Biodegradable polymer

Abstract

A bioremediating composition including a consortial culture with at least one fungal culture and at least one bacterium culture, a growth medium with a waste material that includes synthetic rubber and method for producing a biodegradable byproduct from a waste material that includes synthetic rubber, preparing the growth medium with the waste material, and incubating the growth medium with at least one bioremediating composition with at least one fungal and one bacterium culture.

Description

BIODEGRADABLE POLYMERBACKGROUND

[0001] Synthetic rubber is generally nonbiodegradable. Some recycling and reuse is possible, such as crumb rubber derived from end-of-life tires. Crumb rubber has diverse applications such as sports surfaces, asphalt modifications, and playground flooring. Even though crumb rubber is recycled, it is sometimes difficult to reuse further and is largely disposed of in landfills after it has reached its end of use, and concerns have been raised about potential health and environmental risks of crumb rubber.

[0002] Semi-volatile organic compounds (sVOCs), polycyclic aromatic hydrocarbons (PAHs), phthalates and other components of crumb rubber can be noxious or toxic. Studies about the environmental impact of crumb rubber consider it to be a significant source of microplastics pollution. (Fort J, KobeticovaK, Bohm M, Podlesny J, Jelinkova V, Vachtlova M, Bures F, Cerny R. Environmental Consequences of Rubber Crumb Application: Soil and Water Pollution. Polymers (Basel). 2022 Mar 30; 14(7): 1416.).

[0003] The effects on the environment are still being studied, but there is a clear need for environmentally safe disposal and degradation of synthetic rubber. In some situations, fungi and bacteria can live together in consortia or co-microbial cultures. With additional research, fungi and bacteria consortia may have the potential to assist each other to breakdown synthetic rubber and its environmental contaminants, or facilitate conversion of contaminants to nonthreatening or even beneficial resources.BRIEF SUMMARY

[0004] This disclosure relates to methods, compositions, and products of bioremediation including a method for producing a biodegradable byproduct, the method including: obtaining a waste material, wherein at least a portion of the waste material includes synthetic rubber; preparing a growth medium, wherein at least a portion of the growth medium includes the waste material; incubating the growth medium with at least one bioremediating composition, wherein the at least one bioremediating composition includes at least one fungal culture and at least one bacterium culture.

[0005] In an aspect, the waste material in the growth medium consists essentially of synthetic rubber.

[0006] In an aspect, a portion of the waste material includes rubber crumb.

[0007] In an aspect, the method further includes reducing toxicity of the waste material.

[0008] In an aspect, the method further includes degrading polycyclic aromatic hydrocarbons and phthalates in the waste material.

[0009] In an aspect, the method further includes wherein the at least one bioremediating composition is adapted to break C-S bonds and / or carry out desulfurization.

[0010] In an aspect, the waste material includes the main source of carbon for fungi growth.

[0011] In an aspect, the method the waste material does not include soil.

[0012] In an aspect, the waste material comprises benzo[a]anthracene and the method further includes reducing the benzo[a]anthracene in the waste material.

[0013] In an aspect, the waste material includes Butylbenzyl Phthalate and the method further includes reducing the Butylbenzyl Phthalate in the waste material.

[0014] In an aspect, the waste material includes Di-n-octyl Phthalate and the method further includes reducing the Di-n-octyl Phthalate. in the waste material.

[0015] In an aspect, the waste material includes Bis(2 -butoxyethyl) Phthalate and the method further includes reducing the Bis(2 -butoxy ethyl) Phthalate in the waste material.

[0016] In an aspect, the waste material includes Chrysene and the method further includes reducing the Chrysene in the waste material.

[0017] In an aspect, the waste material includes Pyrene and the method further includes reducing the Pyrene in the waste material.

[0018] In an aspect, the waste material includes Diethyl Phthalate and the method further includes reducing the Diethyl Phthalate in the waste material.

[0019] In an aspect, the waste material includes Fluoranthene and the method further includes reducing the Fluoranthene in the waste material.

[0020] In an aspect, the waste material includes Phenanthrene and the method further includes reducing the Phenanthrene in the waste material.

[0021] In an aspect, the method includes sterilizing the waste material.

[0022] In an aspect, at least one strain of bacteria produces enzymes for desulphurization of sulfur containing contaminants in synthetic rubber, wherein the sulfur containing content includes Dibenzothiophene.

[0023] In an aspect, the growth medium includes Modified Saline Medium.

[0024] In an aspect, the one strain of bacteria includes Rhodococcus erythropolis.

[0025] In an aspect, at least one strain of bacteria includes Cupriavidus basilensis.

[0026] In an aspect, the method includes catalyzing the enzymatic reactions with nanoparticles prepared using cultures of Chlorella.

[0027] In an aspect, the method includes culturing in a humidity controlled environment for more than about one month, alternatively more than about two months, or alternatively more than about three months.

[0028] In an aspect, the method includes a sonication pretreatment.

[0029] In an embodiment, the disclosure presents a consortial culture comprising at least one fungal culture and at least one bacterium culture; a growth medium comprising a waste material, wherein at least a portion of the waste material comprises synthetic rubber.

[0030] In an aspect, the portion of the waste material comprising synthetic rubber includes rubber crumb.

[0031] In another aspect, the composition also includes mycelium growth which penetrates the synthetic rubber, forms aggregates with synthetic rubber, and / or adheres to the synthetic rubber.

[0032] In an aspect, the growth medium consists essentially of synthetic rubber.

[0033] In an aspect, the growth medium does not comprise soil.

[0034] In an aspect, the fungi and bacteria of the composition exhibit high enzyme activity and / or growth together.

[0035] In an aspect, at least one fungal culture includes Pleurotus ostreatus, Purpureocillium lilacinum, Rhodotorula mucilaginosa yeast, Meripilus giganteus, Trametes hirsute, Hypholoma fasciculare, Chaetomium cuniculare, Dactylonectria macrodidyma, Ganoderma sinense, Cladosporium clodosporioides, Rhodotorula mucilaginosa, Acremonium or Pseudomonas putida,' and the bacterium culture includes Cupriavidus basilensis or Rhodococcus erythropolis.

[0036] In an aspect, the composition is configured for degradation of sulfur compounds and / or breakdowns sulfur cross-linkages when the growth medium comprises a source of sulfur.

[0037] In an aspect, the bacterium culture metabolizes polycyclic aromatic hydrocarbons during cultivation with the rubber crumb.

[0038] In another aspect, the composition accelerates biological degradation of the waste material.

[0039] In an aspect, the composition renders the waste material less toxic.

[0040] In another aspect, the composition renders the waste material biodegradeable.

[0041] In an aspect, the portion of the waste material comprising synthetic rubber is pretreated by sonication.

[0042] In an aspect, the composition further includes Modified Saline Medium.

[0043] In an aspect, the growth media has a pH range from about 4 - 10 pH; a pH range from about 5 - 9 pH; a pH range from about 6 - 8 pH; and a pH range from about 7 - 7.5 pH.

[0044] In an aspect, the composition stored in a controlled environment wherein the temperature range is held between 20°C to 35°C.

[0045] In an aspect, the composition produces polyhydroxyalkanes (PHAs).

[0046] In an embodiment, this disclosure presents a biodegradable byproduct from a method of bioremediation including: providing a growth medium, wherein at least a portion of the media growth medium comprises a waste material, wherein the waste material comprises rubber crumb; inoculating the media growth medium with a bioremediating composition comprising at least one strain of fungi, wherein the at least one strain of fungi adheres or penetrates the rubber crumb; and inoculating the media with at least one strain of bacteria, wherein the at least one strain of bacteria produces a biofilm; and, the at least one strain of fungi and the at least one strain of bacteria form a co-culture.

