Microbial Biomass-Based Feed Products

Abstract

High protein and high nutritional products comprising a mix of specific bacterial species, and methods of producing the same, are provided. Products can be produced via a co-culture of axenic source strains grown on simple gas feedstocks, such as carbon dioxide, hydrogen and oxygen, as can be provided by industrial waste gases. A consortium of bacterial strains specially selected for this purpose is cultured in an aqueous culture medium.

Claims

1. A method for producing a biomass, the method comprising: providing a gas mixture to a consortium of bacteria and microbes in a bioreactor, the consortium including at least three of Cupriavidus necator, Rhodobacter sphaeroides, Rhodopsuedamonas palustris, Rhodobacter capsulatus, Bacillus subtilis, and Bacillis magaterium, the gas mixture including each of hydrogen gas, carbon dioxide gas, and oxygen gas, whereby hydrogen is metabolized by at least some of the bacteria of the consortium and growth of the consortium occurs solely by the fixation of carbon dioxide; harvesting cells of the bacteria and microbes from the bioreactor; and drying the solids to yield the biomass.

2. The method of claim 1 further comprising separately receiving hydrogen gas and flue gas, the flue gas including both the carbon dioxide gas and the oxygen gas, and mixing the hydrogen gas and flue gas to produce the gas mixture.

3. The method of claim 2 wherein, mixing the hydrogen gas and flue gas comprises diluting the flue gas approximately 5-fold with the hydrogen gas.

4. The method of claim 1 wherein one of the hydrogen gas, carbon dioxide gas, and oxygen gas is introduced separately into the bioreactor from the other two gases.

5. The method of claim 1 wherein the consortium of bacteria consists solely of Cupriavidus necator, Rhodobacter sphaeroides, Rhodopsuedamonas palustris, Rhodobacter capsulatus, and Bacillus subtilis.

6. The method of claim 1 wherein at least one of the bacterium of the consortium is of a strain that has been adapted over multiple generations to be tolerant of a flue gas, and wherein the gas mixture includes the flue gas.

7. The method of claim 1 further comprising, before providing the gas mixture to the consortium, adapting a bacterium of the consortium to a flue gas by growing multiple successive generations of the bacterium in the presence of the flue gas to produce an adapted bacterium, wherein the gas mixture includes the flue gas.

8. The method of claim 1 wherein one of the bacterium of the consortium expresses a carotenoid.

9. A product made by the method of claim 1.

10. A method for producing a feed product comprising: producing a biomass by providing a gas mixture to a consortium of bacteria and microbes in a bioreactor, the consortium including at least three of Cupriavidus necator, Rhodobacter sphaeroides, Rhodopsuedamonas palustris, Rhodobacter capsulatus, Bacillus subtilis, and Bacillis magaterium, the gas mixture including each of hydrogen gas, carbon dioxide gas, and oxygen gas, whereby hydrogen is metabolized by at least some of the bacteria of the consortium and growth of the consortium occurs solely by the fixation of carbon dioxide, harvesting cells of the bacteria and microbes from the bioreactor, and dewatering, concentrating or drying the solids to yield the biomass; and blending the biomass with an additive.

11. A food or feed additive comprising: a consortium including Cupriavidus necator, a species from the genus Rhodobacter, a species from the genus Rhodopseudomonas, and a species from the genus Bacillus.

12. The food or feed additive of claim 11 wherein the species from the genus Rhodobacter comprises Rhodobacter sphearoidies.

13. The food or feed additive of claim 11 wherein the species from the genus Rhodopseudomonas comprises Rhodopseudomonas palustris.

14. The food or feed additive of claim 13 wherein the species from the genus Rhodobacter comprises Rhodobacter sphearoidies.

15. The food or feed additive of claim 14 wherein the species from the genus Bacillus comprises Bacillus subtilis.

16. The food or feed additive of claim 15 further comprising Rhodobacter capsulatus.

17. The food or feed additive of claim 14 further comprising Rhodobacter capsulatus.

18. The food or feed additive of claim 11 wherein the consortium is characterized by an enhanced attractiveness to fish.

19. The food or feed additive of claim 11 wherein the consortium is characterized by an enhanced palatability to fish.

20. The food or feed additive of claim 11 wherein the C. necator comprises over 85% of a dry weight of the consortium of the food or feed additive.

