still remaining in
the bioreactor after drainage is buffered by adding finely powdered
limestone or chalk powder (CaCO3). The volume of water added to the
bioreactor equals the volume of primary leachate that has been
extracted. The amount of CaCO.sub.3 powder in this water is
calculated based on the volume of the material still remaining in
the bioreactor, the pH of the mature leachate and assuming a
concentration of .about.80% water in the wet solid residue left in
the bioreactor. The time needed to obtain mature primary leachate
in a primary processing bioreactor depends on the type of materials
and time passed from the initial introduction. If raw materials are
rich in sugars and starch, the first primary leachate crop can be
harvested after 3-4 days. Subsequent incubations take longer to
become acidic.
Solid Fermentate
[0224] Solid fermentate is the mixture of organic particles
remaining in the primary processing bioreactor after the primary
leachate has been drained. The residence time of solids in the
primary processing bioreactor ranges between 3 and 6 weeks
depending on composition, temperature and how the processor is
used. The finite solid fermentate resembles pickled vegetables and
contains primary leachate with pH <5 (more frequently closer to
pH 4.0-4.5). During fermentation, cellulosic materials become
increasingly more hydrolyzed. The method for assessing the level of
hydrolysis of acid soluble polysaccharides such as cellulose and
hemicellulose is known in the art (Green and Popa, 2011).
[0225] If incubation requires more than 7 days for the pH to change
with more than 0.1 unit toward acidic or for the COD to increase by
10% relative to the previous value, then changes are preferably
made to the input feedstock material or the solid fermentate should
be considered as spent. Spent solid fementate is preferably removed
from the primary processing bioreactor to create space for adding
more raw materials. If COD increases but the pH does not turn more
acidic, an additive such as a low-cost sugar rich solution (e.g.,
molasses) can be added to the input water. If the pH changes but
the COD does not increase, then more CaCO3 can be added to the
input water or raw materials.
[0226] BSF nurseries (FIG. 2) are facilities using adult BSF flies
to produce BSFL. In nurseries, the larvae are fed with food waste
(or solid fermentate produced in primary bioreactors), are exposed
to solar light (or artificial life with spectral properties similar
to solar light), and are kept at a temperature of 25-35.degree. C.
and 60-90% humidity. BSF eggs are collected and hatched under
controlled conditions as described earlier (Green and Popa, 2012;
Popa and Green, 2012).
[0227] BSFL fed with a diet that is rich in vegetal waste and poor
in animal waste are very efficient in removing secondary
metabolites, and produce little odor in nurseries. BSFL that are
fed excessively with animal-derived products mature fast but are
sloppy eaters and food-selective. This will result in excessive
animal decay odor and creates health risk problems. In nurseries,
it is preferred that animal-derived products and vegetal oil
represent less than 10% by mass based on wet weight. Manure is
preferably not added in large amounts in nursery incubators as it
aggravates both odor and pest problems.
[0228] The larval incubator in the nursery preferably has a
drainage system. Liquid is continuously eliminated and the larvae
and food sprinkled daily with fresh water, otherwise the larval
incubator will produce excessive bad odor, stunted larval growth,
small-sized mature BSF adult flies and very little egg deposition
by the adult BSF. If properly managed, and used in combination with
air filters BSF nurseries will not cause odor problems. The
presence of parasites, predators and competitors (e.g., ants,
rodents, gnats, houseflies, hornets and ichneumonids) is preferably
controlled in the nursery using methods known in the art.
Secondary Processing Bioreactor
[0229] A secondary processing bioreactor is also provided herein.
An embodiment of a secondary processing bioreactor is shown in FIG.
5. The secondary processing bioreactor contains a solid substrate
that decays very slowly and that is not toxic. In one embodiment,
the solid substrate is wood shavings. Other solid, slowly decaying
and non-toxic substrates may be used. The secondary processing
bioreactor also contains primary leachate and premature larvae. The
secondary processing bioreactor is preferably kept at room
temperature and is preferably allowed to function continuously by
removing mature secondary leachate and adding fresh primary
leachate and 7-10 day old larvae.