[0047] In an embodiment, this disclosure presents a method of mitigating climate change by reducing and / or preventing additional greenhouse gas emissions and / or monitoring, tracking, and / or verifying greenhouse gas emission reductions, and / or reducing water contamination and reuse of waste materials, the method including: providing a media, wherein at least a portion of the media comprises a waste material, wherein the waste material includes rubber crumb; inoculating the media with at least one strain of fungi; and inoculating the media with at least one strain of bacteria.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Various aspects of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0049] FIG. 1 presents photographs of mycelial growth observed in exemplary use of the composition in multiple cultures resulting in the adhesion of rubber.

[0050] FIG. 2 presents a chart showing lower sulphur concentrations in the medium after cultivation of selected fungi and the bacterium Rhodococcus erythropolis.

[0051] FIG. 3 shows spectra obtained from rubber samples using ATR-FTIR.

[0052] FIG. 4A presents photomicrographs taken with scanning electron microscopy of interaction of Purureocillium lilacinum with selected bacteria that had relatively high enzymatic activity in presence of rubber crumb after one week (left) and two weeks (right).

[0053] FIG. 4B presents photomicrographs taken with scanning electron microscopy of showing interaction of Trametes with selected bacteria that had relatively high enzymatic activity in presence of rubber crumb after one week (left) and two weeks (right).

[0054] FIG. 4C presents photomicrographs taken with scanning electron microscopy of showing interaction of Purpur eocillium lilacinum with selected bacteria that had relatively high enzymatic activity in presence of rubber crumb after one week (left) and two weeks (right).

[0055] FIG. 5 presents photomicrographs taken with scanning electron microscopy of rubber crumb at different magnification and different pretreatment conditions: nontreated (left raw), sonicated (right) and after SonoFenton treatment (middle).

[0056] FIG. 6 photomicrographs taken with scanning electron microscopy showing mycelium of Pfypholoma showing growth outside and on the surface of rubber crumb.

[0057] FIG. 7 photomicrographs taken with scanning electron microscopy showing mycelium of Acremonium growing inside the rubber crumb and forming coni dial heads on conidiophores; the left raw show crystals that probably originated from the medium as it contains P according to (Energy-dispersive X-ray spectroscopy) ED AX spectrum.

[0058] FIG. 8 photomicrographs taken with scanning electron microscopy showing Purpureocillium mycelium strongly colonizing the rubber and forming conidiophores.

[0059] FIG. 9 photomicrographs taken with scanning electron microscopy showing Pleurotus ostreatus mycelium colonizing the rubber.

[0060] FIG. 10 presents a chart showing benz[a]anthracene (BAAN) concentrations (in ppb) pretreated in rubber following growth of fungi and bacteria.

[0061] FIG. 11 presents a chart showing Dibuthyl phthalate (DBPH) concentrations (in ppb) in pretreated rubber following growth of fungi and bacteria.

[0062] FIG. 12A presents a chart showing Chrysene concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0063] FIG. 12B presents a chart showing Chrysene concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0064] FIG. 13 A presents a chart showing Pyrene concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0065] FIG. 13B presents a chart showing Pyrene concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0066] FIG. 14 A presents a chart showing Dimethyl hydrogen phosphate (DMPH) concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0067] FIG. 14B presents a chart showing Dimethyl hydrogen phosphate (DMPH) concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0068] FIG. 15A presents a chart showing Bis(2-ethylhexyl) phthalate (DEPH) concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0069] FIG. 15B presents a chart showing Bis(2-ethylhexyl) phthalate (DEPH) concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0070] FIG. 16A presents a chart showing Benzo[b]fluoranthene BBFL concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0071] FIG. 16B presents a chart showing Benzo[b] fluoranthene BBFL concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0072] FIG. 17A presents a chart showing Bis(acridine) pyrazole (BAPY) concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0073] FIG. 17B presents a chart showing Bis(acridine) pyrazole (BAPY) concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0074] FIG. 18 presents a chart showing BBPH concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0075] FIG. 19A presents a chart showing DNOPH concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control.

[0076] FIG. 19B presents a chart showing DNOPH concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control.

[0077] FIG. 20 presents a chart showing a comparison of biomass obtained in the presence of PAHs and phthalates in comparison with the control

[0078] FIG. 21 presents a chart showing a comparison of biomass between two species of fungi in the presence of individual substances

[0079] FIG. 22 presents a chart showing PAH and Phthalates (Fluoranthene, Anthracene, and Dimethyl phthalate) concentrations in the control (without the addition of fungi) and the medium in which Pleurotus ostreatus or Purpureocillium lilacinum were grown; data in ppb

[0080] FIG. 23 presents a chart showing PAH and Phthalates (Chrysene, Diethyl phthalate, and Benzyl-buthyl phthalate) concentrations in the control (without the addition of fungi) and the medium in which Pleurotus ostreatus or Purpureocillium lilacinum were grown; data in PPb

[0081] FIG. 24 presents a chart showing PAH and Phthalates (Pyrene, Dibuthyl phthalate, and DNOP) concentrations in the control (without the addition of fungi) and the medium in which Pleurotus ostreatus or Purpureocillium lilacinum were grown; data in ppb.

[0082] FIG. 25A shows photographs under a microscope of the rubber samples after the treatment with bacteria and fungi and the bacteria growing on the rubber forming a biofilm.

[0083] FIG. 25B shows photographs under a microscope of the rubber samples after the treatment with bacteria and fungi and the bacteria growing on the rubber forming a biofilm stained with Nile Blue.

[0084] FIG. 26 shows photomicrographs taken with scanning electron microscopy showing mycelium on a latex glove.DETAILED DESCRIPTIONI. Introduction

[0085] This disclosure relates to compositions, methods, and products of bioremediation with a co-culture of fungi and bacteria, sometimes called a consortium or consortial culture. A consortial culture, or co-culture, of fungi and bacteria refers to a controlled environment where both two or more interacting fungi and bacteria populations are grown together. Fungi and bacteria can coexist and interact with each other in natural ecosystems forming complex microbial communities sometimes including multiple fungi and multiple bacterial cocultures. Contemporary studies have revealed that fungi and bacteria often form physically and metabolically interdependent consortia that harbor properties distinct from those of their single components. Interactions can be mutualistic, where both organisms benefit, but relationships can be also be antagonistic, involving competition or inhibition.

[0086] Not all fungi and bacteria can grow in culture together. Differences in nutritional requirements, competition for resources, and the production of inhibitory substances can contribute to a lack of compatibility. Fungi and bacteria can compete for the same resources, such as nutrients and space. Some microorganisms produce substances (e.g. antibiotics or secondary metabolites) that inhibit the growth of others, leading to competition for dominance in the culture. This can result in the suppression or inhibition of one group by the other. Often, certain strains of a microbial community dominate the environment leading other strains to extinction.

[0087] On the other hand, a symbiotic relationship can take advantage of differences in nutritional requirements by nutrient cycling, breaking down organic matter and releasing nutrients, to the benefit of the other organism. FIG. 26 shows photomicrographs taken with scanning electron microscopy showing mycelium on a latex glove. The mix of different mycelium shows that the different fungi can grow together. In some embodiments, multiple fungi grow together in the co-culture. In this type of relationship, fungi and bacteria can work together in decomposition processes, even breaking down substances believed to be non- biodegradable waste, including potentially hazardous material or toxic substances.

[0088] In this process, an innocuous biodegradable byproduct can be formed that may sequester or break down potentially hazardous waste material or toxic substances. In addition, the biodegradable byproduct may have useful properties and structure.II. Definitions

[0089] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0090] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. And, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0091] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0092] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0093] The term "comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0094] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0095] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless thecontent clearly dictates otherwise. The term "and / or" means any one or more of the items in the listjoined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0096] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0097] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0098] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / - 10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalentsto the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0099] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0100] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0101] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0102] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.III. Exemplary Methods and Compositions

[0103] In one embodiment, this disclosure describes producing a biodegradable byproduct and a composition including a consortial culture including at least one fungal culture and at least one bacterium culture; and, a growth medium including a waste material, wherein at least a portion of the waste material includes synthetic rubber. Synthetic rubber is generally not biodegradable, and it is believed to be a source of microplastics pollution and other toxins / irritants.