Description

BRIEF DISCRIPTIONS OF THE DRAWINGS

[0021] FIG. 1 is a schematic representation of an exemplary bioreactor according to various embodiments of the invention.

[0022] FIG. 2 is a bright field micrograph of a consortium grown on CO.sub.2, H.sub.2, and O.sub.2 showing multiple species of microbes, according to various embodiments of the invention.

[0023] FIG. 3 is a tabular comparison of amino acid profiles between an exemplary final product according to an embodiment of the present invention and three products of the prior art.

[0024] FIG. 4 is a tabular comparison of amino acid profiles between the exemplary final product of FIG. 3 and data Bacillus subtilis grown heterotrophically on sugars.

[0025] FIG. 5 is a graphical representation of weight gain per period for fish fed either an exemplary final product according to an embodiment of the present invention or a fishmeal control diet.

[0026] FIG. 6 is a graphical representation of total weight gain for fish fed either the exemplary final product or the fishmeal control diet of FIG. 5.

DETAILED DESCRIPTION

[0027] Microbial strains. C. necator has the advantage that it can grow very rapidly and to high density on a mixture of H.sub.2, CO.sub.2, and O.sub.2, and it can be continuously cultured for long periods of time without contamination. Bacterial strains for the consortium are selected for ability to grow on H.sub.2 and CO.sub.2, being naturally-occurring (i.e., non-GMO), generally recognized as safe (‘GRAS’) organisms, or because of their apparent beneficial qualities and apparent lack of negative characteristics, so that they will be broadly suitable for feed and food processing, although GMO organisms designed for a specific purpose (e.g., metabolite production via an engineered pathway) can also be included, if desired.

[0028] Experimental results for exemplary products of the invention are discussed herein. Strains used for such experimental results were obtained as pure, axenic type-cultures from culture collections. Further selection was made based on a series of experiments where different bacteria of interest were added to a consortium which was then grown on gas, and after a period of time, subjected to metagenomic analysis via the 16s technique to identify species presence and amount in the final product. These final products we subjected to further analyses including feeding trials with live animals to determine digestibility, palatability, and whether anti-nutritional properties were present. During this work the final products were found to be nutritionally adequate or superior to existing high protein feed and food additives. Surprisingly, products produced through the cultivation of the disclosed consortia also exhibited much higher palatability in aquaculture diets than the industry standard high protein ingredient, fishmeal. The products were also found to be more attractive to fish, and fish ate it at an increased feed consumption rate.

[0029] Gas supply. The CO.sub.2, H.sub.2 and O.sub.2 can be supplied from either flue gas collected from an industrial emitter (designated as ‘flue gas’) or from pure stocks of compressed gas obtained from a gas supplier (designated as ‘lab gas’). These gases can also be derived from process gas produced by an industrial process, from gasifier or pyrolysis output gas, from syngas, from electrolysis of water, from a steam methane reformer, via separation from air, from the manufacture of cement or lime such as quicklime, from a combustion process, or from any industrial, natural, or other process which produces one or more of the desired gases.

[0030] An example of CO.sub.2 produced by an industrial process is the production of CO.sub.2 during the manufacture of cement or quicklime where limestone (CaCO.sub.3) is heated to form CaO and CO.sub.2, in a process sometimes called calcination. For production of feed, food, nutraceuticals, biologicals, and the like, an industrial source of waste CO.sub.2 that is free of toxic elemental contaminants (e.g., mercury) is preferred. Examples of such sources include CO.sub.2 from breweries and bioethanol plants. Hydrogen can be supplied as part of the gas composition of pyrolysis gas, syngas, as an industrial side product from activities such as propylene manufacture, as a component of a mixed gas stream from an oil refinery, or in gas created by steam methane reformation (SMR) process, from compressed gas, or from electrolysis of water. Oxygen can be obtained from atmospheric gas, as a product of electrolysis, as a by-product of ammonia manufacture, or as a component of industrial by-product gas such as cement flue-gas.

[0031] In some embodiments, where the CO.sub.2 and O.sub.2 are derived from either lab gas or flue gas, these gases are further diluted approximately 5-fold with H.sub.2 to supply bacteria with a feedstock mixture that is optimized for growth. For a commercial-scale operation, the fermentation plant can be located near the gas production site, or the gas can be transported by vehicle or pipeline to the biomass production site.

