INSECT-BASED REMOVAL OF ORGANIC SOLUTES FROM LIQUID

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Application No. 13/010,654, filed January 20, 2011, which is incorporated by reference herein in its entirety.

FIELD

The disclosure relates generally to a process for removing organic solutes from a liquid by incubating fly larvae with a liquid containing organic solutes, and apparatus designed for such processing of liquids. Certain embodiments relate to increasing larval biomass.

BACKGROUND

Composting technologies for processing organic material, including animal manure, sewage, agricultural and forest residues, industrial byproducts, produce and food scrap are well-known. For example, microbial-dependent aerobic and anaerobic composting, vermicomposting and processes combining microbial and vermicomposting technologies are useful to dissimilate organic material. However, technologies concerning detoxification and treatment of liquids containing organic solutes, including liquid produced by the use of known composting technologies, are underdeveloped. During these processes numerous byproducts are released, including gases (C0 ₂ , CH ₄ , N ₂ 0, NH ₃ , NO, H ₂ S, methyl sulfides), alcohols (ethanol and methanol), volatile organic acids (VOAs, acetic, propionic, butyric, valeric and isovaleric), amines, mercaptans, sugars, proteins and many other biochemicals. Many of these compounds are also present in the organic material before

composting.

Fly larvae will feed on a variety of organic materials, are known to be useful as a manure management tool and they have economic value as a feedstock (Newton et al., J. Anim. Set, 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). One example is Black Soldier Fly (BSF) larvae. However, known methods of using BSF larvae for processing of organic material call for the use of organic material with a moisture content of less than -80% water by weight (Fatchurochim et ah, J. Entomol. Set, 24:224-231, 1989).

SUMMARY

Disclosed herein is the unexpected discovery that larvae {e.g., fly larvae) can consume and assimilate organic solutes present in a liquid and gain biomass based on this nutrient source. Thus, surprisingly, larvae can be used to remove organic solutes from a liquid and incubating larvae with liquid containing organic solutes will cause the larvae to gain biomass. An apparatus for performing these processes is disclosed herein.

A method of removing organic solute from a liquid is provided. The method comprises selecting a liquid nutrient source containing organic solute, wherein the nutrient source comprises at least 80% water or other liquid and is substantially free of solid nutrients; and incubating fly larvae for a period of time with the nutrient source, thereby removing organic solute from the liquid. In some embodiments, the method further comprises harvesting the larvae.

Also provided is a method of producing larva biomass, comprising incubating fly larvae for a period of time with a liquid nutrient source containing organic solute, wherein the nutrient source comprises at least 80% water or other liquid and is substantially free of solid nutrients; and harvesting the fly larvae, thereby producing larva biomass.

In some embodiments, the fly larvae used in the methods provided herein are

Hermetia illucens larvae.

Also provided is an apparatus for removing organic solute from a liquid or for producing larva biomass, the apparatus comprising an enclosed tank, which enclosed tank comprises a liquid entry port, a liquid exit port, a gas entry port, a gas exit port, an inner reservoir capable of holding liquid, wherein the liquid entry and exit ports are operably linked to the inner reservoir, and access means for accessing the inner reservoir of the tank. In some embodiments, the inner reservoir contains one or more fly larvae, such as Henrietta illucens (black soldier fly; BSF) larvae.

The foregoing and other aspects, objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a schematic diagram illustrating an embodiment of the disclosed apparatus for removing organic solute from a liquid nutrient source or increasing larva biomass.

FIG. 2 is a graph illustrating that BSF larvae clear short chain alcohols and VOAs from compost tea. Short chain alcohol and VOA concentrations in control and BSF larvae treated compost tea were measured by gas chromatography.

Samples were drawn from each condition at the beginning of the experiment, and two days later after incubation with or without BSF larvae, and test outcomes compared. Concentrations of ethanol (Et), propanol (nP), acetic acid (Ac), propanoic acid (Pr), butyric acid (Bu) and isovaleric acid (Is) were measured. These results show that BSF larvae feeding on compost tea can clear short chain alcohols and VOAs from the liquid nutrient source. Errors bars are + 1SD of duplicates using 20 BSF larvae in 10 ml compost tea.

FIG. 3 is a graph illustrating the turnover of the dominant VOAs and alcohols in sterile vs. non-sterile compost tea. Concentrations of ethanol (Et), propanol (nP), acetic acid (Ac), propanoic acid (Pr), butyric acid (Bu) and isovaleric acid (Is) were measured. Errors bars are + 1SD of triplicates in 3 ml of compost tea per sample.

FIG. 4 is a digital image illustrating that BSF larvae clear ninhydrin-positive amines from compost tea. Ninhydrin-positive amines in control and BSF larvae treated compost tea were analyzed by thin layer chromatography. Samples were applied as follows (left to right): lane 1, glycine; lane 2, glycyl-glycine; lane 3, glutamic acid; lane 4, compost tea control; lane 5, compost tea processed with BSF larvae; lane 6, leucine. The results of this experiment show that BSF larvae can clear nitrogen metabolites from compost tea. FIG. 5 is a graph illustrating the effect of BSF larvae on the pH and chemical oxygen demand (COD) of compost tea. pH without BSF larvae (open triangles) and with BSF larvae (filled triangles) and well as COD without BSF larvae (open squares) and with BSF larvae (filled squares) was measured. Errors bars are +1 SD of duplicates using 20 BSF larvae in 10 ml compost tea.

FIG. 6 is a digital image illustrating BSF larvae feeding on compost tea. Darker larvae are at the prepupa stage; arrows show shed exoskeletons.

FIGs. 7A and 7B are a graph and a digital image (respectively) illustrating the growth of BSF larvae in several different liquid nutrient sources as well as a device for assaying BSF larvae growth in a liquid nutrient source. FIG. 7A shows the relative growth of the larvae in one week on different liquid wastes (sewage (Sw), whey (Wh), compost tea (CL) and 2 % milk (Mk)). Each data point is the average of 20 larvae. FIG. 7B illustrates an assay device showing twenty BSF larvae feeding on compost tea in a tilted test tube (bottom tube) and appearance of compost tea in comparable tilted tube (top tube) in which larvae were omitted.

Perforated push caps were used to provide air to the larvae and for access in monitoring larval feeding of liquids presented to the BSF larvae.

FIG. 8 is a graph illustrating the growth of BSF larvae (% weight gain) over a one week interval while they fed on various filtered liquid nutrient sources, including urine (Ur), sewage water (SW), water extracted Gainesville House Fly diet (GF), chicken broth (CB), orange juice (OJ), compost tea (CT), chicken manure extract (CME), whey (Wh); and milk (Mk).

FIG. 9 is a digital image illustrating a Modular Larvae Incubation Unit (MLIU), showing drain holes, used to incubate BSF larvae with circulating fluids as described in Example 6.

FIG. 10 is a digital image illustrating a side view of an operating array of MLIUs housed inside an enclosed tank as described in Example 6.

FIG. 11 is a digital image illustrating a top down view of the uppermost MLIU in an array of MLIUs as described in Example 6. The BSF larvae are incubating in compost tea which enters the array of MLIUs from a liquid entry port and drips through the holes of the uppermost MLIU into the next MLIU in the array. FIG. 12 is a digital image illustrating the bottom MLIU, which lack drainage holes, for use in an array of MLIUs, as described in Example 6. A tube for draining liquid nutrient source from the bottom MLIU is shown.

FIG. 13 is a digital image illustrating an assembled apparatus as described in Example 6, and for use with the methods described herein. The apparatus including an enclosed tank having an inner reservoir comprised of a single array of MLIUs. Each of the MLIUs (except the bottom MLIU) contains BSF larvae which are feeding on compost tea pumped through the apparatus.

FIGs. 14A and 14B are a set of graphs illustrating the evolution of N0 ₃ ^~ , N0 ₂ , NH ₄ ⁺ and pH in a solution of 10 mM NaN0 ₃ made up in tap water with or without BSF larvae. FIG. 14A shows the evolution of N0 ₃ and N0 ₂ . FIG. 14B shows the evolution of NH ₄ ⁺ and pH. BSF larvae were first suspended in the nitrate solution, from which control solutions free of BSF larvae were drawn within 1-2 minutes. This time interval was sufficient for the larvae to release a small amount of NH ₄ ⁺ in solution along with frass. The experiments were done in duplicates, the measurements were done in triplicates, and the error bars represent +1 SD.

FIGs. 15A and 15B are a set of graphs illustrating the evolution of N0 ₃ ^" , N0 ₂ , NH ₄ ⁺ and pH in a solution of tap water with or without BSF larvae. FIG. 15A shows the evolution of N0 ₃ ^" and N0 ₂ . FIG. 15B shows the evolution of NH ₄ ⁺ and pH. BSF larvae were first suspended in tap water, from which control solutions free of BSF larvae were drawn within 1-2 minutes. This time interval was sufficient for the larvae to release a small amount of NH4 ⁺ in solution (~1 mM) along with frass. The experiments were done in duplicates, the measurements were done in triplicates, and the error bars represent +1 SD.

FIGs. 16A 16B are a set of graphs illustrating the evolution of N0 ₃ ^" , N0 ₂ ,

NH ₄ ⁺ and pH in compost tea with or without BSF larvae. FIG. 16A shows the evolution of N0 ₃ and N0 ₂ . FIG 16B shows the evolution of NH ₄ ⁺ and pH. BSF larvae were first suspended in compost tea, from which control solutions free of BSF larvae were drawn within 1-2 minutes. The experiments were done in duplicates, the measurements were done in triplicates, and the error bars represent + 1SD.

FIG. 17 is a graph illustrating the relationship between the concentration of ammonium and the change in the pH of the compost tea incubated with larvae. The curved line is a polynomial fit through all data points in the pH range -4.4-8.8. The straight line represents the linear regression of the data points in the pH range -7.4- 8.8. pH _f = final pH; p¾ = initial pH. DETAILED DESCRIPTION

/. Terms and Abbreviations

BSF black soldier fly

COD chemical oxygen demand

DNRA dissimilatory nitrate reduction to ammonia

FWT fresh weight

MC microaerobic

MLIU modular larvae incubation unit

VOA volatile organic acid The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. The singular forms "a," "an," and "the" refer to one or more than one, unless the context clearly dictates otherwise. For example, the term "comprising a Black Soldier Fly larva" includes single or plural Black Soldier Fly larvae and is considered equivalent to the phrase "comprising at least one Black Soldier Fly larva." The term "or" refers to a single element of stated alternative elements or a combination of two or more elements, unless the context clearly indicates otherwise. As used herein, "comprises" means "includes." Thus, "comprising A or B," means "including A, B, or A and B," without excluding additional elements.

Unless explained otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting. For example, conventional methods well known in the art to which a disclosed invention pertains are described in various general and more specific references, including, for example, Smith and March, March' s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley- Interscience, 2001; Vogel, A Textbook of Practical Organic Chemistry, Including Qualitative Organic Analysis, Fourth Edition, New York: Longman, 1978; and International Union of Pure and Applied Chemistry, Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Oxford: Pergamon, 1979. Additional terms commonly used in chemistry can also be found in these references.

Biomass: A mass of organic material formed by a living organism. Larva biomass is a mass of fly larvae.

Compost: The decomposed or decomposing remnants of organic material, such as plant materials, food scraps, or animal feces and urine.

Composting: The process of decomposition that allows organic material to decompose into compost. Many types of composting are known, including aerobic, anaerobic and insect-based composting.