[0104] In one aspect, the synthetic rubber may include rubber crumb. Rubber crumb, also known as crumb rubber, is generally made from used, discarded tires. The tires are mechanically shredded, oftentimes other components of tires like steel and tire cord (fluff) are removed, and the rubber components are ground into smaller particles. The crumb-like smallerparticles give the recycled rubber its namesake. The specific composition of rubber crumb may vary depending on the source and the processing methods used by the manufacturer. Manufacturers may tailor the production process to meet the requirements of different applications, but the final rubber crumb product can vary in particle size, purity, and characteristics.

[0105] In an aspect, the crumb rubber in the waste material may have a particle size of diameter of approximately 2 cm, 1 cm, 5 mm, 1 mm, 0.5 mm, 0.1 mm, 0.05 mm, 0.03 mm, or 0.01 mm. In an aspect, some impurities found in the crumb rubber of the waste material may include, among other substances, arsenic, heavy metals, polycyclic aromatic hydrocarbons (PAHs), phthalates, volatile organic compounds (VOCs) sulphenamides, guanidines, thiazoles, thiuams, dithiocarbamates, sulfur donors, and phenolics.

[0106] Crumb rubber may be classified into different grades. For example, Grade No. l : Tire Granule consists of granulated tire crumb, black only, guaranteed metal free, sized (magnetically separated materials are not acceptable, and fluff from tire cord removed). Grade No.2: Tire Granule consists of granulated tire crumb, black and white, guaranteed metal free, sized to minus 40 mesh (magnetically separated materials are not acceptable, and fluff from tire cord removed). Grade No.3: Tire Granule consists of granulated tire crumb, black only, magnetically separated, sized (fluff from tire cord removed). Grade No.4: Tire Granule consists of granulated tire crumb, black and white, magnetically separated, sized, fluff from tire cord removed). Grade No.5: Tire Granule consists of unclassified granulated tire crumb, sized, unseparated, not magnetically separated, fluff from tire cord not removed. In an aspect, the crumb rubber may have a selected parade or other characteristics.

[0107] In an embodiment, the crumb rubber may be pretreated. There are different methods of pretreating the crumb rubber, this may include, but is not limited to, cleaning, sanitizing, and sterilizing. In an aspect, the crumb rubber may be washed with detergents and rinsed; soaked with deionized or distilled water; soaked in water and an alkaline; soaked in diluted bleach or hydrogen peroxide solution; or, heat sterilized using dry heat or steam.

[0108] The crumb rubber of the waste material may also be pretreated with ozonification, sonication, and a Sono-Fenton process among other techniques. Ozonification refers to the process of treating or reacting a substance with ozone, which is a highly reactive form of oxygen. Ozone is known as a powerful oxidizing agent and can be used to disinfect as well as oxidize organic and inorganic materials. In an embodiment, the crumb rubber is pretreated byexposure to ozone for about 30 minutes, about 20 minutes, about 10 minutes, or about five minutes.

[0109] In an embodiment, the portion of the waste material comprising synthetic rubber is pretreated by sonication. Sonication can vary in intensity and duration. In some embodiments, the crumb rubber is subjected to a pretreatment of sonication at 10-20 kHz, 20-40 kHz, 40-60 kHz, 60-80 kHz, 80-100 kHz, 100-1000 kHz, or 1000-5000 kHz. In some embodiments, a pretreatment of sonication may be for a period of 30 minutes, 20 minutes, 10 minutes, or five minutes.

[0110] Sonication refers to the use of sound waves, typically ultrasonic sound waves, to agitate particles in a sample. Agitation may happen in many ways: ultrasound waves develop compression and rarefaction while transmitting through a medium. During this process, microbubbles are formed — cavitation and collapse causes strong hydro-mechanical shear forces that can cause breakdown of chemical bonds. Pretreatment of the synthetic rubber by sonication may allow the substances to be more bioavailable in solution and chemical breakdown. For example, scanning electron microscope (SEM) analysis showed changes in problem rubber morphology.[OHl] In Example 1, ATR-FTIR spectroscopy, performed using a Thermo-spectrometer, was used to analyze the samples. FIG. 3 displays two spectra: one for the original rubber and another for the rubber treated with sonication for ten minutes. A third spectrum represents rubber in the standard solution. The sonicated sample spectrum exhibited a new band at approximately 960 cmA(-l), likely associated with out-of-plane trans(-CH=CH-) butadiene or C-H wagging butadiene vibrations. The band at 907 cmA(-l) indicated out-of-plane bending vibrations of (C- H) vinyl groups, while the band at 1075 cmA(-l) suggested (C-S-C) stretching vibrations. Plane trans(-CH=CH-) butadiene, C-H wagging butadiene vibrations, and (C-S-C) stretching vibrations are indications that there are bonds that can be chemically attacked and broken. In addition, for the concentration levels of PAH and phthalates in co-cultures, gas chromatography analysis was performed and statistical analysis was conducted solely on samples subjected to sonication. In Example 1, the experimenters observed successful rubber breakdown, with contributions from both sonication and mycelial activity.

[0112] Sono-Fenton processes combine sonication with a Fenton reaction, and advanced oxidase station process. For example, a conventional Fenton reaction may combine H2O2 with Fe2 +ion at acidic pH to produce ferric ions and hydroxyl radicals that can oxidize organiccompounds. This process has been used to treat recalcitrant contaminants. At the same time, the radicals and other byproducts produced in this process may end up in the liquid media affecting growth or sequestration of chemicals of interest. In Example 2, Sono-Fenton treatment resulted in the release of some PAHs and phthalates from the rubber, potentially compromising fungal growth in extreme cases. While some embodiments allow for the inclusion of pretreatment with Sono-Fenton processes, other embodiments omit this pretreatment. In an embodiment, the portion of the waste material of the composition comprising synthetic rubber has not been pretreated by Sono-Fenton reaction.

[0113] In an aspect, the composition includes mycelium growth. Mycelium describes a network of fungal threads or filaments (hyphae). Hyphae have a firm cell wall made of chitin, and grow from a tip and extend out in search of food sources. Hyphae may release enzymes, absorb nutrients, branch out, and form new, growing mycelium networks. Mycelium growth occurs when hyphae adheres to the synthetic rubber and / or hard tips of the hyphae penetrate the synthetic rubber and aggregate the rubber with filaments of the mycelium networks.

[0114] Synthetic rubber includes derivatives from petrochemicals sources, which may include polymers such as styrene-butadiene rubber (SBR), polybutadiene rubber (BR), and polyisoprene rubber (IR), among others. These long polymers have bonds and structures that are relatively stable and less prone to degradation by chemicals or enzymatic action. Most natural organisms cannot break down or degrade these compounds. In addition, synthetic rubber includes PAHs and phthalates that pose the same challenge to these natural organisms. (Patel AB, Shaikh S, Jain KR, Desai C, Madamwar D. Polycyclic Aromatic Hydrocarbons: Sources, Toxicity, and Remediation Approaches. Front Microbiol. 2020 Nov 5; 11 :562813. doi: 10.3389 / fmicb.2020.562813. PMID: 33224110; PMCID: PMC7674206.). Adhesion to the synthetic rubber and penetration demonstrate mycelial growth. In addition, mycelial adhesion to synthetic rubber results in aggregation of rubber crumb and formation of a biodegradable biopolymer byproduct. FIG. 1 shows photos of mycelial growth observed in multiple fungal cultures resulting in the adhesion of rubber. FIGS. 6, 7, 8, and 9 show the growth of fungi within rubber crumb with scanning electron microscopy (SEM).