[0032] For injection into the fermenter, in the experimental processes, the gas supply was filtered through 0.2 um filters to remove particles and microorganisms. For these experiments, compressed H.sub.2, CO.sub.2 and O.sub.2 were each regulated to 20 psi. The gases were delivered to a flow proportioner, which set the relative fraction of the gases, and to a variable area flow meter to control the mixture and flow rate into the fermenter.

[0033] Nutrient monitoring. The compositions of the input and output gases can be measured and monitored to determine the gas uptake rates, the mass balances, and the mass transfer efficiency for dissolution of the gas into the solution and the biomass. Key nutrients (such as NH.sub.4, PO.sub.4 and SO.sub.4), can also be monitored and replenished to prevent nutrient limitations that might restrict bacterial growth.

[0034] Microbial inocula. The inocula for fermenter runs can be prepared in many ways; each microbial strain may be grown separately, or two or more may be grouped together in a single fermentation. Heterotrophic species are always grown up from pure cultures on an heterotrophic medium that is suitable for propagating the particular species (or group of species) being grown. Chemoautotrophic species can be grown on gases, or, in some cases, on heterotrophic media. Photoautotrophic species may be grown using light or heterotrophic media. Some photoautotrophic species are also chemoautotrophic, and thus may be grown on gaseous substrates. In various embodiments inocula comprise axenic strains of each microbe, that is, they are cultivated in a closed bioreactor where other microbes are kept out such that the final product contains just these bacteria. Inoculating the bioreactor can further involve the sterile addition of a culture containing one or more inocula into the bioreactor.

[0035] In some embodiments, all of the cultures for the consortium are added to the bioreactor, in a short period of time, at the beginning of the fermentation procedure or run. In other embodiments, chemoautotrophic microbes are added to the bioreactor at the beginning of the fermentation, and the inoculum cultures containing other species are added at later points. In further embodiments, the timing of the addition, amount and density of culture additions, and method of preparing inocula can be altered to affect the qualities, composition, and/or value of the final product. In some embodiments, additional inoculations of one or more strains used in the consortia can be added at later times.

[0036] In the experimental runs, cultures were prepared by growing C. necator and the other chemoautotrophic species on H.sub.2/CO.sub.2/O.sub.2 to an OD620 ˜1 in small bottles of media equipped with gas fittings, or on heterotrophic media such as YT, LB, or on minimal salts media supplemented with an organic carbon source such as sugars, gluconate, glycerol or organic acids. Non-chemoautotrophic species were grown in liquid yeast-tryptone medium (YT medium), a well-known and commercially available medium. The bioreactor was inoculated to OD ˜0.1. A ca. 5% inoculum is ideal. The pH was controlled with 2N NH.sub.4OH, or with NH.sub.4OH when ammonia is also desired in order to supply nitrogen. Fermentation runs were carried out over a period of days, resulting in OD620 of 1-100 or greater.

[0037] The recovered biomass was analyzed for protein and lipid content and the composition of each product. Proprietary strains of C. necator and/or R. capsulatus, or other microbes were sometimes used in addition to type strains. Several of these proprietary strains of chemoautotrophic species are adapted to flue gas and therefore tolerant to various toxic gas components, which can be included in some complex industrial flue gases. In some cases, additional inoculations with one or more of the consortium strains were carried out at later times.

[0038] Table 1 provides a consortium disclosed in U.S. patent application Ser. No. 15/641,114. Cultivation of this consortium generates biomass which yielded favorable nutritional analysis and performed well in field studies with rainbow trout that were conducted by the US Fish and Wildlife Service, Bozeman Montana Fish Technology Center.