[0230] Secondary bioreactors function based on the capacity of the
larvae to consume microorganisms and secondary metabolites and thus
to change the primary leachate into secondary leachate. BSFL
increase the pH of the primary leachate as they convert it to
secondary leachate (FIG. 8a), decrease its COD, lower the
concentration of ethanol and organic acids (FIG. 8b) and increase
the concentration of ammonium (Green and Popa, 2012). During this
incubation, the primary leachate being processed into secondary
leachate by the BSFL turns progressively darker and becomes
enriched in melanin. Bioreactors containing 7 L of primary
leachate, 2 kg of larvae, incubated for 12-14 days at 20.degree.
C., produce 4-5 L of secondary leachate with 0.75% melanin (i.e.,
30-37.5 g melanin yield).
[0231] Extraction of melanin from the secondary leachate. Secondary
leachates have alkaline pH (pH 8-9), low COD (<10,000 ppm), high
ammonia content (>1 mM), low concentration of organic acids,
alcohols, sugars and amines (Green and Popa, 2011; 2012) and are
rich in melanins (>0.75%). The method for extracting melanin may
vary based on the quality of the products and chemical composition.
For this study the following method was used to produce
pyomelanin-rich melanin.
[0232] The secondary leachate was evaporated in a hot airstream at
90-100.degree. C. or by exposure to outdoors warm dry air.
[0233] The solid residue was treated with a hot solution of NaOH
(>90.degree. C.) for 24 hours.
[0234] The residue was filtered to obtain the supernatant
containing dissolved pyomelanin and to eliminate insoluble
materials.
[0235] The supernatant was acidified with HCl and titrated to pH
.about.2.
[0236] The acidified liquid was incubated for 24 hours at room
temperature to fully precipitate the pyomelanin.
[0237] The supernatant was decanted and the sediment is filtered
and washed twice with a solution of HCl dissolved in H.sub.2O.
[0238] The washed sediment was evaporated in a warm to hot air
stream (40-90.degree. C.) resulting in a black powder (the
pyomelanin-rich melanin mixture).
[0239] This powder can be diluted in a hot pH 12-14 NaOH or KOH
solution and characterized by art-known methods of
spectrophotometry at 400 nm and by art-known methods of Fourier
Transform Infrared (FTIR) spectroscopy.
Conclusion
[0240] Pyomelanin is a polymer with great electricity storage
properties but high production costs make it impractical for
constructing commercially sustainable batteries. This example
describes a method for producing pyomelanin-rich melanin mixtures
starting from organic waste rich in vegetal materials. Raw organic
materials were first incubated in a primary bioreactor with
microorganisms from the genera Clostridium, Saccharomyces and
Acetobacter. The primary leachate produced in the primary
bioreactor was then converted in a secondary bioreactor into a
secondary leachate rich in melanin through the use of BSFL. Using
this method, it was possible to obtain more than 2 kg of
pyomelanin-rich melanin from one metric ton of food waste and
vegetal biomass. Relative to the surface area of the secondary
bioreactors used in this study, the melanin yield was determined to
be approximately 150 g m.sup.-2 week.sup.-1. An industrial facility
with an installed surface area of secondary bioreactors of 10,000
m.sup.2 will therefore produce 1.5 tons of melanin per week.
6.4. Example 4: Producing Melanin from Sugar-Rich Mixtures and
Mineral Salts
[0241] In one embodiment, melanin is produced using BSFL and
microorganisms using combinations of sugar-rich mixtures (as a
source of carbon and energy) and complex mineral mixtures (as a
source of macro- and micro-nutrients). Sugar-rich primary leachates
can be, in certain embodiments, molasses, extracts from sugar cane
or sugar beets, hydrolyzed starch or hydrolyzed cellulose, residues
of alcohol fermentation, corn syrups, honey, or fruit or vegetable
syrups.
[0242] Mineral and organic nutrients can be obtained from
fermentation processes, sea salts, synthetic mixtures of chemicals,
leachates from manure, soil and compost tea.
[0243] This example describes an assessment that was made of the
method disclosed herein for producing a secondary leachate for use
in producing microbial melanin and/or a microbial
melanin-associated protein. In step (e), i.e., the step of
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, a
liquid mixture of 5 w % molasses, 5 w % sea-salts and 1 and 10 mM
nitrate was tested as an additive. The starting liquid mixture for
the additive had a Brix % value of 4-10 and yellow-orange
color.