[0115] In an aspect, the fungal culture and the bacterial culture of the composition exhibit synergy - demonstrating high enzyme activity and growth despite the challenges that synthetic rubber poses to degradation. For example, the fungal culture and the bacterial culture of the composition produce enzymes, such as laccases and peroxidases, which play crucial roles in catalyzing oxidative reactions. Laccases are the versatile enzymes which catalyze oxidationreactions and peroxidases catalyze the oxidation of a substrate by hydrogen peroxide or an organic peroxide. Generally, the presence of these enzymes indicate high enzyme and oxidative activity. These enzymes are involved in the breakdown of complex organic compounds and are often associated with processes like synthetic polymer degradation and other redox reactions.

[0116] Enzyme activity and growth can be demonstrated by measuring the oxidation of 2, 20- azino-bis(3-ethylbenzothiazoline- 6-sulfonate) (ABTS) or measuring levels of hydrogen peroxide added to the growth medium. In an embodiment, the growth media demonstrates the presence of laccases and / or peroxidases. FIGs. 4A, 4B, and 4C show compositions that had relatively high enzymatic activity. FIG. 4A presents photomicrographs taken with scanning electron microscopy showing Purpureocillium lilacinum cultured with selected bacteria in presence of rubber crumb after 1 week (left) and two weeks (right). FIG. 4B presents photomicrographs taken with scanning electron microscopy showing Trametes cultured with selected bacteria that had relatively high enzymatic activity in presence of rubber crumb after 1 week (left) and two weeks (right). FIG. 4C presents photomicrographs taken with scanning electron microscopy showing of Purpureocillium lilacinum cultured with selected bacteria that had relatively high enzymatic activity in presence of rubber crumb after 1 week (left) and two weeks (right).

[0117] Some combinations of fungi and bacteria exhibit exceptional growth and enzyme activity. In an aspect, the composition includes: at least one fungal culture of Pleurotus oslrealus. Purpureocillium lilacinum, Rhodotorula mucilaginosa yeast, Meripilus giganteus, Trametes hirsute, Hypholoma fasciculare, Chaetomium cuniculare, Dactylonectria macrodidyma, Ganoderma sinense, Cladosporium clodosporioides, Rhodotorula mucilaginosa, Acremonium and Pseudomonas putida; and at least one bacterium culture of Cupriavidus basilensis and Rhodococcus erythropolis . As further explained in Example 1, a screening process provides a selection of compatible fungi and bacteria that exhibit exceptional growth and enzyme activity together in co-culture.

[0118] In an aspect, the method and composition break down sulfur compounds when the growth medium includes a source of sulfur. Some synthetic rubber tires contain sulfur compounds as a contaminant in the fossil fuels used to make them. In addition, some rubber tires may go through a vulcanization process that adds sulfur to improve the material's mechanical properties, such as elasticity and durability. Vulcanization creates cross-links between polymer chains, making the rubber more resistant to chemical and biologicalbreakdown. In some embodiments, the waste material contains synthetic rubber that contains sulfur or has been vulcanized.

[0119] Some fungal and bacterial species have the ability to break C-S bonds or desulphurization reactions and derive sulfur from complex organic sulfur compounds for their growth and vital activities. In an embodiment, the method and composition is configured to break carbon sulfur bonds and reduce the amount of sulfur in the growth medium. FIG. 2 presents a chart showing lower sulfur concentrations in a medium (including dibenzothiophene as a source of sulfur) after cultivation of selected fungi and the bacterium Rhodococcus erythropolis. Reduction in the percentage of sulfur can be seen (from 0.6% to 0.3% with certain species— approximately a 50% reduction in sulfur in the medium). Example 1 further explains testing compatible fungi and bacteria of the composition exhibit desulphurization capabilities.

[0120] In an aspect, the co-culture metabolizes or sequesters polycyclic aromatic hydrocarbons (PAH) and phthalates during cultivation with the rubber crumb. PAHs can be composed of two or more fused aromatic rings with carbon and hydrogen atoms. There are many different types of PAH compounds, which includes light-molecular weight PAHs (LMW-PAHs, 2 or 3 aromatic rings) and high-molecular weight PAHs (HMW-PAHs, 4 or more aromatic rings). The LMW-PAHs are highly volatile compounds with relatively low toxicity, while HMW- PAHs are more stable and thereby more resistant to degradation. The stability of these compounds may result in bioaccumulation and as a result are considered to be toxic. Several HMW-PAHs have been associated cancer. Phthalates are a group of chemical compounds commonly used as plasticizers, substances added to plastics to increase their flexibility, transparency, and durability. While HMW-PAHs have been widely used for decades, concerns have been raised about their potential health effects.

[0121] In some embodiments, the co-culture metabolizes or sequesters LMW-PAHs, HMW- PAHs, and / or phthalates to reduce the amounts detectable in the growth medium. Concentration levels of PAHs and phthalates after being in culture with the composition are generally reduced to nearly undetectable levels (-250 ppb or less). FIGs. 10-18 presents charts comparing concentration levels of PAHs and phthalates in synthetic rubber, and concentration levels of PAHs and phthalates after being in culture with the composition. In FIGs. 10 and 11, Benz [a] anthracene (BAAN) and Dibuthyl phthalate (DBPH) concentrations were present in very low concentrations before culture and barely detectable.

[0122] FIG. 10 presents a chart showing benz [a] anthracene (BAAN) concentrations (in ppb) in the medium with rubber crumb following growth of fungi and bacteria in comparison to control. Benz[a]anthracene is a member of polycyclic aromatic hydrocarbons with the chemical formula CisHn, and comprises an aromatic hydrocarbon consisting of four fused benzene rings. It is believed to be a carcinogenic. In addition, it is very toxic to aquatic life. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 5954, Benz(a)anthracene. Retrieved January 6, 2024 from pub chem . ncbi . nlm . nih . gov / compound / B enz a anthracene . )

[0123] FIG. 11 presents a chart showing Dibuthyl phthalate (DBPH) concentrations (in ppb) in co-cultures with rubber crumb following growth of fungi and bacteria in comparison to control. Dibuthyl phthalate is a phthalate ester that is the diester obtained by the formal condensation of the carboxy groups of phthalic acid with two molecules of butan-l-ol. Although used extensively as a plasticiser, it is a ubiquitous environmental contaminant that poses a risk to humans. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 3026, Dibutyl Phthalate. Retrieved January 6, 2024 from pub chem . ncbi . nlm . nih . gov / compound / Dibuty 1 -Phthal ate . )

[0124] FIG. 12A presents a chart showing Chrysene concentrations (in ppb) in pretreated, control, and rubber crumb medium following growth of fungi and bacteria in comparison to control, and FIG. 12B presents a chart in rubber following growth of fungi and bacteria in comparison to control. Chrysene is a polycyclic aromatic hydrocarbon, specifically an orthofused polycyclic arene found commonly in the coal tar. Chrysene is probably a human carcinogen. It able to induce aryl hydrocarbon hydroxylase (AHH) in cultured human lymphocytes. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 9171, Chrysene. Retrieved January 6, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Chrysene). In some co-cultures, the levels of Chrysene drop more than 95% or more (e.g. from Fig. 12B, 1900 ppb were reduced to less than 50 ppb with P. lilacinum and R. Erythropolis).

[0125] FIG. 13 A presents a chart showing Pyrene concentrations (in ppb) in pretreated and control rubber media following growth of fungi and bacteria in comparison to control, and FIG. 13B presents a chart showing Pyrene concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control. Pyrene is a polycyclic aromatic hydrocarbon (PAH) consisting of four fused benzene rings, resulting in a flat aromatic system. The chemical formula is CieHio. There was an interaction between pyrene and the thyroid system in fish, butis not classifiable as to its carcinogenicity to humans. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 31423, Pyrene. Retrieved January 6, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Pyrene.) In some co-cultures, the levels of Pyrene were reduced by 85% or more (e.g. from 875 ppb to 100 ppb with P. lilacinum and C. basilensis in Fig. 13B).