Compost tea: A liquid produced by separating liquid from a compost/liquid mixture. For example, compost tea can be produced by adding a liquid, such as water, to compost to make a compost/liquid mixture, followed by separating the liquid from the mixture. Compost tea can also be produced by separating liquid from organic matter having a liquid content; for example, by separating liquid from a mixture comprising animal feces and urine. An alternative term for "compost tea" is "compost leachate."

Control: Samples believed to be normal (e.g., representative of an activity or function in the absence of the variable being tested), as well as laboratory values, even though possibly arbitrarily set, keeping in mind that such values can vary from laboratory to laboratory. A control group is practically identical to the treatment group, except for the single variable of interest whose effect is being tested, which is only applied to the treatment group.

Contacting: Placement in direct physical association, including in solid, liquid and gas form. Contacting includes contact between one molecule and another molecule and also includes contact between a larva and a liquid nutrient source.

Fly larvae: Also known as maggots, fly larvae are a stage of the life cycle of a fly, which progresses through (in order) egg, larva, pupa and adult fly stages. Several intermediate stages are also identified, including the pre-pupa stage, a stage in between the larva and pupa stages when the larva moves away from the nutrient source to find a pupation site. Examples of fly larvae include Musca domestica larvae, Muscina stabulans larvae, Fannia canicularis larvae, Fannia femoralis larvae, Ophyra aenescens larvae, or Hermetia illucens larvae. As used herein, "larvae" includes fly larvae, such as Hermetia illucens and other fly larvae.

Enclosed tank: An enclosed container made of a substance that is substantially impermeable to air. Such a container may have openings designed to allow liquid, solid or gas to enter and/or exit the container, for example liquid entry and exit ports, or gas entry and exit ports. The container may be of any size. In some examples, the container has a lid or door that opens to allow access to the interior of the container.

Gas scrubber: A device that can be used to remove particulates and/or gases from air. Gas scrubbers include scrubbers designed for wet scrubbing and dry scrubbing.

Harvesting: The collection of something, by any means. For example, harvesting larvae includes collecting larvae by hand or by machine, among other methods.

Hermetia illucens: Commonly known as the Black Soldier Fly or Privy Fly, Hermetia illucens is a fly of the family Stratiomyidae.

Incubating: A term that includes a sufficient amount of time for a larva, such as a BSF larva, to interact with something, such as a liquid nutrient source.

Liquid nutrient source: A liquid containing organic solutes. A liquid nutrient source is substantially free of solid nutrients. A liquid nutrient source is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent liquid by weight. Though some or all of the liquid portion of a "liquid nutrient source" can be water, water is not the only liquid that is contemplated. Other non-limiting examples of liquids that may be components of a "liquid nutrient source" include alcohols and other organic solvents. A liquid has the property of dripping and flowing, and is capable of being pumped through a pipe. Non-limiting examples of liquid nutrient sources include whey, compost tea, liquid produced from sewage, grey water, liquid produced from food processing and combinations of two or more thereof. A liquid nutrient source may contain microorganisms, for example aerobic and anaerobic bacteria. A liquid nutrient source stream is a liquid nutrient source that is flowing.

Manure: Material, especially barnyard or stable dung, often with discarded animal bedding, used to fertilize soil. For example, manure includes organic material excreted by animals feeding on varying feeds, which may be mixed with feces and urine, compost and plant material.

Modular Larvae Incubation Unit (MLIU): A partially or fully enclosed container made of a substance that is substantially impermeable to liquid, e.g., metal, rubber, wood or plastic, which is used for incubating larvae with a liquid nutrient source. Some MLIUs comprise means for liquid to pass through the bottom of the MLIU, for example, an MLIU may comprise holes in the bottom surface of the MLIU.

In some embodiments, the MLIUs are included in an array of MLIUs, wherein the MLIUs are positioned, such that at least one MLIU is above at least one other MLIU. In some embodiments, the uppermost module in an array of MLIUs includes a lid. Typically, each MLIU in an array (except the lowest unit in an array) has means for liquid nutrient source to pass through its lower surface. The lowest MLIU in an array of MLIUs usually lacks drain holes, allowing liquid nutrient source traveling through the array of MLIUs to collect at least temporarily in the lowest unit. Any number of MLIUs may be included in an array of MLIUs. In some cases, multiple MLIUs will be located above one or more MLIUs. In each array of MLIUs, liquid nutrient source may pass from higher MLIUs to lower MLIUs. An array of MLIUs may include MLIUs stacked directly above one another, or may include MLIUs stacked partially above one another, or a combination thereof. Some MLIUs are constructed to be capable of connecting to other MLIUs, others are not capable of connecting to other MLIUs. For example, an array of MLIUs may include MLIUs capable of interconnecting vertically to form vertical arrays of MLIUs, e.g., by snapping together snuggly one on top of the other in vertical stacks of repeating units.

Organic compound: A gaseous, liquid, or solid chemical compound, the molecules of which contain carbon, nitrogen, sulfur, phosphorous or a combination of two or more thereof. Various organic compounds are listed in International Union of Pure and Applied Chemistry, Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Oxford: Pergamon, 1979. Examples include gases (C0 ₂ , CH ₄ , N ₂ 0, NH ₃ , NO, H ₂ S, methyl sulfides), alcohols (ethanol and methanol), volatile organic acids (VOAs, acetic, propionic, butyric, valeric and isovaleric), amines, mercaptans, sugars, proteins, nitrite (N0 ₂ ^~ ), nitrate (NO ₃ ^" ), ammonium (NH ₄ ⁺ ) and many other biochemicals.

Organic material: Solid and/or liquid matter composed of organic compounds.

Organic solute: An organic compound dissolved in fluid, forming a solution.

Sewage: Water-based fluid containing organic matter and solutes. Sewage may include feces and urine from human and non-human animals. Sewage may include waste from human activities, for example, blackwater (e.g., toilet and dishwasher waste) and grey water (e.g. waste water generated from washing activities). Residential, institutional, commercial and industrial establishments may produce sewage, including waste from toilets, baths, showers, kitchens, sinks, etc. Typically, sewage is waste intended to be carried away from the source of the waste, for example, carried to a sewage treatment facility.

Vermicompost: The product of composting utilizing various species of worms, usually to create a heterogeneous mixture of decomposing vegetable or food waste, bedding materials, and vermicast. Vermicast, also known as worm castings, worm humus or worm manure, is the end-product of the breakdown of organic matter by species of earthworms. The earthworm most often used is the Red Wiggler (Eisenia foetida or Eisenia andrei). Containing water-soluble nutrients, vermicompost is a nutrient-rich organic fertilizer and soil conditioner. Methods of using worms to produce vermicompost are well known (see, e.g., U.S. Pat. No. 6,223,687, 6,838,082, 6,890,438, 7,141,169). //. Overview of Several Embodiments

Provided here in are methods of reducing and/or removing organic solutes from a liquid and methods of increasing larval biomass, as well as apparatuses and devices for performing these methods.

A method of removing organic solute from a liquid is provided herein. The method comprises selecting a liquid nutrient source containing organic solute, wherein the nutrient source comprises at least 80% water or other liquid and is substantially free of solid nutrients; and incubating fly larvae for a period of time with the nutrient source, thereby removing organic solute from the liquid. In some embodiments, the method further comprises harvesting the larvae.

Also provided herein is a method of producing larva biomass, comprising incubating fly larvae for a period of time with a liquid nutrient source containing organic solute, wherein the nutrient source comprises at least 80% water or other liquid and is substantially free of solid nutrients; and harvesting the fly larvae, thereby producing larva biomass.

In some embodiments, the liquid nutrient source comprises compost tea, liquid produced from urine, whey, sewage, liquid produced from manure, or a combination of two or more thereof. In some embodiments, the pH of the liquid nutrient source is about 3.2 to about 9.4.

In some embodiments, the fly larvae used in the methods provided herein are selected from the group consisting of Musca domestica larvae, Muscina stabulans larvae, Fannia canicularis larvae, Fannia femoralis larvae, Ophyra aenescens larvae, Hermetia illucens larvae or a combination of two or more thereof.

In some embodiments, the fly larvae are incubated at about 2x10 to about 2xl0 ⁵ larvae per liter of liquid nutrient source, at a temperature of about 20°C to about 40°C, with the liquid nutrient source for about 1, 6, 12, 24 or 48 hours, or about 1 day, 1 week or 1 month, or a combination of two or more thereof.

In some embodiments, harvesting the larvae comprises harvesting mature larvae or larvae entering the pupa stage of the larva life cycle, or both. In various embodiments, harvesting the larvae comprises passing the larvae through a filter having a pore size of about 0.3 cm to about 0.5 cm. Also provided herein is an apparatus for removing organic solute from a liquid or for producing larva biomass. In some embodiments, the apparatus comprises an enclosed tank, which enclosed tank comprises a liquid entry port, a liquid exit port, a gas entry port, a gas exit port, an inner reservoir capable of holding liquid, wherein the liquid entry and exit ports are operably linked to the inner reservoir, and access means for accessing the inner reservoir of the tank. In some embodiments, the inner reservoir contains one or more fly larvae, such as Henrietta illucens larvae. In some embodiments, the inner reservoir comprises one or more arrays of modular larvae incubation units.

In some embodiments of the apparatus, the gas exit port is operably linked to a gas pump. Optionally, the apparatus is operably linked to at least one gas trapping scrubber means.

In some embodiments, the apparatus further comprises means for maintaining fly larva at a depth of about 0.5 to about 3 cm of liquid in the inner reservoir, means for preventing fly larvae from passing through the liquid exit port, means for removing larvae from the inner reservoir, means to monitor organic solute concentration of the liquid in the liquid entry port, the inner reservoir, the liquid exit port or a combination of two of more thereof, means for heating or cooling a liquid in the inner reservoir, or a combination of two or more thereof.

It will be further understood that the methods of reducing organic solute in a liquid or producing larva biomass, as well as the apparatus for performing these methods disclosed herein are useful beyond the specific circumstances that are described in detail herein, and for instance are expected to be useful for any number of situations where it is desirable to remove organic solute from liquid or increase insect larva biomass.

///. Larvae

The larval growth stage is a stage of the life cycle of a fly, which progresses from egg to larva to pupa to adult fly stages. Several intermediate stages are also identified, including the pre-pupa stage, a stage in between the larva and pupa stages when the larva moves away from the nutrient source to find a pupation site. The skilled artisan is familiar with larvae generally, and with methods of breeding and propagating larvae. For example, methods of breeding and propagating Musca domestica larvae, Muscina stabulans larvae, Fannia canicularis larvae, Fannia femoralis larvae, Ophyra aenescens larvae, or Hermetia illucens larvae (see, e.g., Fatchurochim et al., J. Entomol. ScL, 24:224-231, 1989).

Some embodiments use BSF larvae. BSF larvae feed on a variety of vegetal and manure wastes of varying extreme pH ranges and 0 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 45 ^th degree latitude (Newton et al, J. Anim. ScL, 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 αΙ., Αηη. 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). Adult BSF do not need to eat; they survive on the fat stored from the larva stage. BSF larva consume organic matter, including kitchen waste, spoiled feed, and manure, and assimilate organic compounds in the organic matter into larva biomass.

Methods of breeding and propagating BSF larvae, including methods of breeding BSF larvae in captivity, as well as methods of using BSF larvae to process solid wastes, 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). Additionally, BSF larvae can be purchased commercially, for example BioGrubs™ BSF larvae (Prota Culture, LLC, Dallas, TX) and Phoenix Worms™ BSF larvae (Insect Science Resource, LLC, Tifton, GA). Alternatively, BSF larvae, 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.