TABLE-US-00001 TABLE 1 Strain Species Source B-3226 Rhodospirillum rubrum, (ARS NRRL Type Strain) B-1727 Rhodobacter sphaeroides, (ARS NRRL Type Strain) B-4276 Rhodopseudomonas palustris, (ARS NRRL Type Strain) B-14308 Bacillus megaterium, (ARS NRRL Type Strain) B-356 Bacillus subtilis, (ARS NRRL Type Strain) B-354 Bacillus subtilis, (ARS NRRL Type Strain) B-14200 Bacillus subtilis subspecies subtilis, (ARS NRRL Type Strain) B-41406 Bifidobacterium animalis subspecies animalis, (ARS NRRL Type Strain) B-4495 Lactobacillus acidophilus, (ARS NRRL Type Strain) B-1922 Lactobacillus casei subspecies casei, (ARS NRRL Type Strain) B-4383 Cupriavidus necator, (ARS NRRL Type Strain) B-14690 Cupriavidus necator, (ARS NRRL Type Strain) Cupriavidus necator strain H16 (ATCC Type Strain) Rhodobacter capsulatus strain SB-1003 (ATCC Type Strain) OB213 Rhodobacter capsulatus, Oakbio Proprietary Strain OB311 Cupriavidus necator, Oakbio Proprietary Strain

[0039] Surprisingly, a metagenomic analysis (conducted by Zymo Research, Irvina, Calif.) showed that the majority of the biomass product was composed of a subset of the original species of the consortium. Subsequent experiments confirmed this.

[0040] Table 2 provides exemplary microbes for use in combinations according to various consortia embodiments.

TABLE-US-00002 TABLE 2 Strain Species Source B-1727 Rhodobacter sphaeroides, (ARS NRRL Type Strain) B-4276 Rhodopseudomonas palustris, (ARS NRRL Type Strain) B-14308 Bacillus megaterium, (ARS NRRL Type Strain) B-356 Bacillus subtilis, (ARS NRRL Type Strain) B-354 Bacillus subtilis, (ARS NRRL Type Strain) B-14200 Bacillus subtilis subspecies subtilis, (ARS NRRL Type Strain) B-4383 Cupriavidus necator, (ARS NRRL Type Strain) B-14690 Cupriavidus necator, (ARS NRRL Type Strain) H16 Cupriavidus necator strain H16 (ATCC Type Strain) SB-1003 Rhodobacter capsulatus strain (ATCC Type Strain) OB213 Rhodobacter capsulatus, Oakbio Proprietary Strain OB311 Cupriavidus necator, Oakbio Proprietary Strain

[0041] Consortia are not required to include all of these strains, and additional microbes not listed can be employed as well. A typical fermentation can thus comprise, for example, microbes from each of: [0042] Cupriavidus necator [0043] Rhodobacter sphaeroides [0044] Rhodopseudomonas palustris [0045] Rhodobacter capsulatus [0046] Bacillus subtilis.

[0047] Bioreactor Fermentation. In various embodiments a bioreactor for chemoautotrophic synthesis can be used, though many types and designs of bioreactor are suitable. For the cultivation of the product discussed herein a suitable bioreactor includes a vessel at least partially filled with a liquid medium in which the microbes are dispersed. The liquid medium comprises chemicals required for growth of the microbes, examples of which are described below. At least one port exists for introducing the gaseous substrates into the liquid in the bioreactor. The vessel may have a headspace into which gases collect after traversing the fluid in the vessel. An exhaust port allows gases to exit the vessel. Additional ports are present as needed for sensors, addition of liquids or chemicals and removal of product, liquids, or samples for testing, as would be expected to be found on common bioreactors, which are well known in the field of fermentation, cell culture and microbe cultivation. A minimal design bioreactor is shown in FIG. 1.

[0048] In FIG. 1, bioreactor 100 includes a vessel 105 that in operation holds a quantity of a liquid medium 110 containing the chemoautotrophic micro-organisms and other microbes in culture. The bioreactor 100 also includes a substrate port 115 through which a gaseous substrate 120 can be introduced into the vessel 105 for introduction into the liquid medium 110, a media inlet port 125 through which fresh media 130 can be introduced into the vessel 105 for introduction into the liquid medium 110, and a media outlet port 135 through which the medium 110 can be removed, for example, to harvest biomass and/or chemical products. The bioreactor 100 can also comprise a headspace 140 and a gas release valve 145 to vent gases from the headspace 140. In some embodiments, the media outlet port 135 and the media inlet port 125 are connected via a system which harvests biomass and reconditions the medium 110 for recirculation. In some embodiments, the gas release valve 145 is attached to a system which re-circulates the gaseous substrate back to the substrate port 115, and may make additions or subtractions to optimize the gas composition.