[0126] FIG. 14A presents a chart showing Dimethyl hydrogen phosphate (DMPH) concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control, and FIG. 14B presents a chart showing Dimethyl hydrogen phosphate (DMPH) concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control. Dimethyl phthalate (DMP) is an organic compound and phthalate ester. It has been used as a plasticizer (added to polymers to effect flexibility and / or toughness). Phthalate esters are endocrine disruptors, but is not a known human carcinogen. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 8554, Dimethyl Phthalate. Retrieved January 6, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Dimethyl-Phthalate.)

[0127] FIG. 15 A presents a chart showing Bis(2-ethylhexyl) phthalate (DEPH) concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control, and FIG. 15B presents a chart showing Bis(2-ethylhexyl) phthalate (DEPH) concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control. Diethyl phthalate (DEP) is a phthalate ester. It has also been used as a plasticizer, and is known as endocrine disruptors but not classifiable as to human carcinogenicity. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 6781, Diethyl Phthalate. Retrieved January 7, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Diethyl-Phthalate.) In some co-cultures, the levels of DEPH were reduced by about 80% (from 250 ppb 20 50 ppb in FIG. 15b with / < ostreatus and R. erthropolis).

[0128] FIG. 16A presents a chart showing Benzo[b]fluoranthene BBFL concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control. FIG. 16B presents a chart showing Benzo[b]fluoranthene BBFL concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control. Benzo(b)fluoranthene (B(b)F) has no commercial use. Acute toxicity is rarely reported, but it is a probable human carcinogen. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 9153, Benzo[b]fluoranthene.Retrieved January 7, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Benzo_b_fluoranthene.) In some co-cultures, the levels of BBFL were reduced by about 95% (from 1250 ppb to about 50 ppb in FIG. 16b with P. lilacinum and R. erthropolis).

[0129] FIG. 17A presents a chart showing Bis(acridine) pyrazole (BAPY) concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control and FIG. 17B presents a chart showing Bis(acridine) pyrazole (BAPY) concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to contro. Benzo[a]pyrene (BaP or B[a]P) is a five-ring polycyclic aromatic hydrocarbon. It is not commercially used in the US and is carcinogenic to humans. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 2336, Benzo[a]pyrene. Retrieved January 7, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Benzo_a_pyrene.) In some co-cultures, the levels of BAPY were reduced by about 95% (from 3250 ppb to about 200 ppb in FIG. 17b with P. lilacinum and C. basilensis).

[0130] FIG. 18 presents a chart showing BBPH concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control. Benzyl butyl phthalate (BBP) is an organic compound historically used a plasticizer, but which has now been largely phased out due to health concerns. Benzyl butyl phthalate (BBP) is an organic compound historically used a plasticizer, but which has now been largely phased out due to health concerns. A range of toxicity tests with aquatic organisms has indicated the adverse effects occur at exposure concentrations greater than 100 ug / L. (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 2347, Benzyl butyl phthalate. Retrieved January 7, 2024 from pubchem.ncbi.nlm.nih.gov / compound / Benzyl-butyl-phthalate.) In some cocultures, the levels of BAPY were reduced by about 50% or more (from about 300 ppb to 150 ppb with P. lilacinum andR. erthropolis ).

[0131] FIG. 19A presents a chart showing DNOPH concentrations (in ppb) in pretreated and control rubber following growth of fungi and bacteria in comparison to control and FIG. 19B presents a chart showing DNOPH concentrations (in ppb) in rubber following growth of fungi and bacteria in comparison to control. 2,4-Dinitrophenylhydrazine (DNOPH) is the organic compound C6H3(NO2)2NHNH2. It is a dangerous explosive and it is used as a reagent for aldehydes and ketones (National Center for Biotechnology Information (2024). PubChem Compound Summary for CID 3772977, 2,4-Dinitrophenylhydrazine. Retrieved January 7,2024 from pubchem.ncbi.nlm.nih.gov / compound / 2_4-Dinitrophenylhydrazine.) In some cocultures, the levels of DNOPH were reduced by about 80% (from 700 ppb to about 150 ppb in FIG 19b with P. lilacinum and C. basilensis).

[0132] The reduction in levels of PAHs and phthalates renders the waste material less toxic. In an aspect, the composition, in particular the application of the composition, renders the waste material less toxic. The ability of the method and composition to break down or degrade sulfur bonds, PAHs, and phthalates demonstrate acceleration of biological degradation of the waste material. In an aspect, the method and composition accelerate biological degradation of the waste material. This is a unique and unexpected result of the disclosed composition as it is well known that waste tires have low biological degradability. Samesova, D., Ponist, J., Hybska, H. et al. Determination of aerobic and anaerobic biological degradability of waste tyres. Environ Sci Eur 35, 19 (2023). doi.org / 10.1186 / sl2302-23-00723-6 (“Biodegradability tests confirmed the low biological degradability of waste tyres... The ecotoxicity tests performed confirmed the toxic effect of tyre leachate on selected tested organisms...” (Abstract)).

[0133] Biological degradation, or biodegradability, includes biological processes by which a substance is broken down by oxygen and microorganisms. Whether a substance is biodegradable may depend on the “completeness” to which the substance is broken down and its potential environmental impact. A substance can be considered to biodegrade completely when the substance is broken down into CO2, H2O and biomass and are essentially benign to the environment. Different organizations offer different standards and tests for biodegradability. For example, the Organization for Economic Co-operation and Development (OECD), ASTM, international, formally known as American Society for Testing and Materials, and International Organization for Standardization have tests for biodegradability, (e.g. OECD 301 tests).

[0134] In an aspect, the method and composition renders the waste material mostly biodegradeable. For example, certain metal impurities may not be broken down into CO2, H2O and biomass in the conventional sense, but can be integrated into biomass in such a way as to prevent them from being released into the environment. In Example 2, the increase in mycelial biomass in the presence of PHA and phthalates indicates the ability of the fungi to break down these substances and use these substances as a substrate for growth. See FIGs. 20 and 21. In addition, the adhesion of rubber crumb particulate sequesters the waste material protecting the environment and the method prevents leachates from being released into the environment. Thepresent disclosure includes an increase in mycelial biomass using waste material mostly consisting of synthetic rubber as its source of carbon.

[0135] In an embodiment, the waste material in the growth medium consists essentially of synthetic rubber where the main source of carbon for fungi growth comes from synthetic rubber. The growth medium should not include soil and depend on carbon dioxide (and / or plant growth) for increase in biomass. Limiting the source of carbon to the waste material substrate with synthetic rubber allows more of the biomass increase to come from breaking down and consumption of the waste material including synthetic rubber. In an aspect, consumption of the waste material describes colonization of substantially all of the waste material within a coculture (or a container in which the coculture has been inoculated). In some embodiments, the consumption of the waste material may take about 2 months, alternatively about 3 months, alternatively about 4 months, alternatively about 5 months, alternatively about 6 months, alternatively about 7 months, alternatively about 8 months, alternatively about 9 months, alternatively about 10 months, alternatively about 11 months, or alternatively about 12 months.

[0136] Selecting appropriate co-culture conditions, adjusting nutritional components, and understanding the specific interactions between microorganisms can determine the success of co-cultures. In an embodiment, the growth medium of the composition includes a Modified Saline Medium. A Modified Saline Medium, generally, refers to a minimal media including saline that supplies elements required for growth. Non-limiting examples of growth medium include a Modified Saline Medium for Fungi (MSM-F) and Modified Saline Medium for Bacteria (MSM-B). The MSM-F may further include NaNO3, K2HPO4, MgSO4x7H2O, KC1, and / or yeast extract, and the MSM-B may further include (NH4)2xSO4, MnSO4xH2O, K2HPO4, KH2PO4, MgSO4x7H2O, NaCl, Ca(NO3)2, Na2MoO4xH2O, CaCl2x2H2O, and / or FeSO4x7H2O.

[0137] In another aspect, the growth media has a pH range from about 4 to about 10, alternatively a pH range from about 5 to about 9, alternatively a pH range from about 6 to about 8, or alternatively a pH range from about 7 to about 7.5. In another aspect, the composition is stored in a controlled environment wherein the temperature is held between about 20°C to about 35°C.