BSF eggs take approximately 4 days to hatch and are typically deposited in crevices or on surfaces above or adjacent to the food source. BSF larvae

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.

IV. Liquid Nutrient Sources

Embodiments described herein utilize liquid nutrient sources. The liquid nutrient sources are substantially free of solid nutrients (such as solid organic matter). However, particulates of solid nutrients may be included in a liquid nutrient source. In the embodiments described herein, the liquid nutrient source is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent liquid by weight.

The liquid nutrient sources disclosed herein comprise liquids containing organic solute, e.g., dissolved organic compounds, including, gases (e.g., C0 ₂ , CH ₄ , N ₂ 0, NH ₃ , NO, H ₂ S, methyl sulfides), alcohols (e.g., ethanol and methanol), volatile organic acids (e.g., acetic, propionic, butyric, valeric and isovaleric), amines, mercaptans, sugars, proteins and many other biochemicals. Liquids containing organic solutes will be apparent to the skilled artisan. Examples include liquid produced from food processing (e.g., whey), liquid industrial waste or liquid produced from industrial waste, sewage (e.g., toilet waste), liquid produced from sewage and sewage treatment facilities, liquid produced from manure and compost tea. In some embodiments, the liquid nutrient source may contain microorganisms, for example, aerobic and anaerobic bacteria.

In some embodiments, the liquid comprises sewage or a liquid produced from sewage. The skilled artisan understands methods used to identify and obtain sewage and liquid produced from sewage. For example, sewage includes both blackwater (toilet and dishwasher waste) and grey water (wastewater that does not include human waste, e.g. waste water generated from washing activities).

Residential, institutional, commercial and industrial establishments produce sewage, including waste liquid from toilets, baths, showers, kitchens, sinks and so forth. Liquid produced from sewage is typically obtained by filtering or separating sewage into solid and liquid phase (e.g., by physical treatment of sewage at a wastewater treatment facility). Though some or all of the liquid portion of a liquid nutrient source can be water, water is not the only liquid that is contemplated. Other non-limiting examples of liquids that may be components of a liquid nutrient source include alcohols and other organic solvents.

The liquid nutrient sources disclosed herein may range in pH. The liquid nutrient source may be any pH in which a larva is capable of surviving for a period of time sufficient to gain biomass or remove organic solute from the liquid nutrient source. For example, the liquid nutrient source may have a pH of about 3.0 to about 10, including about 3.2 to about 9.4, or higher or lower pH. In some embodiments, the liquid nutrient source has a pH of about 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or about 10.0. In some embodiments the pH of the liquid nutrient source is not uniform throughout the liquid nutrient source, or the pH of the liquid nutrient source may change over the time that the larvae are incubated in the liquid nutrient source. The pH of a liquid nutrient source may be altered by adding acid or base to the liquid nutrient source.

The liquid nutrient sources disclosed herein may range in temperature. The liquid nutrient source may be any temperature in which a larva is capable of surviving for a period of time sufficient to gain biomass or remove organic solute from the liquid nutrient source. For example, the liquid nutrient source may have a temperature of about 10°C to about 40°C, for example about 15°C to about 40°C, about 20°C to about 40°C, or higher or lower temperatures. In some embodiments, the liquid nutrient source has a temperature of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 20, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C. In some embodiments the temperature of the liquid nutrient source is not uniform throughout the liquid nutrient source, or the temperature of the liquid nutrient source may change over the time that the larvae are incubated in the liquid nutrient source. The temperature of the liquid nutrient source may be altered by cooling or heating.

Selecting a liquid nutrient source that may be useful in one of the provided methods usually involves identifying a liquid containing organic solute and selecting the liquid, thereby selecting a liquid nutrient source. Methods of identifying that a liquid contains organic solute are known to the skilled artisan, and representative examples are described herein. For example, a sample of a liquid may be obtained and tested for organic solute. If the sample contains organic solute, then the liquid is a liquid nutrient source and selecting the liquid is selecting a liquid nutrient source. In other examples, it will be apparent to the skilled artisan that a liquid contains organic solute, and is therefore a liquid nutrient source. For example, certain liquids (e.g., whey, compost tea, sewage, liquid produced from food processing) are known to contain organic solute. Selecting a liquid known to contain organic solute is a means of selecting a liquid nutrient source. V. Methods for Removing Organic Solutes and Producing Larva Biomass

Disclosed herein are methods of producing larva biomass as well as methods of removing organic solute from a liquid. In some embodiments, the method of producing larva biomass includes incubating larvae for a period of time with a liquid nutrient source containing organic solute, wherein the liquid nutrient source comprises at least about 80% water and is substantially free of solid nutrients and harvesting the larvae, thereby producing larva biomass. In other embodiments, the method of removing organic solute from a liquid includes selecting a liquid nutrient source containing organic solute, wherein the nutrient source comprises at least about 80% water and is substantially free of solid nutrients; and incubating larvae for a period of time with the nutrient source, thereby removing organic solute from a liquid.

Incubation with a liquid nutrient source for a period of time

The embodiments described herein involve incubating larvae with a liquid nutrient source, for example incubating BSF larvae with compost tea. Incubation of larvae with a liquid nutrient source involves contacting a liquid nutrient source with one or more larvae. The skilled artisan will understand methods of contacting a larva with a liquid nutrient source.

In several embodiments described herein, the depth of the liquid nutrient source that the larvae are exposed to is limited. For example the depth of the liquid nutrient source that the larvae are exposed to may be altered by changing the depth of the liquid nutrient source, or by adjusting the depth that the larvae may descend into the liquid nutrient source, e.g., by placing a barrier in the liquid nutrient source, below which the larvae cannot descend. In the embodiments described herein, the maximum depth of the liquid nutrient source that the larvae are exposed to is about 0.5, 1, 2, 3, 4 or 5 cm. In some embodiments, the maximum depth of the liquid nutrient source that the larvae are exposed to is about 3 cm. In some embodiments, the maximum depth of the liquid nutrient source that the larvae are exposed to varies between about 0 and 5 cm, 0 and 3 cm, 0.5 and 5 cm, or 0.5 and 3 cm deep.

Incubating larvae with a liquid nutrient source includes incubating various densities of larvae with the liquid nutrient source. In some embodiments, the density of the larvae in the liquid nutrient source is a minimum of about 2x10 larvae per liter of liquid nutrient source. In some embodiments, the density of the larvae in the liquid nutrient source is a maximum of about 2xl0 ⁵ larvae per liter of liquid nutrient source. In other embodiments, the density of the larvae in the liquid nutrient source ranges from about 2x10 3 to about 2x105 larvae per liter of liquid nutrient source.

The period of time that larvae are incubated with a liquid nutrient source may be determined based on numerous factors, for example, the particular liquid nutrient source the larvae are incubated with, the consumption rate of organic solute by the larvae, or the growth stage of the larvae. Once a particular determining factor is reached, the larvae may be removed from the liquid nutrient source. Methods of removing larvae from a liquid nutrient source are known to the skilled artisan and further described herein, for example as described for methods of harvesting larvae. Additionally, mixed populations of larvae of varying growth stage and size may be used. In many examples, the period of time that larvae are incubated with a liquid nutrient source will be apparent to the skilled artisan based on previous incubation of particular larvae species with particular types of liquid nutrient sources.

In some embodiments, the larvae are incubated with the liquid nutrient source for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 36 or 48 hours, or about 1 day, 1 week, 2 weeks, 3 weeks, 1 month, time periods in between these time, or even more or less time. Optionally, the amount of time the larvae are incubated with the liquid nutrient source may be conveniently measured by the amount of size/mass gain desired in the larva - that is, by measuring size/mass gain in order to achieve a goal larval biomass production level. By way of example, the larvae may be incubated with the liquid nutrient source for a sufficient length of time to increase in mass by 10%, by 20%, by 30%, by 40%, by 50% or more, including a 2-fold increase in mass, a 3-fold increase in mass, a 4- or 5-fold increase in mass, and so forth.

In some examples, the period of time that the larvae are incubated with the liquid nutrient source is determined based on the growth stage of the larvae. For example, a larva can be incubated with the liquid nutrient source until it reaches the pre-pupa stage, or pupa stage. When at least two larva are incubated with the liquid nutrient source, the larvae can be incubated with the liquid nutrient source until at least one larva reaches a particular growth stage, or all larvae reach the particular growth stage, for example the pre-pupa stage. In some embodiments when larvae are incubated with the liquid nutrient source, the larvae are incubated for a period of time until at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the larvae have reached a particular growth stage, for example the pre-pupa or pupa stage. The skilled artisan is familiar with identifying a particular growth stage of a larva.

In some examples, the period of time that the larvae are incubated with the liquid nutrient source is determined based on the size of the larvae. For example, a larva can be incubated with the liquid nutrient source until it reaches a particular size. When at least two larvae are incubated with the liquid nutrient source, the larvae can be incubated with the liquid nutrient source until at least one larva reaches a particular size, or all larvae reach the particular size, for example 0.3 cm in diameter. In some embodiments when at least two larvae are incubated with the liquid nutrient source, the larvae are incubated for a period of time until at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the larvae have reached a particular size, for example 0.3 cm in diameter. Larvae 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. Thus, various sizes in between these ranges, or even more or less than these ranges, may be used to determine the period of time that the larvae are incubated with a liquid nutrient source. For example the larvae may be incubated with a liquid nutrient source until they reach about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, or 2.0 cm in length, or about 0.2, 0.3, 0.4, or 0.5 cm in diameter. The skilled artisan is familiar with identifying a particular size of a larva.

In some examples, the period of time that the larvae are incubated with the liquid nutrient source is determined based on the organic solute concentration in the liquid nutrient source, for example, the concentration of a particular organic solute, such as protein. For example, a larva can be incubated with the liquid nutrient source until the organic solute concentration reaches a particular level, or until the organic solute concentration is reduced by a particular percentage. A reduction in the organic solute concentration can be measured by determining the organic solute concentration of the liquid nutrient source before incubation with the larvae at various times during incubation with the larvae and after incubation with the larvae. Additionally, the organic solute concentration of the liquid nutrient source may be continuously monitored before, during and after contact with the larvae. The period of time that the larvae are incubated with the larvae can be adjusted in response to the measured organic solute concentration. In some instances, the larvae are incubated with the liquid nutrient source for a period of time until a particular reduction in the concentration of an organic solute concentration is reached, for example an about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or even 100% reduction in the concentration of the organic solute. In some embodiments, the larvae are incubated with the liquid nutrient source for period of time until a particular reduction in the concentration of two or more organic solutes is reached. In some embodiments, the larvae are incubated for a period of time until one organic solute concentration is reduced by a particular amount, and another organic solute concentration is reduced by a different amount. The skilled artisan understands how to measure organic solute

concentrations.

The skilled artisan will appreciate that certain variables may be considered to optimize the rate of organic solute removal or production of larva biomass, including the chemical properties of the organic solute of interest to be removed from the waste stream, the efficiency of the larvae in assimilating specific organic solutes, the average age and density of larvae in the liquid waste stream, the period of time the larvae are incubated with the organic solute, and the depth, temperature and pH of the liquid nutrient source. The skilled artisan will appreciate that these variables can be adjusted to optimize removal of organic solute from the liquid nutrient source, or production of larva biomass, based upon a comparison of the amount of organic solute, including particular organic solutes present in the liquid nutrient source before and after incubation with the larvae. Hence, the depth of liquid, density of larvae, period of time, etc., can be adjusted as needed in maximizing organic solute removal or production of larva biomass.