[0244] A substrate can be used in the secondary processing
bioreactor that is preferably glass, pebbles or wood shavings.
Coarse sand can also be used. Very fine sand makes it difficult to
extract the liquid, and in tests, it was observed that BSFL had
difficulty burrowing through the sand. The conversion of primary
leachate into secondary leachate enriched in melanin, during the
step of culturing the BSFL with the primary leachate in the
secondary processing bioreactor, was observed to be very poor in
fine sand.
[0245] During step (e), i.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, the following steps were
taken.
[0246] During the culturing step, water loss by evaporation was
replaced by adding tap water. During the first week of culturing,
the BSFL did not lose weight and little change in color was
observed in the secondary leachate. The culturing step was stopped
after two weeks when the secondary leachate turned brown,
indicating that melanin had been produced. At this point, the Brix
value had reached 0-1% and larvae had lost 9-38% of their initial
weight. The conditions for incubation were: 0.014 m.sup.2, 600 g
solid substrate (=43 kg/m.sup.2), 150 ml liquid mixture (=10.7
L/m.sup.2), 50-150 g of white-yellow BSFL (>100 mg in weight)
(=3.6-10.7 kg/m.sup.2) where the depth of the culture layer is
approximately 4 .+-.2 cm, room temperature. Under these conditions,
after two weeks of incubation, the Brix% dropped from 10 to 1, and
the liquid residue contained approximately 0.6-2% DW, 0.3-1%
melanin. The BSFL lost as much as 9-25% of their initial
weight.
[0247] In certain embodiments, after the secondary leachate is
extracted, feed can be added to feed the BSFL to increase their
weight and restore the larval biomass. The BSFL can then be used to
produce melanin again.
[0248] In other embodiments, after one or more cycles, the BSFL can
be used as animal feed or allowed to grow and metamorphose into
pupae.
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[0281] A sample of methods that are described herein are set forth
in the following numbered paragraphs:
[0282] 1. A method for producing a secondary leachate for use in
producing microbial melanin and/or a microbial melanin-associated
protein comprising the steps of: [0283] (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; [0284] (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; [0285] (c)
isolating or removing the primary leachate from the primary
processing bioreactor; [0286] (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 and/or mineral substrate;
[0287] (e) culturing the BSFL in an aerated culture with the
primary leachate isolated or removed from the primary processing
bioreactor and the cellulose-based and/or mineral 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; [0288] (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 [0289] (g) extracting or isolating
the melanin or melanin-associated protein from the secondary
leachate.
[0290] 2. The method of paragraph number 1 wherein the melanin is
selected from the group consisting of pyomelanin, eumelanin and
pheomelanin.
[0291] 3. The method of paragraph number 1 wherein the
melanin-associated protein is associated with the melanin.
[0292] 4. The method of paragraph number 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.
[0293] 5. The method of paragraph number 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.
[0294] 6. The method of paragraph number 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.
[0295] 7. The method of paragraph number 1 wherein the step of
culturing the BSFL with the primary leachate proceeds for 10-20
days.
[0296] 8. The method of paragraph number 1 wherein the fermentation
medium is organic waste.
[0297] 9. The method of paragraph number 8 wherein the organic
waste is food waste, plant waste, compost, cellulosic residues,
cellulose-rich waste, starch-rich waste, or protein-rich waste.
[0298] 10. The method of paragraph number 1 wherein the step of
fermenting the fermentation medium with the microbial culture is
conducted under anaerobic or microaerobic conditions.
[0299] 11. The method of paragraph number 1 wherein the
microorganisms in the microbial culture are bacteria, yeast and/or
fungi.
[0300] 12. The method of paragraph number 1 wherein the
microorganisms in the microbial culture further comprise
Clostridium and/or Acetobacter bacteria.
[0301] 13. The method of paragraph number 1 wherein the providing
step (a) comprises providing a microbial culture that is a mixed
microbial culture.
[0302] 14. The method of paragraph number 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.
[0303] 15. The method of paragraph number 1 comprising the step of
monitoring the chemical composition of the primary leachate.