[0049] For the purposes of the experimental results described herein, bioreactors comprised custom-built 250 ml, 1 L, and 4 L, glass flask-based bioreactors or a commercially manufactured New Brunswick Scientific Bio Flo 4500 with the 4-gas handling option. Some experiments were conducted in 5 L and 55 L looping gas bioreactors. Fermentations were run at a constant or varied temperatures between 15 and 70° C., for example at 30° C. Additional bioreactor designs that can be used in conjunction with the present invention can be found in U.S. patent application Ser. No. 13/204,649 filed on Aug. 6, 2011 and entitled “Chemoautotrophic Bioreactor Systems and Methods of Use” which is incorporated herein by reference. Bioreactors for various embodiments can comprise one or more vessels and/or towers or piping arrangements, and can comprise, for example, a Continuous Stirred Tank Reactor (CSTR), an Immobilized Cell Reactor (ICR), a Trickle Bed Reactor (TBR), a Bubble Column, a Gas Lift Fermenter, a Static Mixer, a Fluidized Bed, an Up-flow or Down-flow, a continuous, batch or loop reactor, or any other vessel or device suitable for maintaining suitable gas-liquid contact. In some embodiments, the bioreactor may comprise a first growth vessel and a second chemoautotrophic synthesis vessel, while in other embodiments a single vessel is used throughout both of the growth and synthesis stages.

[0050] In some embodiments, a gas recirculation system can be used to improve the conversion efficiency, particularly during a continuous process, in order to reduce the total gas requirement. Continuous harvesting of the cell mass can be advantageous for commercial production processes, and can be implemented through the continuous removal of cell broth and the continuous replenishing of medium, in order to maintain the culture volume and cell density.

[0051] Monitoring cell growth and species diversity. To monitor the progress of cell growth and verify the species diversity of a culture, samples can be periodically removed for analysis, or the bioreactor system can comprise analytic equipment. Removal allows for characterization techniques such as microscopy to determine cell morphology, an example of which is shown in FIG. 2 (from the completion of a run), a brightfield micrograph showing that the species diversity was maintained. Four different species are numbered 210, 220, 230, and 240 in the micrograph. Species diversity can also be monitored and quantified using methods well known in the art, such as analysis of 16S rRNA genes, 23S rRNA genes, or other genetic markers and phenotypic indicators (Jovel et al., 2016). Metagenomic analysis of exemplary fermentation products was carried out by Zymo Research, Irvine, Calif. Growth behavior was characterized by optical density (OD) measurements at 620 nm using an ICN TiterTek 96-well plate reader. Aliquots of each fermenter sample (200 ul) were measured in duplicate to plot the growth curves.

[0052] Carbon capture. Carbon capture from a new source of flue gas can be verified by performing headspace gas analysis, as well as growth experiments that use the flue gas as the sole carbon source for bacterial biomass production. The dry weight of each culture can also be determined by centrifuging the culture, washing the pellet, drying the cells in a lyophilizer, and weighing the lyophilized cells.

[0053] Gas mixing. For hydrogen fermentations typically, the CO.sub.2 feedstock or raw flue gas can be diluted with hydrogen gas to achieve ratios of about 8:1 to 1:1 (H.sub.2:CO.sub.2, v/v), resulting in a final CO.sub.2 concentration of about 50%-1% or less. The O.sub.2 concentration is ideally 3-15%, or 3-12%, or 8-15%, or 8-12%. For methane fermentations, typically the methane concentration is between 80% to 5%, CO.sub.2 is between 40%-1%, and the O.sub.2 concentration is 50% to 5%. In either system, CO can be up to 10%, and a variety of other gases may be present, including sulfur oxides, nitrogen oxides, hydrogen sulfide, molecular nitrogen or other gases found in the gas source.