Harvesting larvae

In some embodiments, larvae are harvested after incubation with the liquid nutrient source. The skilled artisan will understand methods of harvesting larvae, including methods of harvesting larvae from a liquid nutrient source. For example, harvesting larvae includes collecting larvae by hand or by machine, among other methods. Harvested larvae can be dried (if need be for shipping and storage), for example before sale as animal feedstock.

In some examples, the larvae are harvested from the liquid nutrient source following the period of time for incubating the larvae with the liquid nutrient source, as described herein. For example, the larvae may be harvested when the organic solute concentration reaches a particular level, when the organic solute

concentration has been reduced by a particular amount, when the larvae reach a particular size, when the larvae reach a particular growth stage or after a designated period of time that the larvae are incubated with the liquid nutrient source. In additional embodiments, the larvae self -harvest from the liquid nutrient source (i.e., the larvae migrate out of the liquid nutrient source) and are then harvested from the position migrated to.

In particular embodiments, harvesting takes place when at least one larva or all larvae reach a particular size, for example 0.3 cm in diameter. In some embodiments when at least two larva are incubated with the liquid nutrient source, the larvae are incubated for a period of time until at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the larvae have reached a particular size, for example the 0.3 cm in diameter.

In some embodiments, harvesting the larvae involves passing the liquid nutrient source in which the larvae are incubating through a filter. Larvae 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. Thus, various filter sizes may be used to harvest larvae, depending on the size of larvae to be harvested. For example, filters with pore sizes of about 0.2, 0.3, 0.4, or 0.5 cm in diameter, or smaller or greater pore sizes may be used to harvest larvae.

Thus, larvae of particular sizes can be periodically harvested from the liquid nutrient source by using a filter with appropriate pore size (e.g., 0.3 to 0.5 cm), leaving smaller larvae (i.e. , immature larvae) in the liquid nutrient source. In other embodiments, larvae can be periodically harvested by passing the waste stream through continuous flow centrifugal processing equipment. Filters and continuous flow centrifugal processing equipment are known to the skilled artisan.

Alternatively, the larvae may be constantly harvested. For example, mixed populations of larvae of varying ages, size and/or growth stage may be used in the methods described herein, and larvae can be continuously added and removed from the apparatus as necessary in maintaining operation of the apparatus. In some embodiments, the larvae are constantly harvested by constantly cycling the liquid nutrient source that the larvae are incubating in through a filter or continuous flow centrifugal processing equipment.

Synergistic interactions

In some embodiments, use of microbes in combination with larvae, and the inclusion of nutrients beneficial to the well-being of the larvae, may be expected to enhance larval capacity to process a liquid nutrient source stream. Thus the methods disclosed herein, and the apparatus for use with these methods, and its optimization in processing a liquid nutrient source stream, include exploration of optimal conditions that depend upon the nature of the material being processed. This includes pH, chemical composition, synergistic interactions that may be exploited such as the presence or absence of microbes in the liquid nutrient source stream, the period of time the larvae are incubated with the liquid nutrient source, etc. As noted earlier, optimization can be measured by comparing the organic solute

concentration, including concentration of particular organic solutes before and after incubation with the larvae by any of a variety of measuring techniques known to the skilled artisan.

Synergism between larval processing of liquid wastes and microbes present or growing in the liquid waste may occur, e.g., when a specific microbe, itself growing in the liquid waste, provides a nutrient source that serves as a feed source for the larvae. Alternatively, synergism between larvae and microbes present in the liquid waste may arise as a result of microbes producing vitamins, essential amino acids, or lipids (or other compounds) as byproducts of the microbes' growth in the liquid, one or more of which compounds stimulate the growth of the larvae.

Synergism in some embodiments results in the removal of byproducts present in the liquid waste which the microbes alone cannot clear from the liquid.

VI. Representative Apparatuses for Performance of Methods Described Herein Provided herein are apparatuses and devices that can be used to perform the disclosed methods of removing organic solute from a liquid nutrient source and increasing larva biomass.

In some embodiments, the apparatus disclosed herein includes an enclosed tank, comprising a liquid entry port, a liquid exit port, a gas entry port, a gas exit port, a gas pump, as well as, an inner reservoir capable of holding liquid, wherein the liquid entry and exit ports are operably linked to the inner reservoir, and access means for accessing the inner reservoir of the tank. The enclosed container is made of a substance that is substantially impermeable to air, for example, concrete, metal, rubber, wood or plastic. The enclosed tank may be any shape or size, for example the enclosed tank may have a box configuration. The skilled artisan will understand methods and materials for the construction of the enclosed tank.

In some embodiments, the apparatus includes liquid entry and exit ports and gas entry and exit ports. Such ports will be familiar to the skilled artisan. In additional embodiments, the ports described herein form a gas impermeable seal with the enclosed tank. Such seals are also familiar to the skilled artisan.

In some embodiments, the apparatus includes means for maintaining the interior of the enclosed tank at a negative air pressure relative to the external air pressure. For example, in some embodiments an air pump is positioned inside enclosed tank, drawing air from inside the tank and forcing it outside the tank through the gas exit port. Alternatively, the air pump can be positioned outside the enclosed tank with its air intake communicating inside the enclosed tank so as to maintain a net negative air pressure inside the enclosed tank relative to the external air pressure. In such embodiments, maintenance of negative air pressure inside the enclosed tank is important so that volatile greenhouse gases and odors can be drawn through the air pump and directed subsequently to scrubbing tanks designed to remove these components from the gas stream before the air, cleared of these elements, is vented to the outside atmosphere.

In some embodiments, the apparatus includes an inner reservoir capable of holding a liquid nutrient source. For example, the inner reservoir may have an open box configuration. In some embodiments, the inner reservoir has a box

configuration, including a lid. The liquid entry and exit ports are operably linked to the inner reservoir such that liquid nutrient source can flow from the liquid entry port, into the inner reservoir, and out the liquid exit port. In some embodiments, the inner reservoir forms a serpentine path for the flow of liquid nutrient source between the liquid entry and exit ports. One of skill in the art will be familiar with methods and materials for the construction of the inner reservoir.

In some embodiments, the inner reservoir comprises one or more arrays of interconnected modular larvae incubation units (MLIUs). Any number of MLIUs may be included in each array. The MLIUs are made of a substance that is substantially impermeable to liquid, for example, metal, rubber, wood or plastic. In some embodiments, the uppermost module in an array of MLIUs comprises a lid. MLIUs are further described herein (e.g. , see FIG. 6). Additionally, the skilled artisan will understand methods and materials for the design and construction of MLIUs.

In some embodiments, the MLIUs are included in an array of MLIUs, wherein the MLIUs are positioned such that at least one MLIU is above at least one other MLIU. Any number of MLIUs may be included in an array of MLIUs. In some cases, multiple MLIUs will be located above one or more MLIUs. An array of MLIUs may include MLIUs stacked directly above one another, or may include MLIUs stacked partially above one another, or a combination thereof. Some MLIUs are constructed to be capable of connecting to other MLIUs, others are not capable of connecting to other MLIUs. For example, an array of MLIUs may include MLIUs capable of interconnecting vertically to form vertical arrays of MLIUs, e.g., by snapping together snuggly one on top of the other in vertical stacks of repeating units.

In each array of MLIUs, liquid nutrient source may pass from higher MLIUs to lower MLIUs. Each MLIU in an array of MLIUs (except the lowest unit or units in an array) includes means for liquid to pass to a lower MLIU in the array, e.g. , an MLIU may include drain holes spanning longitudinally across the floor of the module which allows a liquid nutrient source passing through the module to drain into an identically constructed module positioned under the upper module to catch the liquid nutrient source passing into it from the overhead module. In some embodiments, the drain holes are up to about 0.2 cm in diameter, or even larger. The drain holes may be positioned anywhere along the floor of a modular unit. In some embodiments, the drain holes are positioned to allow a temporary shallow reservoir of liquid nutrient source to accumulate in the area free of drain holes, e.g. , by positioning the drain holes in the base floor of the MLIU approximately one- quarter of its floor width from wall to wall. The lowest MLIU in an array of MLIUs usually lacks drain holes, allowing liquid nutrient source traveling through the array of MLIUs to collect at least temporarily in the lowest unit.

The angle of an array of MLIUs may be tilted from a flat horizontal position before or while a liquid nutrient source passes through the array, thereby allowing for an increase or decrease (depending on the angle) in the depth of liquid nutrient source in the area free of drain holes.

In some embodiments involving MLIUs, the modular units include holes in the side walls of the units. For example, holes may be included in the sidewalls up to about 0.2 cm in diameter, or even larger. The holes provide for air in supporting respiration of any BSF larvae housed in the modular units. For example, the holes may be placed about 0.5 to 2.5 cm beneath its top rim of a modular unit.

In some embodiments including an inner reservoir comprising one or more arrays of MLIUs, the liquid entry port is operably linked to at least one MLIU that is higher than the lowest MLIU in the one or more arrays of MLIUs. The liquid exit port is operably linked to at least one of the MLIUs. For example, the liquid entry port may be operably linked to the highest MLIU in the one or more arrays of MLIUs and the liquid exit port may be operably linked to the lowest MLIU in the one or more arrays of MLIUs. In some embodiments including more than one array of MLIUs, one or more MLIUs in an array of MLIUs may be operably linked to one or more MLIUs in the same or a different array of MLIUs, for example by connecting the MLIUS with a tube.

In some embodiments, the lowest module in an array of MLIUs is operably linked to a tube which is connected to a MLIU that is higher than the lowest the uppermost module(s) in the array, thereby allowing recycling of the liquid nutrient source from the lowest module to the uppermost module using a circulating pump designed to feed the liquid back into the upper unit of the array.

In embodiments including an inner reservoir comprising multiple arrays of MLIUs, the uppermost module(s) in the uppermost array of modular units is operably linked to the liquid entry port. The lowest module in the uppermost array of modules lacks drain holes and is operably linked to the uppermost module in the second-highest array. The lowest module in the second highest array is operably linked to the uppermost module in the next highest array, etc. The lowest module in the lowermost array of MLIUs is operably linked to the liquid exit port. In some embodiments, the lowest module in an array is operably linked to a tube which is connected to the uppermost module in the array, thereby allowing recycling of the liquid nutrient source from the lowest module to the uppermost module using a circulating pump designed to feed the liquid back into the upper unit of the modular stack.

For example, to start the processing and cascading of liquid nutrient source through the units, larvae are first added to each MLIU (except the lowermost MLIU), and liquid nutrient source is then allowed to infuse into the top MLIU via the liquid entry port. The liquid begins to fill the top MLIU, and as it reaches the drain holes, trickles into the MLIU snapped into position below it, in turn filling up to the drain holes of the second MLIU, passing then to the unit below, etc. Larvae can be added to and harvested relatively easily from the individual MLIUs by simply opening and closing the snap-lock connections. Furthermore, the drain holes in the modules make it possible to use the MLIUs as sieves in washing the larvae free of liquid nutrient source at harvest.

In some embodiments, the apparatus includes access means for accessing the inner reservoir of the enclosed tank. In some embodiments, the access means has opened and closed states. When open, the access means allows for access to the inner reservoir of the apparatus. When closed, the access means forms a seal with the enclosed tank and does not allow access to the inner reservoir of the apparatus. The seal is substantially impermeable to air. For example, in some embodiments, the access means may be a lid or door that opens and closes, allowing access to the inner reservoir of the apparatus when in the open state.