[0304] 16. The method of paragraph number 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.
[0305] 17. The method of paragraph number 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.
[0306] 18. The method of paragraph number 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.
[0307] 19. The method of paragraph number 1 wherein the step of
culturing the BSFL comprises the step of adding an additive to the
secondary processing bioreactor.
[0308] 20. The method of paragraph number 19 wherein the additive
comprises carbohydrate, cellulose and/or starch.
[0309] 21. The method of paragraph number 20 wherein the
carbohydrate is a sugar.
[0310] 22. The method of paragraph number 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, or 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.
[0311] 23. The method of paragraph number 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.
[0312] 24. A method for producing a secondary leachate for use in
producing inorganic fertilizer from the secondary leachate
comprising the steps of:
[0313] (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;
[0314] (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;
[0315] (c) isolating or removing the primary leachate from the
primary processing bioreactor;
[0316] (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 and/or mineral substrate;
[0317] (e) culturing the BSFL in an aerated culture with the
primary leachate isolated or removed from the primary processing
bioreactor and the cellulose-based and/or mineral 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
[0318] (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
[0319] (g) extracting or isolating the inorganic fertilizer from
the secondary leachate.
[0320] 25. The method of paragraph number 24 wherein the inorganic
fertilizer comprises ammonium, phosphate, carbonate and/or
micronutrients.
[0321] 26. The method of paragraph number 24 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.
[0322] 27. The method of paragraph number 24 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.
[0323] 28. The method of paragraph number 24 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.
[0324] 29. The method of paragraph number 24 wherein the step of
culturing the BSFL with the primary leachate proceeds for 10-20
days.
[0325] 30. The method of paragraph number 24 wherein the
fermentation medium is organic waste.
[0326] 31. The method of paragraph number 30 wherein the organic
waste is food waste, plant waste, compost, cellulosic residues,
cellulose-rich waste, starch-rich waste, or protein-rich waste.
[0327] 32. The method of paragraph number 24 wherein the step of
fermenting the fermentation medium with the microbial culture is
conducted under anaerobic or microaerobic conditions.
[0328] 33. The method of paragraph number 24 wherein the
microorganisms in the microbial culture are bacteria, yeast and/or
fungi.
[0329] 34. The method of paragraph number 24 wherein the
microorganisms in the microbial culture further comprise
Clostridium and/or Acetobacter bacteria.
[0330] 35. The method of paragraph number 24 wherein the providing
step (a) comprises providing a microbial culture that is a mixed
microbial culture.
[0331] 36. The method of paragraph number 24 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.
[0332] 37. The method of paragraph number 24 comprising the step of
monitoring the chemical composition of the primary leachate.
[0333] 38. The method of paragraph number 37 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.
[0334] 39. The method of paragraph number 24 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.
[0335] 40. The method of paragraph number 24 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.
[0336] 41. The method of paragraph number 24 wherein the step of
culturing the BSFL comprises the step of adding an additive to the
secondary processing bioreactor.
[0337] 42. The method of paragraph number 41 wherein the additive
comprises carbohydrate, cellulose and/or starch.
[0338] 43. The method of paragraph number 42 wherein the
carbohydrate is a sugar.
[0339] 44. The method of paragraph number 24 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, or 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.
[0340] 45. The method of paragraph number 24 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.
[0341] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description. Such modifications are intended to fall
within the scope of the appended claims.
[0342] While embodiments of the present disclosure have been
particularly shown and described with reference to certain examples
and features, it will be understood by one skilled in the art that
various changes in detail may be effected therein without departing
from the spirit and scope of the present disclosure as defined by
claims that can be supported by the written description and
drawings. Further, where exemplary embodiments are described with
reference to a certain number of elements it will be understood
that the exemplary embodiments can be practiced utilizing either
less than or more than the certain number of elements.
[0343] All references cited herein are incorporated herein by
reference in their entirety and for all purposes to the same extent
as if each individual publication, patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes.
[0344] The citation of any publication is for its disclosure prior
to the filing date and should not be construed as an admission that
the present invention is not entitled to antedate such publication
by virtue of prior invention.
* * * * *
References