[0138] In another embodiment, the method and composition are configured to produce polyhydroxyalkanes / polyhydroxybutyrates (PHA / PHBs) and polyhydroxyalkanoates. PHA molecules appear in a variety of structures and are naturally occurring biodegradable polymers. PHAs are also a source material for bioplastics and may be a replacement for petroleum-derivedplastics. More than 150 monomers can be combined within this family of polymers to produce materials with different, useful properties Polyhydroxyalkanoates are polyesters that contain a characteristic bond of esters, which are accumulated as carbon and energy reserve along and assist in providing energy. Polyhydroxybutyrate (PHB) is used in versatile fields as it is a biodegradable, biocompatible, and ecologically safe thermoplastic.

[0139] Some bacteria, such as Cupriavidus basilensis. can produce polyhydroxyalkanes and polyhydroxyalkanoates (PHAs) for energy storage, and fungi have been known to metabolize PHA / PHBs. Different combinations of fungi and bacteria may produce more PHA / PHBs if desired, or combinations can be used to break down or use PHA / PHBs as intermediates in bacteria fungi interactions. Moreover, the amount of PHA / PHBs produced can be manipulated by the ratio of the bacteria and the presence of catalysts, such as iron oxide nanoparticles. In some embodiments, the method and composition include Cupriavidus basilensis and iron oxide nanoparticles.

[0140] FIG. 25A shows photographs of the rubber samples under a microscope after the treatment with bacteria and fungi. The bacteria growing can be seen forming a biofilm. FIG. 25B shows photographs of the same rubber samples under a microscope after the treatment with bacteria and fungi and the bacteria. The samples have been stained with Nile blue. Nile blue stains PHA granules and fluoresce bright orange on the irradiation of UV light. In FIG. 25B, droplets of fluorescing oily material can be seen indicating the presence of PHA / PHBs. The mechanical properties and biocompatibility of PHA can be changed by blending, modifying the surface or combining PHA with other polymers, enzymes and inorganic materials, making it possible for a wide range of applications. PHAs can be either thermoplastic or elastomeric materials, with melting points ranging from 40°C to 180°C. PHA also has a good resistance to moisture and aroma barrier properties.

[0141] Biopolymer composite can comprise a matrix of biomass that is polymer reinforced. Mycelia of filamentous fungi provide mechanical strength to panels and boards, fire resistance, acoustical and thermal insulating properties. Biopolymer composites have good acoustic properties because of their inherent damping properties, porous structures, and lower densities. Biopolymer composites may also be useful in various applications like building and construction industry— window frames, isolation boards, and wall panels.

[0142] In an embodiment, the composition and byproduct of the method may have several applications such as, but not limited to, landscape, trails and walkways, road pavement, ground cover under playground equipment, running track material, and as a soil additive in sports andplaying fields. In an embodiment, the composition and byproduct could be added into mixes like cement or asphalt.In an embodiment, the composition and byproduct may be dried, shaped, pressed, compressed, molded formed, and / or finished with post-processing. In an embodiment, the composition and byproduct could be formed or pressed into bricks or tiles. In another embodiment, the composition may find use in roofing materials, insulation, and floor substrates.

[0143] In another embodiment, the composition and byproduct may be added in construction materials modifying and improving its properties. In an aspect, the composition and byproduct may provide desired properties, for example, to attenuate the vibrations, annoying noises and sounds caused by the transit of trains and trams in areas near buildings in walls or barriers. In another embodiment, the composition may be used for its resistance to moisture and aroma barrier properties. In another embodiment, the composition and the byproduct of the method may form a biocomposite with mechanical, thermal, or other properties to take any form or shape or modular piece desirable for construction. These applications would further include more environmentally friendly options compared to untreated crumb rubber.

[0144] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0145] EXAMPLES

[0146] Example 1

[0147] Screening fungi and bacteria

[0148] First, 200 samples of rubber (2g each) were prepared in sterilized 100 ml Erlenmeyer bottles and inoculated with 50 strains of bacteria and 150 strains of fungi from the laboratory of Prof. Katarzyna Turnau, Institute of Environmental Sciences (head of Plant-Microbial Interaction Group), Jagiellonian University. Specific culture media for the bacteria and fungi were selected, namely Modified Saline Medium for Bacteria (MSM-B) and Modified Saline Medium for Fungi (MSM-F). The pH of MSM-B was adjusted to 7.5, while MSM-F had a pH of 7.0. The composition of MSM-B included various compounds such as (NH4)2xSO4,MnSO4xH2O, K2HPO4, KH2PO4, MgSO4x7H2O, NaCl, Ca(NO3)2, Na2MoO4xH2O, CaC12x2H2O, and FeSO4x7H2O. MSM-F consisted ofNaNCh, K2HPO4, MgSO4x7H2O, KC1, and yeast extract. The yeast extract (10 g / 1) was added in the yeast pre-culture phase. The MSM-B and MSM-F media were sterilized at 121 °C for 20 minutes, and added to the samples. The samples were kept in Memmert cultivation chamber at 25°C for 2 month, without shaking.

[0149] During the following 2 month the cultures were regularly observed. In several cultures of fungi, the growth of mycelium was detected by visual inspection. Selection of the fungi for further analysis was done on the basis of rubber aggregation and enzymatic activity. Measurements of pH values in cultures were carried out twice by sampling 100 pl of the medium and depositing it on the piece of indicator paper. The pH value was checked by comparing the color of the standard chart.

[0150] Enzyme activity

[0151] Laccase and peroxidase activities were quantified spectrophotometrically (BioTek EPOCH2 microplate reader, Agilient technology, CA, USA) at 415 nm. This allowed to detect the oxidation of 2,20-azino-bis(3-ethylbenzothiazoline- 6-sulfonate) (ABTS). For laccase activity, 100 pL of sample were mixed with a solution containing 900 pL of 10 mM ABTS and 0.2 M sodium acetate pH 5.0. For peroxidase activity, 100 pL of sample were mixed with a solution containing 800 pL of lOmM ABTS and 100M sodium acetate pH 5.0 and 100 pL of 20 mM H2O2. These measurements were done twice.

[0152] Through a systematic screening process, the initial list of species was narrowed down to the top eleven candidates. The top fungi candidates were selected based on rubber aggregation and enzymatic activity: Hypholoma fasciculare; Pleurotus ostreatus; Chaetomium cuniculare; Meripilus giganteus; Dactylonectria macrodidyma; Trametes hirsuta; Ganoderma sinense; Purpureocillium lilacinum; Cladosporium clodosporioides; Rhodotorula mucilaginosa and Pseudomonas putida.

[0153] Desulphurization test

[0154] To evaluate the desulphurization capabilities of the selected fungal strains, a standard basal salt medium supplemented with dibenzothiophene (DBT) as the sole sulfur source was utilized. Rubber crumb was not included in this cultivation medium. Cultivation medium consisted of the following components: KH2PO4 - 2.44 g / L, Na2HPO4 - 5.47 g / L, NH4CI - 2 g / L, MgCl26H2O - 0.2 g / L, CaCl22H2O - 0.001 g / L, FeCl36H2O - 0.001 g / L, MnCl24H2O - 0.004 g / L, and glycerol - 1.84 g / L. A total of 100 ml of the medium was transferred to 250 mlflasks and sterilized at 121°C for 20 minutes. The solution of DBT in ethanol was added to the sterilized medium, the concentration of DBT in the medium was 0.5 mM.

[0155] A 5 mL sample of the medium was obtained from the fungal cultures using a sterile syringe. The samples were then filtered through a syringe filter with a pore size of 0.45 pm. The volume of 4,000 mL of liquid was transferred into a Polystyrene (PS) container and frozen in liquid nitrogen. The samples were then lyophilized at -50 °C in vacuum overnight. Solid samples were weighed to the accuracy of 0.001 mg and analyzed by CHNS analyzer.

[0156] FIG. 2 shows the sulfur concentration in the medium after cultivation of fungi and bacterium, Rhodococcus erythropolis.