In some embodiments, the apparatus includes means for maintaining larvae at a depth of about 0.5 to about 3 cm from the top surface of the liquid nutrient source. In some embodiments, the depth of liquid nutrient source accumulating in the inner reservoir may be determined by how high above the floor of the inner reservoir the operable linkage to the liquid exit port is positioned. For example, the height may be set at 0.5 to 3 cm above the floor of the inner reservoir, thereby maintaining the larvae in the inner reservoir at a depth of about 0.5 to about 3 cm of liquid nutrient source. Some embodiments include an operable linkage to the liquid exit port or a liquid exit port that is adjustable. These embodiments allowing for adjustment of the height of the operable linkage to the liquid exit port or liquid exit port over the floor of the inner reservoir; thus allowing for adjustment of the depth of liquid nutrient source in the inner reservoir. In other embodiments, a pump is used to maintain the level of liquid nutrient source in the inner reservoir.

Alternatively, means for adjusting the flow of liquid nutrient source into or out of the inner reservoir may be included to maintain the height of the liquid in the inner reservoir, for example by including a valve at the liquid entry or exit ports. In some embodiments, the depth of liquid nutrient source in the inner reservoir can be set at more than 3 cm, providing for a larger processing reservoir. In this instance, a net or screen having a pore size small enough to contain immature larvae (i.e., less than 0.1 cm in diameter) is included in the apparatus at about 0.5 to about 3 cm from the top of the liquid nutrient source. The height of the net or screen may be adjustable, for instance to maintain a constant larva depth when the depth of the liquid nutrient source is altered.

In some embodiments, a means for preventing larvae from passing through the liquid exit port is included. For example, a filter positioned at the inner reservoir or at the liquid exit port or in between the inner reservoir and the liquid exit port keeps larvae from escaping the apparatus as liquid exits the apparatus. Such a filer has a pore size of less than 0.1 cm in diameter (i.e. , a pore size smaller than an immature larva).

In some embodiments, the apparatus includes means for monitoring the organic solute concentration of the liquid in the liquid entry port, in the inner reservoir, in the liquid exit port, or a combination of two of more thereof. For example, the apparatus may be designed for samples of liquid to be removed for testing and measurement of organic solute concentration as described herein. In other examples, the apparatus includes instruments capable of detecting organic solute concentration. Such instruments are known to the skilled artisan.

In some embodiments, the apparatus includes means for recycling a liquid nutrient source through the apparatus. For example a pump and piping system may be included to cycle liquid exiting the liquid exit port back to the liquid entry port.

In some embodiments, the apparatus includes a means for removing larvae from the inner reservoir (e.g., means for removing mature larvae and those entering the pupa stage). For example, in some embodiments, the means for accessing the inner reservoir is sized appropriately to allow mechanical removal of larvae (e.g. , scooping of the larvae by hand). In other embodiments, the apparatus includes a vacuum capable of removing larvae from the inner reservoir. Other embodiments include a filter or screen with a pore size that allows immature larvae to pass, but not mature larvae (e.g. a pore size of 0.3 to 0.5 cm in diameter). In some such embodiments, the filter is positioned in the inner reservoir such that it can be passed through the liquid nutrient source in the inner reservoir to remove larvae that are bigger than the pore size of the filter.

In some embodiments, the apparatus is operably linked to an air pollution control device, such as a gas scrubber. For example, a gas scrubber may be operably linked to the gas exit port. The use and manufacture of gas scrubbers is known to those of skill in the art; gas scrubbers are available commercially. For example, wet, dry and semi-dry gas scrubbers may be used, among others. Thus, a train of Ca(OH) ₂ and acidic (H ₂ S0 ₄ ) sparging towers, biofilters, bio-nitrification towers, wet electroscrubbers or other devices may be used as a gas scrubber for purging and scrubbing gases (such as VOAs, volatile amines, C0 ₂ , N ₂ 0, etc.; see, e.g., Schifftner and Hesketh, Wet Scrubbers (2 ^nd Ed.), Lancaster: Technomic Publishing, 1996; Devinny et ah, Biofiltration for Air Pollution Control, Boca Raton: Lewis

Publishers, 1999 ; Jaworek et ah, Environ. Sci. Technol., 40:6197-207, 2006;

Amlinger et al, Waste Manag. Res., 26:47-60, 2006; U.S. Pat. No. 6,013,512).

In some embodiments the apparatus includes means for cooling or heating the liquid nutrient source in the inner reservoir. For example, the apparatus may include a heating element that heats the liquid nutrient source before it enter the inner reservoir, for example the heating element may be positioned at the liquid entry port. In other examples, the apparatus may include a heater that warms the interior of the apparatus. Additionally, the apparatus may include an air

conditioning unit to cool the interior of the apparatus.

FIG. 1 illustrates an embodiment of the apparatus having an open inner reservoir 1 for processing a liquid nutrient source enclosed inside enclosed tank 2. Enclosed tank 2 is made of material substantially impermeable to gases, has a gas entry port 3 where air is drawn inside enclosed tank 2, and a gas exit port 4, where air exits enclosed tank 2. Airflow through enclosed tank 2 provides larvae 5 with air needed for respiration as they incubate with the liquid nutrient source passing through inner reservoir 1. A liquid nutrient source stream is introduced into inner reservoir 1 by way of a liquid entry port 6 passing through the wall of enclosed tank 2, filling inner reservoir 1 to a depth of about 0.5 cm to about 3 cm liquid nutrient source. A liquid exit port 7 provides a means for larvae -processed liquid nutrient source to exit the apparatus. The shallow liquid depth in inner reservoir 1 allows larvae to respire as they feed on the liquid nutrient source. Enclosed tank 2 is constructed with gas entry port 3 near its topside so that entering air mixes with gases given off during fermentation and microbial and BSF respiration inside enclosed tank 2. Air is forcibly drawn inside enclosed tank 2 through gas entry port 3 by slightly lowering the air pressure in enclosed tank 2 relative to the external air pressure. In some embodiments, as illustrated in FIG. 1, air pump 8, which draws air from inside enclosed tank 2 and forces it out exit port 4, is included.

Alternatively, the air pump can be placed outside the enclosed tank 2. However, in the latter instance, the air intake for the air pump must be communicated inside the enclosed tank so that the net air pressure inside the enclosed tank remains negative relative to the external air pressure.

EXAMPLES

The following examples are provided to illustrate certain particular features and/or embodiments. These examples should not be construed to limit the invention to the particular features or embodiments described.

Example 1: Materials and Methods

This example describes materials and methods used in Examples 2-8, except as indicated otherwise below.

Sulfuric acid and 89% phenol were obtained from Mallinckrodt (St. Louis, MI, USA). Nessler's reagent was obtained from VWR (Radnor, PA, USA).

Microaerobic (MC) fermentors (20 L and 210 L capacity) were obtained from Bokashicycle, LLC (Henderson, NV, USA). Stabilwax-DA and RtQPLOT 30 m x 0.32 mm ID capillary GC columns, for separation of volatile organic acids (VOAs), alcohols and ketone metabolites, respectively, were obtained from Restek

(Bellefonte, PA, USA). Standards for VOA analyses (acetic, propanoic, butyric, isovaleric, valeric and caproic acid), alcohols and ketones (ethanol, acetone, acetylacetone, methanol, propanol, butanol and pentanol) were obtained from Sigma- Aldrich (St. Louis, MO, USA) and J.T. Baker (Austin, TX, USA). All other chemicals were reagent grade or better.

BSF larvae were hatched from egg clutches laid by mating adult flies in an insect nursery 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 described previously (Tomberlin et ah, Environ. Entomol., 38: 930-934, 2009). The eggs were hatched in wheat bran moistened with tap water. At approximately one week of age, larvae were freed of wheat bran by washing them in a stainless steel mesh colander (mesh size ~ 1 mm) with several liters of tap water. The larvae were then suspended in compost tea, whey (prepared by acidification of 2 % milk with HC1 to pH ~2, followed by boiling and filtration), raw sewage effluent (obtained from the local water treatment plant, Portland, OR, and freed of particulate matter by centrifugation), or 2 % pasteurized milk. Larvae were added to solutions at two larvae per ml in replicate culture tubes. Compost tea was prepared as previously described in 20 L and 210 L MC fermentors following two-week micro-aerobic fermentation of food scrap residues (Green and Popa, Appl. Biochem. Biotech., 165:270-278, 2011). Sterile compost tea was prepared by centrifugation of harvested compost tea at 14 K rpm for 20 minutes and filter- sterilized by passage of the supernatant through a 0.2 μιη pore size filter.

BSF larvae incubation assays were carried out in 10 ml volumes in culture tubes tilted at a 20° angle capped with gas permeable plastic lids in replicate (n=3); they were maintained between sampling times at 30° C in the dark. Results are reported as the average of experiments run in triplicate. Chemical analysis results were calculated by averaging test results obtained on aliquots drawn from each culture tube, assayed in triplicate, by calculating the average of the nine aggregate test results per data point (3 replicate culture tubes x 3 replicate assays per culture tube). Averages of each data set are reported +1 standard deviation (SD) of the data set. For assigning significance to differences in test treatment between control compost teas and that processed by BSF larvae, data comparisons were evaluated by ANOVA iteration of the variables measured as reported in the text. Measured variables in comparing test outcomes on control compost tea with that of BSF treated compost tea with P scores <0.01 were considered highly significant.

pH was measured using a Model 410 Thermo Orion pH meter. Soluble saccharides were measured by bichromatic analysis, at 485 and 570 nm on an HP Model 8452A diode array spectrophotometer, in glucose equivalents using a modified phenol sulfuric acid assay and glucose standards made up in deionized water to known concentrations (Green and Popa, J. Polym. Environ., 18:634-637, 2010). Ammonium (NH ₄ ⁺ ) was measured by a modified micro titer 96 well plate assay based upon the Nessler method (Jenkins, Adv. Chem. Ser., 73:265-280, 1967) with readings taken at 415 nm on a Model FLx800 BioTek™ plate reader using BioTek™ KC4 software. Plates were set up by mixing Rochelle reagent (Na,K- tartrate tetrahydrate, 25 mg ml ^"1 made up fresh in deionized water) with Nessler reagent in a ratio of 3.2: 1, respectively, and mixing 20 μΐ of this latter working reagent with 270 μΐ samples and NH ₄ C1 standards prepared fresh (serially diluted to span a calibration concentration range of 62.5 to 1000 μΜ). The concentration of unknown samples was then calculated by linear regression analysis relative to standards. Water was used as a "0" calibrator.

Ascending thin layer chromatography was performed on sample aliquots (-25 μΐ) drawn from test samples that had been applied to 250 μιη silica gel G plates developed in isopropanol:H ₂ 0 (90: 10 ratio). Ninhydrin positive amines present in compost tea samples were detected in the resulting TLCs by spraying plates with freshly prepared ninhydrin reagent made up at -0.5 % ninhydrin in ethanol (WAV) and heating the plates in an oven for - 20 minutes at 100° C.

VOAs, alcohols and ketones were analyzed using a Shimadzu GC-2010 instrument equipped with a splitter (50: 1 setting), flame ionization detector and using the following temperature profile setup: injector at 240°C, iso 60°C for 10 min., ramp 10°C per min. to 240°C and detector at 260°C (Green and Popa, J.

Polym. Environ., 18:634-637, 2010). COD was measured at 600 nm on a Model 8452A diode array spectrophotometer on sample aliquots drawn for analysis digested with a HACH instrument using the HACH 0-1500 COD kit (Cole-Parmer, IL, USA) based on standard wastewater protocols (Greenberg et al. (eds.). Standard methods for the examination of water and wastewater. 18 ^th edition. American Public Health Association Publications, 1992. 5-9.).

Example 2. BSF larvae clear alcohols and VOAs from compost tea.