[0054] Culture medium. Many different mineral media recipes can be used, and varying the media is one of the ways the characteristics of the final product can be influenced. An exemplary mineral salts medium (modified from Repaske & Mayer, 1976) containing no organic carbon or complex nutrients comprises Na.sub.2HPO.sub.4.2H.sub.2O 4.5 g/L, KH.sub.2PO.sub.4 1.5 g/L, NH.sub.4Cl 1.8 g/L, MgSO.sub.4.7H.sub.2O 0.11 g/L, NaHCO.sub.3 0.2g/L, FeSO.sub.4.7H.sub.2O 12 mg/L, CaCl.sub.2.2H.sub.2O 10 mg/L, ZnSO.sub.4.7H.sub.2O 100 μg/L, MnCl.sub.2.4H.sub.2O 30 μg/L, H.sub.3BO.sub.3 300 μg/L, CoCl.sub.2.6H.sub.2O 200 μg/L, CuCl.sub.2.2H.sub.2O 10 Ξg/L, NiCl.sub.2.6H.sub.2O 20 μg/L, Na.sub.2MoO.sub.4.2H.sub.2O 30 μg/L. Systems operating at larger volumes for commercial production can employ different compositions, for example, less phosphate may be required as an active Ph control, limiting the need for a strong phosphate buffer system.

[0055] Concentration and harvesting. Biomass products can be harvested through many methods, such as filtration, gravity separation, or other methods, of which many are industrially practiced. Drying can be achieved by spray drying, freeze drying, thermal drying, desiccation, or many other methods, many of which are currently practiced industrially.

[0056] Heat treatment, radiation exposure, or chemical treatment can be useful if the cells must be made non-viable prior to further processing. The dried material can be easily blended with other ingredients to form a nutritious fish feed that can replace aquafeed products that typically rely on fishmeal for protein, fatty acids, and other nutrients. Exemplary additives that can be blended with the dry material include oils, plant-based ingredients, animal-based ingredients such as fishmeal, feather meal, blood meal, minerals, vitamins, amino acid supplements, attractants, colorants, medicines, flow additives, and preservatives. The amino acid composition of the dried material from a 30 L batch of the cultivated consortium shown in Table 2 compares favorably to that of fishmeal (IAFMM Report, 1970), in that it has similar amino acid distribution.

[0057] Hot water, enzymatic, protein isolation or other treatments may be used to reduce the amount of nucleic acids in the material. Recovered cellular biomass can be subjected to lysis by heat, chemical, enzymatic or mechanical disruption such as freeze thaw, pressure cell, grinding, or shear, to obtain a protein isolate.

[0058] In the below referenced example, cell suspensions were removed from the fermenter via a sterile exit port. The supernatant was then removed by centrifugation to form a cell pellet. The cells were then washed in a low-salt buffer solution, and then re-pelleted. The final cell paste was then freeze dried to a powder using a commercial Labconco lyophilizer.

[0059] A cultivation of the consortium shown in Table 2 was performed at the 55L scale in a custom-built looping gas bioreactor. The cultivated product consisted of chemoautotrophs, photoautotrophs, and probiotic heterotrophs, Amino acid composition analysis was conducted by NP Analytical Laboratories (St. Louis, Mo.) and shown below in Table 3:

TABLE-US-00003 TABLE 3 Amino Acid g/100 g Dried Biomass Aspartic Acid 4.49 Threonine 2.40 Serine 1.79 Glutamic Acid 7.31 Proline 1.67 Glycine 2.15 Alanine 3.24 Valine 3.34 Methionine 1.16 Isoleucine 2.60 Leucine 3.24 Tyrosine 1.71 Phenylalanine 1.99 Histidine 0.930 Lysine 3.48 Arginine 2.45 Cysteine 0.312 Tryptophan 0.519

[0060] At the conclusion of fermentations with the consortia mentioned in Table 2, beginning with all 12 species of bacteria, a metagenomic analysis carried out by Zymo Research, Irvine, Calif. on the final products. Surprisingly, it was found that the bacterial population in the final product represented a subset of the initial species provided. An exemplary final species composition comprised 92.10% C. necator, 4.30% Rhodobacter, 0.20% Bacillus, and a trace amount of Phodopseudamonas. Another exemplary final composition, starting from the same consortium but using a larger gas-fed bioreactor, had a final species composition comprising 93.70% C. necator, 0.30% Rhodobacter, 0.20% Bacillus, and 5.80% others.

[0061] Accordingly, various embodiments of consortia consist of, or consist essentially of, at most six species of bacteria and microbes and in some cases only 3. These six species consist of Cupriavidus necator, Rhodobacter sphaeroides, Rhodopsuedamonas palustris, Rhodobacter capsulatus, Bacillus subtilis, and Bacillis magaterium.