[0157] This desulphurization analysis aimed to determine the efficiency of the selected fungal strains in the removal of sulfur compounds, providing insights into their potential for degradation. Results are shown in FIG. 2. Two fungal species, Pleurotus ostreatus and Purpureocillium lilacinum, emerged as the most promising candidates. These species exhibited robust growth in the presence of rubber and demonstrated significant enzymatic activity relevant to rubber degradation.

[0158] Based on their ability to breakdown sulfur in the samples, a new experiment was designed involving Pleurotus ostreatus and Purpureocillium lilacinum. Enzymatic tests were performed to select bacteria with high enzymatic activity or the ability to produce PHA / PHBs. Through enzymatic tests, bacteria were selected. Combining the action of bacteria with fungi in consortia. These examples demonstrate the tests conducted to assess these interactions.

[0159] Enzymatic tests

[0160] Half of the repetitions for each species were inoculated with the bacterium Rhodococcus erythropolis, can produce polyhydroxy alkanes and / or polyhydroxyalkanoates during cultivation with rubber crumb, while the other half were inoculated with the bacterium Cupriavidus basilensis, another bacteria that would not inhibit the growth of the studied fungi. These bacteria were chosen due to their compatibility with fungal growth and their ability to catalyze enzymatic reactions with the assistance of iron oxide nanoparticles (Fe4O2 NPs).

[0161] Both bacteria were decorated with Fe4O2 NPs (nanoparticles) to catalyze the enzymatic reactions. The Fe4O2 NPs were prepared in our laboratory using cultures of Chlorella. The experimental setup explored the interactions and effects of different microorganisms on rubber degradation, considering the presence of specific bacteria and the catalytic properties of the Fe4O2 NPs.

[0162] The interactions between selected fungi and bacteria introduced into the same cultures are depicted in FIGs. 4A-C. These results provided valuable insights into the interactions between fungi and bacteria within the selected cultures, particularly in the case of Cupriavidus basi lie rise.jwhich utilizes a different carbon source to produce PHA / PHBs. No negative effects were observed between the bacteria and fungi, indicating potential synergistic interactions.

[0163] Pretreatment

[0164] Additionally, each species was replicated six times, and two sets of experiments were conducted using pure rubber and rubber treated with Sono-Fenton. In this case, the pre-cultures, which were cultivated on a standard medium with the addition of 0.3 g / L to stimulate growth in small spheres, were added to the medium containing rubber crumb. This experiment aimed to investigate rubber decomposition using a combination of bacteria and fungi in consortia and various pre-treatment methods.

[0165] Three pre-treatment methods were applied to modify the rubber structure and enhance its susceptibility to microbial degradation and other subsequent processes in the study, including ozonification, sonication, and Sono-Fenton reaction.

[0166] Ozonification involves the treatment of rubber with ozone gas, which can lead to the oxidation and degradation of rubber molecules. To test this pretreatment condition, a portion of dry rubber samples were subjected to ozonification for 20 minutes.

[0167] Sonication is a process that uses high-frequency sound waves to agitate the rubber particles. This mechanical agitation can help in breaking down the rubber structure and improving its accessibility for further processing. To test pretreatment by sonication, rubber samples underwent sonication alone for 10 minutes.

[0168] The Sono-Fenton reaction generates hydroxyl radicals, highly reactive species that can contribute to the degradation and transformation of rubber compounds. This process involves the combination of titanium dioxide (TiO2) catalyst, hydrogen peroxide (H2O2), and ultrasound waves. To test pretreatment by Sono-Fenton reaction, rubber samples were treated using the Sono-Fenton reaction for 1.5 hours.

[0169] Ozonification and sonication did not produce visible changes in the rubber samples, while Sono-Fenton treatment resulted in increased elasticity and a grey appearance. Simple sonication equipment was used in the experiment, suggesting the potential for improved results with professional sonicators.

[0170] Rubber samples were obtained directly from the Falcon tubes after overnight drying at 40°C. ATR-FTIR spectroscopy, performed using a Thermo-spectrometer, was used to analyze the samples. FIG. 3 displays two spectra: one for the original rubber and another for the rubber treated with sonication for ten minutes. A third spectrum represents rubber in the standard solution. The sonicated sample spectrum exhibited a new band at approximately 960 cmA(-l), likely associated with out-of-plane trans(-CH=CH-) butadiene or C-H wagging butadiene vibrations. The band at 907 cmA(-l) indicated out-of-plane bending vibrations of (C-H) vinyl groups, while the band at 1075 cmA(-l) suggested (C-S-C) stretching vibrations. Other observed bands were characteristic of hydroxyl, aliphatic, and other groups commonly found in organic compounds.

[0171] Scanning Electron Microscopy (SEM)

[0172] Preliminary data were obtained using scanning electron microscopy (SEM) to examine the growth of fungi within rubber crumb when rubber served as the sole carbon source in the medium. After a growth period of two months, rubber samples were collected and prepared for analysis. FIG. 5 illustrates the non-colonized rubber samples subjected to different pretreatment methods. Notably, the Sono-Fenton reaction resulted in pronounced changes, causing the rubber to exhibit a strongly polygonal morphology. Simple sonication also showed some effects on the rubber material. These observations provide initial insights into the structural modifications induced by the pre-treatment methods.

[0173] FIGs. 6-9 show photomicrographs from SEM analysis conducted on rubber crumb samples collected after two months of growth. The samples were pretreated using different methods, including sonication and Sono-Fenton reaction. SEM images revealed significant effects on the rubber crumb, particularly with the Sono-Fenton treatment, which resulted in a polygonal appearance. Simple sonication also had noticeable effects.

[0174] Latex Comparison

[0175] In addition to the investigation on SBR, an older sample of a blue latex glove that had been stored first in liquid and then on a water agar surface was analyzed. This experiment was conducted several months before the sample had not been analyzed with SEM. Because the appearance of black structures on the surface of the material after approximately three months was observed, which were not visible during the liquid culture phase. Although the latex glove is not made from turf grass rubber crumb, this sample can help explain the fungal growth observed in the previous figures.

[0176] Interestingly, the rubber glove was initially colonized by mycelium growing within the thin layer of latex. Subsequently, yeast cells began to develop primarily inside the latex layer (FIG. 25). There are also distinct mycelial structures visible, suggesting the presence of two different fungi (the latex was not sterilized). The growth of mycelium and yeast within the thin glove layer is surprising, but similar patterns were observed in the growth of fungi in SBR. In the case of rubber crumb, fungi primarily developed inside the crumb, and the presence of mycelium outside the crumb was mainly observed in samples of Pleurotus ostreatus during liquid culture. In other samples, the external mycelium became visible much later. This indicates that the process of fungal growth may remain invisible to us for a long time, and the growth of mycelium outside the material may not be a reliable indicator when estimating fungal growth rates. We may need to consider alternative measurements, such as enzymatic evaluation or ergosterol estimation, although the presence of rubber in the samples could pose challenges in such assessments.

[0177] Example 2. Degrading PAHs and phthalates in rubber crumb with Pleurotus ostreatus and Purpureocillium lilacinum.

[0178] Rubber samples were soaked in alcohol for 24 hours and rinsed and sterile water. 10 g samples of rubber were placed in sterilized 250 ml Erlenmayer flasks, flooded with 100 ml of MSM-F medium containing: NaNO33 g / 1, K2HPO41 g / 1, MgSO4x7H2O 0.5 g / 1, KC1 0.5 g / 1, 50 pM CuSO4. Pleurotus ostreatus or Purpureocillum lilacinum (approx. 0.15 g from liquid cultures with the addition of 0.3% agar), 3 ml of Rhodotorula mucilaginosa yeast (sonicated for 10 minutes, as a source of carotenoids) and a large loop transfer of bacteria from the genus Cupriavidus or Rhodococcus were added.