This example illustrates that BSF larvae clear short chain alcohols and VOAs from compost tea. Concentrations of ethanol, propanol, acetic acid, propanoic acid, butyric acid and isovaleric acid were measured in control and BSF-larvae treated compost tea. The results show that BSF larvae have the capacity to specifically clear short chain alcohols and VOAs from compost tea. Compost tea derived following fermentation of food scraps consisting of left over coffee grounds, produce, discarded bread, cooked rice, fish bones and skin, egg shells, etc. formed by enclosure of the food scrap in a 20 L incubator maintained at ambient room temperature (~ 21° C) for one month was drawn into cylindrical polyethylene reservoirs set up in triplicate and tilted at 20° angles from a horizontal plane to create a shallow layer of liquid nutrient source in the reservoir open to room air at the surface layer, approximately 1 cm deep at the deepest section of the reservoir and providing a means for BSF larvae (2 per ml liquid nutrient source) to migrate in and out of the liquid nutrient source as they feed. An identical set of reservoirs were set up with compost tea drawn from the same stock but lacking BSF larvae, and all of the reservoirs were placed in an incubator maintained at 30° C.

Aliquots (10 μΐ) of the liquid nutrient source were drawn and analyzed in duplicate for short chain alcohol and VOA concentration as described in Example 1. Results were expressed in mM VOA by measuring peak areas and comparing peak areas for each alcohol and VOA analyzed to that of authentic standards made up in known concentrations. Samples were drawn from each reservoir at the beginning of the experiment, and two days later, and test outcomes compared.

Results are shown in FIG. 2. Addition of BSF larvae to the compost tea markedly accelerated turnover of VOAs in the compost tea evidenced by the sharp fall off in the concentration of the short chain alcohols and all of the principle VOAs detected in the compost tea relative to the control experiments in which BSF larvae were omitted. Concentrations of ethanol (Et), propanol (nP), acetic acid (Ac), propanoic acid (Pr), butyric acid (Bu) and isovaleric acid (Is) were measured.

Relative to controls, the initial concentration of the three dominant VOAs (78 mM acetic, 43 mM propanoic and 94 mM butyric) in the compost tea was reduced 7 to 9 fold in two days (P < 1.5 x 10 ^"4 , < 4 x 10 ^"3 , and < 1 x 10 ^"5 , respectively) (FIG. 2). In the case of the principal alcohols (ethanol and propanol) detected in control and BSF-treated compost tea, there was no significant turnover attributable to BSF (P = 0.34 for ethanol, and P = 0.47 for propanol). The volatility of these two alcohols made it difficult to differentiate between losses of the alcohols from compost tea caused solely by evaporation as opposed to BSF processing of the compost tea. The average reduction in residual concentration of alcohols and VOAs in the compost tea having BSF larvae feeding on the tea relative to control tea lacking BSF larvae averaged from 7- to 10-fold.

In scaled up experiments, we passed compost tea at 8 L day ^"1 through a stack of trays containing a total of 4 kg of larvae at ~20°C (see Example 6). The processing capacity in clearing the compost tea of VOAs was about 1 L of liquid per 1 kg fwt of BSF day ^"1 . Based on VOAs present in the starting compost tea, about 1 kg of larvae removed 55 mmoles of acetic, 60 mmoles of butyric and 30 mmoles of propanoic acid from one liter of compost tea in one day.

Without BSF larvae, VOAs, and alcohols, were recovered in sterilized compost tea even after five days, while turnover was seen in non-sterile samples (FIG. 3). Under the same experimental conditions, about 50% of ethanol and 30% of propanol was lost by evaporation. Relative to the processing capacity of BSF larvae, the microbial flora appeared to have reduced the concentration of VOAs at about half to one-third of the rate seen with BSF included in the compost tea.

These results establish that BSF larvae feeding on compost tea have the capacity to clear short chain alcohols and VOAs from the liquid nutrient source at a markedly accelerated rate over that which occurs in their absence.

Example 3. BSF larvae clear amines and residual protein from compost tea.

This example illustrates that BSF-larvae clear ninhydrin-positive amines from compost tea. Glycine, glycyl-glycine, glutamic acid and leucine were measured in control and BSF larvae treated compost tea by thin layer

chromatography. The results show that BSF larvae can clear nitrogen metabolites from compost tea.

To verify turnover of amines and residual proteins in compost tea, a similar set of assays were set up as in Example 2, but instead of measuring alcohols and VOA content, total protein concentration and ninhydrin-positive amines were studied by comparing compost tea treated with BSF larvae to control tea lacking BSF larvae.

Aliquots of BSF larvae-treated tea and untreated control tea were first compared for protein content following a two day larvae exposure period at 30° C using the standard Bradford protein assay and bovine serum albumin as a protein calibrator. As determined use the Bradford protein assay, protein in control compost tea was 275 ± 7 μg ml ^"1 (+ 2 SD; n=3) compared to 148 + 13 μg ml ^"1 (+ 2 SD; n=3) in compost tea processed with BSF larvae, indicating that the BSF larvae while feeding on the compost at 30° C cleared -50% of the protein from the compost tea relative to control tea in which BSF larvae were excluded.

Aliquots of compost tea derived from control and BSF larvae-treated samples were also examined for changes in ninhydrin-positive amines using thin layer chromatography silica G plates (20x20 cm; 250 μΜ thick). Ninhydrin standards of known amino acids (glycine, glycyl-glycine, glutamic acid and leucine) were applied to the plate in 20 μΐ aliquots and run simultaneously with aliquots of control and BSF-larvae treated compost tea drawn from 14K, 5 minute supernatant fractions recovered from a microfuge also applied to the same plates. Amines were separated using a solvent made up to 90% isopropanol and 10% H ₂ 0, and after air drying thin layer plate the plate was sprayed with ninhydrin reagent (2% made up in acetone), and heated in a 100° C oven for ~ 30 minutes to detect amines present in the samples applied to the plate

The results of the amine assays are shown in FIG. 4, which shows a comparison of the ninhydrin-positive pattern recovered from the thin layer chromatography plate for standards, control and BSF larvae-treated compost.

Samples were applied to the plate as follows (left to right): lane 1, glycine; lane 2, glycyl-glycine; lane 3, glutamic acid; lane 4, compost tea control; lane 5, compost tea processed with BSF larvae; lane 6, leucine. The larvae-treated tea was substantially cleared of amines except for positive material remaining at the point of application. The latter is likely residual protein remaining in the tea which stains positive with ninhydrin reagent but is too polar to migrate from its point of application on Silica gel G plates.

Together, the results of the protein and amine assays confirm that BSF larvae incubated with compost tea markedly reduced nitrogen metabolites in the tea as reflected by the decrease in total protein content and ninhydrin-positive metabolites relative to control compost tea treated similarly but without BSF larvae feeding on the tea. Example 4. BSF larvae grow and assimilate carbon, nitrogen and other essential elements and nutrients while feeding on liquid nutrient sources.

This example illustrates that BSF larvae grow and assimilate carbon, nitrogen and other essential elements and nutrients while feeding on a variety of filtered liquid nutrient sources. Additionally, this example illustrates that incubation of BSF larvae with compost tea decreases the chemical oxygen demand (COD) and increases the pH of compost tea.

BSF larvae were allowed to feed freely on: urine, sewage water (7 fold diluted), water extract of Gainesville House Fly diet (Sheppard et ah, J. Medical Entomology, 39:695-698, 2002), chicken broth, orange juice, compost tea (prepared as in Example 1), water extract of chicken manure ((200 g DW chicken manure extracted in 2 L tap water at 20°C), commercial 2% milk and whey from soured 2% milk. The weight gain of the larvae was measured over the course of 7 days. The results of this experiment establish that BSF larvae feeding solely off liquids with organic solutes thrive and gain weight. In compost tea the larvae were grown in separate experiments to full maturity. Since BSF larvae attain a protein content of ~40+ , and total lipid content in the range of ~30+ growing on solid nutrient sources as they mature into the pupate stage, experiments were set up to measure their ability to grow and assimilate carbon and nitrogen required for protein synthesis and lipid production by tracking their growth on compost tea compared to that of control larvae fed solid food scraps. Additionally, the COD and pH of compost tea incubated with BSF larvae was monitored.

The results of these assays are shown in FIGs. 5-8. While feeding on compost tea, the larvae decreased the compost tea's COD (P< 0.008) and increased its pH (P< 1 x 10 ^"6 ) by day seven (FIG. 5).

Additionally, BSF larvae grew and matured to the pupal stage while feeding solely on compost tea derived from decaying vegetal and food scrap waste. FIG. 6 shows larvae in varying stages of maturation feeding on compost tea. Evidence of larval molting can be seen based upon the presence of exuviae left behind in the compost tea (FIG. 6, arrows). In addition, some of the larvae entering the prepupae stage can be seen, as evidenced by the darkening of their outer integument. The larvae also grew on other liquids containing organic solutes and devoid of particulate matter, including sewage water, whey and milk (FIG. 7 A and FIG. 8). Relative to initial weight, the average larval growth increase (n = 10) in a week was 15.2 % in sewage water (P = 0.44), 39.5 % in whey (P = 0.32), 70.7 % in compost tea (P = 0.17) and 173.7 % in milk (P = 0.008). The larvae were grown in tilted (-20°) test tubes in 5 ml volumes of liquid freed of particulates by centrifugation and paper filtration (FIG. 7B). Larvae grown on compost tea for four weeks reached full maturity, pupated, hatched as adults and laid viable eggs, indicating that they are able to process the compost tea as they assimilated nutrients in biomass sufficient to carry them into the adult stage where they subsequently were able to lay viable eggs.

Larvae fed food scraps (produce and bread, replaced twice weekly) grew from 35 + 5.2 mg fwt per larva at one week of age to the black instar prepupa stage at 280 + 13.7 mg fwt per larva in -20 days at 30°C. Larvae grown on compost tea derived from decaying vegetal matter (replaced twice weekly) reached comparable prepupa stage weight of 260 + 13 mg fwt per larva in -30 days (n = 10, P = 0.42).

Thus, BSF larvae grew on all liquids tested. Further, larvae feeding on compost tea continued to gain weight throughout the full 30 days of the experiment, and by the 30 ^th day reached a comparable weight to that of larvae fed food scrap. At this weight, the BSF larvae fed compost tea were entering the pupate stage, and the experimental measurements were terminated.

Additionally, larval processing of compost tea over a seven day interval caused a significant (~6-fold) rise in ammonium concentration recovered in the compost tea, but had less effect on water soluble saccharides. Control (without larvae) compost tea had an initial average NH ₄ ⁺ concentration of 14.6 + 0.28 mM (+1 SD) and reached 19.9 + 0.69 mM (+1 SD) by the seventh day, while larvae- treated compost tea sampled after seven days treatment reached a concentration of 102 + 2.16 mM (+1 SD) (P < 1 x 10 ^"6 ). In contrast, total water soluble saccharides decreased -3-fold (P < 0.03) in both control compost tea (without larvae) and BSF larvae treated compost tea (control compost tea, 3.4 + 0.07 mg ml ^"1 to 0.86 + 0.09 mg ml ^"1 glucose equivalents; larval treated compost tea, 3.4 + 0.07 mg ml ^"1 to 1.01 + 0.02 mg ml ^"1 glucose equivalents).

These results demonstrate that BSF larvae can assimilate nutrition from metabolites present in liquid nutrient sources, evidenced by their growth on nutrient solutions as in the example shown here, while simultaneously altering the chemical composition of solution they are growing on, evidenced, for example, by changes in the concentration of nitrate and nitrite containing compounds in the nutrient solution caused by their inclusion in the solution.