[0062] In the various experimental final products C. necator comprised in excess of 85% of the total microbial population, independent of which strain of C. necator was in the initial consortium. Likewise, the use of different B. subtilis strains did not have a significant impact on the nutritional composition of the final product or the overall representation of Bacillus in the final product which was always below 5%.

[0063] In further experiments, when consortia were grown on gas, as described above, from starting consortia comprising C. necator, R. sphaeroides, R. palustris, and B. subtilis, each of these microbes were represented in the final product again with C. necator comprising over 85% of the final product and B. subtilis comprising less than 5%. One exemplary final product consisted of 86.10% C. necator, 2.40% Rhodobacter, 0.30% Bacillus, and trace Rhodopseudomonas.

[0064] An exemplary food or feed additive according to some embodiments, comprises, consists of, or consists essentially of a biomass including Cupriavidus necator, a species from the genus Rhodobacter, a species from the genus Rhodopseudomonas, and a species from the genus Bacillus. In various embodiments the species from the genus Rhodobacter comprises Rhodobacter sphearoidies, and/or the species from the genus Rhodopseudomonas comprises Rhodopseudomonas palustris, and/or the species from the genus Bacillus comprises Bacillus subtilis. Any of the exemplary foods or feed additives noted above can additionally comprise, consist of, or consist essentially of that biomass plus Rhodobacter capsulatus. These compositions are characterized by an enhanced attractiveness to fish and/or characterized by an enhanced palatability to fish.

[0065] Table 4, plotted in FIG. 3, provides a comparison of amino acid profiles between an exemplary final product from the chemoautotrophic cultivation of a consortium comprised of four species of bacteria, C. necator, R. palustris, R. sphearoidies and B. subtilis and three other products of the prior art, M. extorquens, fishmeal, and C. necator. The exemplary final product included 86% total protein, M. extorquens comprised 53% total protein, the fishmeal comprised 70% total protein, and C. necator comprised 41% total protein. The C. necator was a monoculture also grown chemoautotrophically by the applicant. The data for M. extorquens is drawn from the GRAS notice AGRN 33 filed on Aug. 16, 2019 with the USDA. Amino acid profiles for the exemplary and C. necator products were performed by Nestle Purina Analytical labs, St. Louis, Mo. the Fishmeal and M. extorquens are currently approved for use in aquaculture diets.

TABLE-US-00004 TABLE 4 Experimental M AA Amino Acid product extorquens fishmeal C. necator Arg Arginine 5.64 3.59 4.38 2.546 His Histidine 1.79 1.08 1.69 0.822 Ile Isoleucine 2.91 1.87 2.97 1.45 Leu Leucine 5.66 3.46 5.08 3.29 Lys Lysine 3.42 2.87 5.30 2.635 Met Methionine 2.00 0.76 1.91 0.89 Phe Phenylalanine 3.56 2.10 2.75 1.543 Thr Threonine 3.60 2.25 2.89 1.682 Trp Tryptophan 1.15 0.29 0.71 0.614 Val Valine 4.36 2.90 3.46 2.1 Ala Alanine 5.69 4.21 4.45 3.85 Asp Aspartic Acid 6.75 4.32 6.42 3.581 Cys Cysteine 0.77 0.36 0.56 0.265 Glu Glutamic acid 8.82 6.77 8.90 4.32 Gly Glycine 3.72 2.66 4.52 2.19 Pro Proline 2.78 2.97 2.97 1.58 Ser Serine 2.73 1.83 2.75 1.476 Tyr Tyrosine 2.75 1.47 2.19 1.392

[0066] It can be seen that the experimental final product had the highest protein content, and generally higher levels of the essential amino acids that the three other products. Where the experimental final product did not exceed the others, it was at least similar, for example, cystine, proline, and lysine. Particularly, methionine is at a level of 2% of cell dry weight in the experimental final product which exceeds the level in fishmeal by a small amount but exceeds the level found in M. extoquens and monocultured C. necator by more than double. Methionine is an essential amino acid which is normally only found in low concentrations in plant proteins, so methionine is often added as a separate amino acid ingredient in formulated diets to compensate for its natural low level.