Example 5. BSF larvae process VOA solutes dissolved in particulate-free liquid.

This example illustrates that BSF larvae can process dissolved VOA in a solution of only a single VOA and water. Tubes containing solutions of ~ 0.9 % v/v butyric, acetic or valeric acid were incubated at 30°C for approximately one week with and without BSF larvae. Acid concentrations were measured by gas

chromatography and used to calculate the turnover rate for each of the VOAs. The results show that each of the VOAs was processed by the BSF larvae, demonstrating that the ability of BSF larvae to process solutes in an aqueous solution and that BSF larvae have the capacity to turnover organic acids commonly found in abundance in decomposing organic matter independent of solids suspended in the liquid nutrient source stream.

Solutions of ~ 0.9 % v/v butyric, acetic or valeric acid and chlorinated tap water were dispensed in paired sets in Hungate culture tubes (capped with a polyurethane open pore plug) similarly as in Example 1. Larvae were omitted from one set of tubes labeled controls, and BSF larvae (approximately 2 weeks old) were added to the other matching set. The tubes were incubated at 30°C for

approximately one week and the concentration of each organic acid was tracked by gas chromatography. During this interval the larvae remained active evidenced by swimming and spiraling about in the liquid and crawling up and down the walls of the Hungate tubes.

From the concentration measurements, the turnover of each VOA was determined (evidenced by a fall in organic acid content as BSF larvae fed on the solution relative to the corresponding organic acid content of the controls). The turnover of butyric, acetic and valeric acids was calculated from the difference in VOA concentrations between the two parallel sets of tubes for each solute tested (control residual organic acid content less that of the corresponding BSF larvae test set) with results expressed as VOA turned over in mmoles L - ^" 1 day - ^" 1 per 103 larvae. The larvae weight was approximately 100-150 mg each. Table 1 shows the results of these calculations. These results show that BSF larvae have the capacity to turnover organic acids commonly found in abundance in decomposing organic matter independent of solids suspended in the waste stream.

Table 1. BSF larvae processing of organic acid from particulate free water containing only one organic acid.

Example 6. Operation of a representative apparatus for removing organic solute from a liquid and/or for producing larva biomass.

This example illustrates construction and operation of a representative apparatus comprising an array of modular larval incubation units (MLIUs) as provided for herein.

The modular units are constructed from identical polyethylene rectangular boxes designed to snap together snuggly one on top of the other in a vertical stack of repeating units in which larvae are retained for incubation with compost tea. Each modular unit, with the exception of the lowest module in the stack of modules has drain holes (0.2 cm in diameter) spanning longitudinally across floor of the module which allows compost tea passing through the module to drain into an identically constructed module positioned under the upper module to catch the fluid passing into it from the overhead module. FIG. 9 shows a digital image illustrating how the drain holes in an upper module are placed into the floor of one example module. By positioning the drain holes in the base floor of the modular unit some distance from the edge of the unit's side walls, in this case approximately one-quarter of its floor width from wall to wall, a temporary shallow reservoir of compost tea accumulates in the area free of drain holes, on which BSF larvae can feed.

BSF larvae were added to each unit, and compost tea allowed to flow into the top unit by means of a pump and tubing carrying the compost tea to the top module. As the compost tea began to accumulate in the top module, it reached the drain holes, trickled into the module snapped into position below the top module, in turn filling up to the drain holes of the second unit, passing then to the unit below, etc. , until finally collecting in the bottom unit. The bottom unit lacks drain holes similar to the other units, but contains a tube and drain assembly providing a means of removing processed compost tea from the modular stack out of the invention for subsequent handling. At this point liquid can be either recycled through the stacked units using a circulating pump designed to feed the liquid back into the upper unit of the modular stack, or carried away from the units in a single pass through operation.

As shown in FIG. 10, to provide for air in supporting respiration of the BSF larvae housed in the modular units, multiple holes, each about 0.2 cm in diameter, were placed around the upper perimeter walls of each modular unit on three of the unit's walls from about 0.5 to 2.5 cm beneath its top rim.

FIG. 10 shows a side view of a series of operating modular units housed inside an enclosed tank as illustrated in FIG. 1. Larvae are present inside each unit (except for the bottom liquid collection unit), feeding on compost tea. Breathing holes can be seen in each of the modular processing units (excluding the bottom liquid collection unit). The modular units, snapped together as in FIG. 10, rest on the floor of the outer box reservoir. The outer box reservoir is furthermore tilted at an angle of approximately 20°, causing compost tea infusing into each modular reservoir to temporarily pool in its lower left basin area as it trickles and flows through the units, allowing BSF larvae retained in the modules to feed on the compost tea as it works its way to the collection module at the bottom of the stack. FIG. 11 shows a top down view of the top module making up a modular stack with its lid removed housing BSF larvae and compost tea delivered into the unit from the infusion inlet port illustrated in FIG. 1.

FIG. 12 shows the bottom module used in the apparatus shown in this example. The bottom modules is equipped with a drainage tube allowing fluid passing into its reservoir to be drawing out through the liquid exit port as illustrated in FIG. 1. The purpose of the bottom module in the stacked modular is to provide a means of collecting processed liquid nutrient source and a mechanism for then passing it out the exit port illustrated in FIG. 1. FIG. 13 shows the assembled and operating apparatus as described in this example, including the stacked modules housed inside the enclosed tank with compost tea entering the device by means of a liquid peristaltic pump passing tea into the enclosed tank a liquid entry port and into the uppermost module of the inner reservoir. BSF larvae were present in each module of the array and were incubated with the compost tea passing through the apparatus. The BSF larvae were added through the lid of each module residing above the collection module at the bottom of the stack. Processed compost tea collected at the bottom of the modular stack passes out of the bottom module and exits the enclosed tank through the liquid exit port inserted in the wall of the enclosed tank.

A vacuum air pump operating inside the enclosed tank and operably connected to a gas exit port maintains a negative air pressure inside the enclosed tank and directs the flow of gases drawn into the enclosed tank, and those generated by metabolic activity through a gas exit port. Gases exiting the enclosed tank through the gas exit port pass directly through sparging tank reservoirs designed to capture residual C0 ₂ , VOAs and volatile amines.

Example 7. Temperature and pH tolerance of BSF larvae feeding on compost tea.

BSF larvae feeding on compost tea larvae tolerated compost tea well at ambient room temperature conditions (approximately 21° C) and up to 35° C.

Between 35° C and 40° C, BSF larvae lost considerable vigor and became extremely sluggish, lost weight, showed excessive molting of their outer exoskeleton, stiffened up and died. In cold tap water (approximately 10 to 15° C), the larvae stiffen up and tend to become immobile, but on warming back up to ambient room temperature conditions resume their normal writhing and crawling activities as they feed on compost tea.

BSF larvae feeding well in processing liquid nutrient sources exhibit a characteristic almost nonstop continuous writhing, rolling, and crawling activity. Evidence that the larvae are thriving on the liquid waste can be easily viewed by their continuous movement and crawling activities as they feed off the liquid concomitant with weight gain and a transition in the color of their pigmented exoskeleton from an initial white-tan yellow color after hatching from eggs to a darker reddish-brown to black-brown color as they mature into their 5 ^th instar pupa state.

BSF larval behavior as a function of the compost tea pH is similar to the effect of temperature on BSF larvae. The BSF larvae remain very active in writhing, crawling and gaining weight while feeding on compost tea ranging in pH from as low as pH 3.0 to as high as approximately 9.0. No long term decrease in larvae survival or growth rates in compost teas residing in this pH range were observed. Example 8. Effect of larval density and liquid depth on the viability of BSF larvae feeding on liquid compost tea.

BSF larvae added to compost tea incubated at room temperature, and under experiments run at 30° C, did well in terms of their behavioral characteristics while feeding on the tea evidenced by their crawling and writhing activities, and by their growth activity when kept under these environmental conditions. They grew to maturity, reaching the pupa stage, between three and four weeks at the very latest following introduction to compost tea when maintained with fresh compost tea at least every third day during their growth cycle at a density of 2 x 10 3 L - ^" 1.

Experiments with larvae densities up to 2 x 10 ⁴ L ^"1 in compost tea were also run successfully evidenced by healthy activity in crawling and feeding of the larvae on compost tea, and clearance of VOAs at shortened intervals as the density of the larvae population increased. The larvae were observed to actually cluster together in close proximity to one another while feeding on the tea as the density increased, and by their behavior to seek out and crawl up and over other larvae in proximity to them as they fed on the liquid. This behavior suggests that the larvae density in liquid waste can be raised to slightly in excess of 2 x 10 ⁴ L ^"1 , depending upon the age and size of the larvae. The major limitation appears to be maintenance of sufficient liquid nutrients that the larvae process so as to not starve them and cause die off and putrefaction of the dead larvae. Example 9: Enhanced Ammonium Content in Compost Tea Processed by BSF Soldier Fly Larvae

This example illustrates the use of BSF larvae to process organic nitrogen and increase ammonium concentration in compost tea.

Nitrogen released during microbial decay of organic matter is poorly retained in the solid phase of compost. Instead, it is released as solutes in the compost tea fraction (mostly as ammonium (NH ₄ ⁺ ), nitrate (N0 ₃ ^~ ), nitrite (N0 ₂ ^~ ) and amines) and as gases (N ₂ , NO, N ₂ 0 and NH ₃ ). The results described below show that BSF larvae are useful to facilitate dissimilatory nitrate reduction to ammonium.

Materials and Methods

Standards for NH ₄ ⁺ , N0 ₂ and NO ₃ ^" were prepared in deionized H ₂ 0 using, respectively, analytical reagent grade NH ₄ C1, NaN0 ₂ and NaN0 ₃ (Sigma, St. Louis, MO). All other reagents were prepared from reagent grade or higher chemicals.

Compost tea was prepared from vegetal feedstock made up of a mixture of grass clippings, leaves and discarded food scraps (primarily bread, vegetables and fruit leftovers from cafeterias and restaurants) fermented at room temperature (see Green and Popa, Appl. Biochem. Biotech., 165, 270-278, 2011). BSF larvae (multiple generations) were raised on the same feedstock in an insect nursery maintained between 30-35°C and lit on a 12 hour day-night cycle with natural light (Sheppard et al., J. Med. Entomol., 39:695-698, 2002).

Approximately 200 white BSF larvae (ranging in size between 100 and 250 mg per larva) were harvested by suspending and washing them in approximately 3-4 liters of tap water to rid them of vegetal debris, then suspended in three separate -200 ml wash solutions of 10 mM NaN0 ₃ made up in tap water, tap water, and compost tea, respectively. Larvae were transferred into duplicate sets of Hungate culture tubes (20 per tube) along with 10 ml of the same solutions they were suspended in. Controls were made up similarly using the same solutions recovered from the larvae washing steps. The tubes were closed at the top with plastic caps which allowed air and gas exchange, tilted at an angle of 20 degrees, and placed in a 30°C incubator for analysis over the course of seven days of incubation in the dark.

Aliquots (100 μΐ) of the growth medium at the beginning of the experiments and thereafter at varying intervals were analyzed. All results for NH ₄ ⁺ , N0 ₂ and NO ₃ ^" are expressed as averages of triplicates + 1SD assays of duplicate sets of experiments. The pH is reported as the average + 1SD of duplicate measurements.

Ammonium (NH ₄ ⁺ ) was measured by a modified microtiter 96 well plate assay based upon the Nessler method (Jenkins, Adv. Chem. Ser., 73:265-280, 1967) with readings at 415 nm on a Model FLx800 BioTek™ plate reader using BioTek™ KC4 software. Plates were set up by mixing Rochelle reagent (Na,K-tartrate tetrahydrate, 25 mg/ml made up fresh in deionized H ₂ 0) with Nessler reagent in a ratio of a ratio of 3.2: 1, respectively, and mixing 20 μΐ of this latter working reagent with 270 μΐ samples and NH ₄ C1 standards prepared fresh (serially diluted to span a calibration concentration range of 62.5 to 1000 μΜ). The concentration of unknown samples was then calculated by linear regression analysis relative to standards. H ₂ 0 was used as a "0" calibrator. N0 ₂ and NO ₃ ^" were measured similarly on microtiter plates using sulfanilamide color reagent (Escalante-Semerena et al. , Appl. Environ. Microbiol., 40:429-430, 1980) adapted for analysis by dispensing and mixing 30 μΐ samples and calibration standards into plate wells prefilled with 90 μΐ of 16.7 μg/ml CuS0 ₄ 5H ₂ 0 made up in 333 μΜ NaOH. For N0 ₂ , each well received an additional 60 μΐ of H ₂ 0. For N0 ₃ ^~ , each well received 60 μΐ hydrazine sulfate made up at 0.67 mg/ml in H ₂ 0. The plates were then incubated with lids for 2 hours at 30°C, and subsequently read at 520 nm after adding 60 μΐ color developing reagent made up atlO mg/ml sulfanilamide and 0.8 mg/ml N-(l-naphthyl)-ethylenediamine dihydrochloride in 3 M H ₃ P0 ₄ to each well. In the case of N0 ₃ ^" , since N0 ₃ ^" converts to N0 ₂ with hydrazine reagent, and because the color read at 520 nm measures N0 ₂ , absorbance readings attributable to N0 ₃ ^" were calculated as the difference in plate readings between readings obtained with hydrazine less the readings with H ₂ 0.

The pH was determined using a Thermo Orion Model 410 pH meter.

Whether or not N ₂ was formed was evaluated in 18 mm culture tubes by submerging inverted Durham tubes inside the test solutions and screening for gas formation. Confirmatory follow-up analysis by gas chromatography was unnecessary because gas did not accumulate in the inverted tubes. Results

Larvae excreted frass from vegetal and food scrap waste they had previously ingested into the suspending solutions commencing within seconds of their transfer into particle free solutions. FIG. 14A shows the evolution of N0 ₃ ^~ , N0 ₂ , NH ₄ ⁺ and pH after suspending BSF larvae in a solution of 10 mM NaN0 ₃ made up in tap water. N0 ₃ ^" was depleted by the fifth day. N ₂ was not generated over the course of these experiments based upon an absence of gas accumulation in inverted culture tubes submerged in the solution in which the larvae were confined. By the end of the seventh day, N0 ₃ ^" reappeared in solution, reaching a concentration of ~2 mM (FIG. 14A).

These results show that the larvae, presented with N0 ₃ ^" , facilitate its reduction (i.e., denitrification). In both BSF and control samples, N0 ₃ ^" reappeared and built up between fifth through the seventh day of the study. This indicates that, apart from N0 ₃ ^" reduction, nitrification was also occurring. One non-limiting explanation is that nitrification is attributable to the activity of NH ₄ ⁺ -oxidizing microorganisms released into solution, for example from frass and using NH ₄ ⁺ introduced at the onset of the experiment from vegetal matter ingested by the larvae (FIG. 14B). The rise in N0 ₂ peaking at ~ 2 mM between the third and fourth day (FIG. 14A), and its decline to ~ 1 mM by the seventh day, also indicates that the BSF larvae facilitate denitrification.

In controls, N0 ₃ ^" bottomed out at ~ 6.5 mM around the second day of incubation, and returned to its initial starting level of 10 mM by the end of the seventh day, while N0 ₂ was not detected (FIG. 14A). One non-limiting possible explanation for the fall in N0 ₃ ^" , and its subsequent climb back to its initial level, is the opposing activity of denitrifying and nitrifying bacteria on N0 ₃ ^" and NH ₄ ⁺ , both ions are present (FIGs. 14A and 14B). There is a limited supply of reducing equivalents in the N0 ₃ ^" solution used in these experiments due to the short interval the washed larvae were temporarily retained in it. This may account for the impaired denitrification in controls relative to that seen with larvae. A non-limiting possible explanation is that in the BSF experiments, additional reducing equivalents were provided through frass and excreta. NH ₄ ⁺ , though not present in freshly prepared stock N0 ₃ ^" solution used for washing and suspending the larvae at the onset of the study, was detected in all of the NO ₃ ^" solutions drawn from the culture tubes (those representing larvae-free controls and those with larvae). The presence and concentration of NH ₄ ⁺ in the tubes is likely the result of a small amount of frass and possibly external carryover of NH ₄ ⁺ brought by the larvae in the NO ₃ ^" solution during the setup of the experiment.

NH ₄ ⁺ started out at ~ 15 mM in controls, fell to zero by the second day, and did not reappear (FIG. 14B). In the larvae experiments, NH ₄ ⁺ concentration rose concomitant with the disappearance of NO ₃ ^" (FIG. 14B). The decrease in NO ₃ ^" (-10 mM), and shift in NH ₄ ⁺ concentration (A _change = [NH ₄ ⁺ ] _fina i - [NH ₄ ⁺ ]i _nitia i = [28.9] - [17.8] ~ Δ 11.1 mM), match. This indicates that the decrease in NO ₃ ^" (when larvae were present in the NO ₃ ^" solution) occurred via dissimilatory nitrate reduction to ammonia (DNRA). DNRA pathways are commonly expressed in decaying vegetal waste, especially when partial fermentation is also occurring as was the case with the feedstock the larvae were grown on in our experiments. The pH with larvae added to the NO ₃ ^" solution furthermore rose from a starting value of 6.3 to 8.4 over the seven day cycle examined. Controls, on the other hand, showed a strikingly smaller pH change of only 0.2 pH units over the same interval (FIG. 14B). One explanation for the difference in pH is that enhanced accumulation of NH ₄ ⁺ in the solutions in which larvae were retained accounts for the larger shift in pH relative to the controls.

In experiments with BSF larvae suspended in tap water, a small amount of NO ₃ ^" , starting at -0.3 mM (originating in carryover), peaked between 0.8 and 0.9 mM by the seventh day (FIG. 15A). N0 ₂ ^~ rose steadily, reaching -0.3 mM by the 7 ^th day. NO ₃ ^" and N0 ₂ ^" were not detected in the corresponding controls (without larvae) except for a small peak of NO ₃ ^" (-0.1 mM) on the sixth day which tapered off by the seventh day, and a small rise in N0 ₂ to -0.05 mM by the 7 ^th day.

No NH ₄ ⁺ was detected in the tap water. A small amount of NH ₄ ⁺ (just under -1 mM) was detected in controls as carryover from the larval wash (as discussed a above in the NO ₃ ^" experiments). Large amounts of NH ₄ ⁺ peaking near -40 mM accumulated however in the BSF experiments (FIG. 15B). The concentration of NH ₄ ⁺ by the seventh day of the experiment with larvae suspended in solution was in excess of that of N0 ₃ ^" and N0 ₂ combined by a ratio >50: 1. NH ₄ ⁺ formed in the larval suspension furthermore shifted the pH upwards, peaking at ~pH 8.2 by the seventh day (FIG. 15B) in a manner similar to that seen earlier in the NO ₃ ^" experiments (discussed above).

In compost tea, NO ₃ ^" started out at -4.5 mM, fell to zero by the fourth day in both control and larval-treated samples, and from the fourth day through to the end of the seventh day reappeared, peaking by the seventh day at ~1 mM (FIG. 16A). N0 ₂ ^" was at -0.1 mM, then rose slightly and held relatively constant in the range of -0.3-0.4 mM in experiments with larvae, and in the range of 0.1-0.2 mM in controls, and tapered off by the seventh day (FIG. 16A). Except for subtle differences in NO ₃ ^" and N0 ₂ concentrations, the overall pattern of

reduction/oxidation of nitrate, and nitrite, were similar between samples and controls. These results indicate that the larvae have no significant effect on the nitrite and nitrate transformations in the compost tea.

However, with larvae in the compost tea, NH ₄ ⁺ rose sharply by the seventh day to -100 mM, a concentration more than 25-fold that of the combined concentrations of NO ₃ ^" and N0 ₂ detected in the compost tea (FIG. 16B).

Concomitant with this change in NH ₄ ⁺ concentration, the pH of the compost tea shifted from -4.4 at the start of the experiment to -8.8 by the seventh day. Controls showed no significant changes in the concentration of NH ₄ ⁺ , and a change in compost tea pH by the seventh day of -0.8 pH units above that at which it started (FIG. 16B).

On a stoichiometric basis, the magnitude of change in NH ₄ ⁺ concentration accumulating in BSF-treated compost tea is unaccountable as a product of NO ₃ ^" or N0 ₂ reduction via a DNRA. Furthermore, since the experiments were conducted in the presence of 0 ₂ , because no N ₂ gas was trapped, and whereas N ₂ fixation occurs mainly in anaerobic conditions, the large amount of NH ₄ ⁺ produced under these experimental conditions cannot be linked with N ₂ fixation.

FIG. 17 shows a nearly perfect fit between the ΔρΗ shift in BSF-treated compost tea and accumulation of NH ₄ ⁺ commencing on the second day of the study, when the accumulation of NH ₄ ⁺ began (FIG. 16B). The consumption of organic acids in the compost tea by the larvae likely accounts for the initial shift in pH from its starting value of 4.4 to -7.4, after which NH ₄ ⁺ accumulation commenced. One non-limiting explanation is that this accounts for the nonlinear relationship between measured NH ₄ ⁺ levels and ΔρΗ seen at the beginning of the experiment, and the subsequent linear fit thereafter.

The NH ₄ ⁺ in the BSF-treated compost tea appears to have come from organic nitrogen within the frass produced by the larvae. Most insects store nitrogen waste in the form of uric acid which precipitates in the insect's rectum (or in some instances the proximal portion of the Malpighian tubule). Several investigators studying Diptera have identified allantoin, the immediate degradation product of uric acid, a water soluble waste product, in insect excreta in addition to lesser albeit measureable quantities of NH ₄ ⁺ . Allantoin, carried into the compost tea with frass, is likely getting broken down into urea, and by the action of urease, into NH ₄ ⁺ . Microbes colonizing the gut of the larvae, subsequently delivered into the compost tea with frass, in addition to those already present in the compost tea, may also contribute to mineralizaton of organic nitrogen.

These results show that BSF larvae initially feeding on decaying vegetal and food scrap waste markedly increase nitrogen-mineralization evidenced by the observation that they elevate the concentration of NH ₄ ⁺ in compost tea. The larvae furthermore facilitate recovery of N0 ₃ ^" present in the compost tea fraction via DNRA. Additionally, the results indicate that BSF larvae-based processing of waste can be used to offset costs incurred with nitrogen fertilization of crops. Moreover, while feeding on decaying waste, BSF larvae assimilate nitrogen and carbon, and other nutrients into insect biomass (useable for instance as animal feedstock), which reduces the amount of carbon and nitrogen that otherwise would have been given up as greenhouse gases to the atmosphere.

It will be understood by those of skill in the art that numerous and various modifications can be made without departing from the spirit of the present disclosure. Therefore, it should be clearly understood that the embodiments disclosed herein are illustrative only. We therefore claim all that comes within the scope and spirit of these claims.