[0067] FIG. 4 shows a further comparison between the same data as above for the chemoautotrophically grown experimental final product and data for Bacillus subtilis grown heterotrophically on sugars. The B. subtilis yielded only 43% total protein, and also produced a large amount of bad smelling polyamine compounds which would make it unsuitable as a feed additive, and also unpleasant to handle.

[0068] In a test conducted by the U.S. Fish and Wildlife Service Fish Technology Center, Bozeman Montana, juvenile Rainbow Trout, (Oncorhynchus mykiss), a commercially relevant salmonid species, were fed diets which compared the experimental final product to a high-quality commercial fishmeal. In this study a very high level of the experimental final product, 53% by weight of the formulated feed, was fed to groups of 100 juvenile salmon and their growth compared to similar groups fed a diet comprising a protein content match of high-quality fishmeal. The very high inclusion rate of the experimental final product was more than twice as high as a normal inclusion rate of fishmeal or protein, was meant to test for antinutritional properties by observing mortality during the study as well as growth.

[0069] The results are illustrated, in part, by FIGS. 5 and 6. As shown in these graphs, the testing found that the fish fed the experimental final product and the fish fed the fishmeal diet were the same weight at the end of the study. The fish fed the experimental final product grew more slowly on average initially, but ultimately exceeded the growth rate of the fishmeal fed fish between weeks 4 and 6. Mortality over a 6-week trial showed statistically identical results with 99 of the 100 fish tested with the experimental final product survived to the end of the trial.

[0070] A subsequent test of digestibility and palatability was carried out by Japan Scientific Feeds Association, JFSA Report No. 2020486, Mar. 11, 2021. For digestibility, 135 rainbow trout with an average body weight of 106.7 g were divided into 9 experimental and control groups of 15 fish each. Three control groups were fed formulated diets comprising fishmeal. The experimental diets consisted of 3 groups which were fed a diet comprising 20% of the experimental final product in place of the fishmeal, and another 3 groups which were fed a diet comprising 40% of the experimental final product in place of the fishmeal. The fish were fed these diets for 26 days. Waste products were collected and analyzed using an international accepted test protocol.

[0071] The results of these experiments showed high digestibility of feed, with 94.8% and 91.4% digested for the 20% and 40% inclusion rate feeds respectively, indicating a high degree of digestibility. These inclusion rates, 20% and 40%, are higher than normally would be included in a formulated diet and were formulated to test whether anti-nutritional properties are present and to see if there is a limit on digestibility. For palatability, the same groups of fish were studied for the time to initiate feeding and also the time to consume the total amount of food presented. Surprisingly, the diets comprising the experimental final product in lieu of fishmeal were consumed significantly faster than the control diets which comprised fishmeal. The results showed that the diets containing the 20% and 40% of the experimental final product were consumed in 66.2% and 35.0% of the time, respectively, relative to the time it took the fish to consume the control diet comprising fishmeal. Stated differently, this means fish will consume a diet which comprises 20% or 40% of the experimental final product in lieu of fishmeal 1.5 times and 2.85 times as fast, respectively, as a diet comprising fishmeal. This shows higher palatability for the experimental final product when compared to diets containing only fishmeal.

[0072] Because feed which is not consumed within a certain period of time is lost in industrial, artisanal, and hobbyist cultivation of aquatic species, the rate of feed consumption is an important metric. Because of this, attractants or substances which comprise improved attractant qualities are much sought after, as are those which encourage rapid consumption of feed due to taste, olfactory, or other characteristics. Likewise, with respect to the wild capture of fish, for sport or commercial purposes, baits with attractant qualities are highly desired, as well as baits which cause fish to strike.

[0073] In the foregoing specification, the invention is described with reference to specific embodiments thereof, but those skilled in the art will recognize that the invention is not limited thereto. Various features and aspects of the above-described invention may be used individually or jointly. Further, the invention can be utilized in any number of environments and applications beyond those described herein without departing from the broader spirit and scope of the specification. The specification and drawings are, accordingly, to be regarded as illustrative rather than restrictive. It will be recognized that the terms “comprising,” “including,” and “having,” as used herein, are specifically intended to be read as open-ended terms of art.