[0179] The co-culture experiments were run in six repetitions. Half of the rubber samples were subjected to the Sono-Fenton process with the addition of Fe, prior to co-culture colonization. After three weeks of incubation at 28°C and shaking at 120 rpm, rubber samples were collected from fungal culture flasks. Then, the samples were washed with distilled water, placed in a polystyrene container, and dried at 40°C for 24 hours. After drying, samples were weighed with analytical precision.

[0180] Next, the samples were immersed in 5 mL toluene and then heated. The hot extraction was carried out for 30 min. After reducing the solvent volume, the toluene (with analytes) was transferred entirely to a volumetric flask. The sample was washed with a fresh portion of toluene added to the first portion. The solution was made up to the mark with toluene. Thesamples were analyzed by GC-MS PerkinElmer Clarus 600 with Mass Spectrometer. Standards from Merck were used to identify individual substances and to evaluate their concentrations. The data were analyzed using the STATISTICA program parametric ANOVA methods after checking for normal distribution and homogeneity of variances.

[0181] The ability of both fungi and bacteria to grow together and to degrade both PAH and phthalates was confirmed. The level of degradation appears dependent on fungi and bacteria type. The growth of Purpur eocillium seems to be less affected in presence of studied substances and thus seems to be more effective in destroying PAH and phtalates. At the same time, in the case of DBPH (Dibutyl phthalate) and DMPH (dimethyl phthalate), there were no significant differences between the concentration of these substances in the control rubber samples (without the addition of microorganisms) and the samples treated with fungi and bacteria, but both cases concern substances present in rubber in very low concentrations (up to 250 ppb). Concerning preparation of the material for fungal growth, SonoFenton was not effective, but sonication probably made the substances to be more available in solution due to maceration.

[0182] Individual Compounds of Interest (PAH and Phthalates)

[0183] Individual treatments of 100 ml Erlenmeyer flasks filled with 50 ml of MSM-F medium containing NaNO3, K2HPO4, MgSO4x7H2O, and KC1 were prepared. The flasks were sealed with sponge stoppers and autoclaved under standard conditions. After cooling, individual PAHs and phthalates at a concentration of approximately 0.0050 mg / L were added to each flask, except for the control without live microorganisms. Fungal inoculum was prepared in PDB liquid cultures with 0.3% agar. The fungal mycelium, in the form of pellets, was rinsed with sterile water prior to application. The cultivation was carried out for ten days at 28°C with shaking at 120 rpm in light-free chambers. After the culture period, the mycelium was removed from the flasks and placed in pre-weighed Eppendorf tubes. The mycelia were then subjected to liquid nitrogen, lyophilized, and weighed. Control samples and solutions obtained from the fungal cultures were analyzed using the GC-MS PerkinElmer system to assess the presence and concentrations of PHAs and phthalates.

[0184] Both fungal species displayed visible growth, particularly on the flask walls. The dark color of the mycelium indicated successful rubber breakdown, with contributions from both sonication and mycelial activity. P. ostreatus, mycelium was seen in aggregates, while P. lilacinum grew in a strongly tearing / jagged form. P. lilacinum, in most cases, showed statistically higher biomass after ten days of growth in the presence of the tested substancesthan in control. It appears both species show a different development strategy in the studied substrate.

[0185] Gas chromatography analysis was performed, and statistical analysis was conducted solely on samples subjected to sonication, as samples treated with Sono-Fenton exhibited substantial standard deviations and were excluded. Sono-Fenton treatment resulted in the release of some PAHs and phthalates from the rubber, potentially compromising fungal growth in extreme cases.

[0186] The statistical analysis (FIGs. 10-19B) revealed that, among the analyzed substances, only DBPH (Dibutyl phthalate) and DMPH (dimethyl phthalate) showed no significant differences in concentration between the control rubber samples (without microorganisms) and the samples treated with fungi and bacteria. It is important to note that these substances were present in the rubber at very low concentrations (up to 250 ppb). With the exception of DNOP, both Pleurotus ostreatus and Purpureocillium lilacinum demonstrated a reduction in the concentration of toxic substances in the solutions.

[0187] For most of the analyzed substances, there were no significant differences in concentrations between the different fungal and bacterial treatments. In cases where differences were observed (between bacterial species), no statistically significant interactions between fungi and bacteria could be confirmed. However, by converting the loss of the tested substances into fungal biomass, it was possible to evaluate the effectiveness of each species. See FIGs 22- 24. P. lilacinum exhibited greater efficacy in degrading chrysene, while P. ostreatus was more effective in degrading fluoranthene. The remaining substances were degraded to a similar extent by both fungal species

[0188] Fungi incorporated the rubber into their growth matrix and exhibited degradation through enzymatic catalysis, confirmed by microscopic analysis of hyphal strands. Macroscopically, it appeared that P. lilacinum had greater growth than P. ostreatus, although biomass measurements revealed a different outcome. P. lilacinum displayed a tendency to produce more polysaccharides on the mycelium surface, enabling it to explore the substrate more efficiently. Therefore, both species exhibited distinct development strategies within the studied substrate. In most cases, P. lilacinum exhibited statistically higher biomass after ten days of growth in the presence of the test substance compared to the control. In the case of P. ostreatus, the differences were less clear due to larger standard deviations. These findings confirm the divergence in growth strategies between the studied species. The increase inmycelial biomass in the presence of PHA and phthalates indicates the ability of the fungi to break down these substances.

[0189] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0190] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMS1. A method for producing a biodegradable byproduct, the method comprising: obtaining a waste material, wherein at least a portion of the waste material comprises synthetic rubber; preparing a growth medium, wherein at least a portion of the growth medium comprises the waste material; incubating the growth medium with at least one bioremediating composition, wherein the at least one bioremediating composition comprises at least one fungal culture and at least one bacterium culture.

2. The method of claim 1, wherein the waste material in the growth medium consists essentially of synthetic rubber.

3. The method of claim 1 or claim 2, wherein a portion of the waste material comprises rubber crumb.

4. The method of any one of claims 1 to 3, further comprising reducing toxicity of the waste material.

5. The method of any one of claims 1 to 4, further comprising degrading polycyclic aromatic hydrocarbons and phthalates in the waste material.

6. The method of any one of claims 1 to 5, wherein the at least one bioremediating composition is adapted to break C-S bonds and / or carry out desulfurization.

7. The method of any one of claims 1 to 6, wherein the waste material comprises the main source of carbon for fungi growth.

8. The method of any one of claims 1 to 7, wherein the waste material does not include soil.

9. The method of any one of claims 1 to 8, wherein the waste material comprises benzo[a]anthracene and the method further comprises reducing the benzo[a]anthracene in the waste material.

10. The method of any one of claims 1 to 9, wherein the waste material comprises Butylbenzyl Phthalate and the method further comprises reducing the Butylbenzyl Phthalate in the waste material.

11. The method of any one of claims 1 to 10, wherein the waste material comprises Di-n- octyl Phthalate and the method further comprises reducing the Di-n-octyl Phthalate in the waste material.

12. The method of any one of claims 1 to 11, wherein the waste material comprises Bis(2- butoxy ethyl) Phthalate and the method further comprises reducing the Bis(2 -butoxy ethyl) Phthalate in the waste material.

13. The method of any one of claims 1 to 12, wherein the waste material comprises Chrysene and the method further comprises reducing the Chrysene in the waste material.

14. The method of any one of claims 1 to 13, wherein the waste material comprises Pyrene and the method further comprises reducing the Pyrene in the waste material.

15. The method of any one of claims 1 to 14, wherein the waste material comprises Diethyl Phthalate and the method further comprises reducing the Diethyl Phthalate in the waste material.

16. The method of any one of claims 1 to 15, wherein the waste material comprises Fluoranthene and the method further comprises reducing the Fluoranthene in the waste material.

17. The method of any one of claims 1 to 15, wherein the waste material comprises Phenanthrene and the method further comprises reducing the Phenanthrene in the waste material.

18. The method of any one of claims 1 to 17, further comprising sterilizing the waste material.

19. The method of any one of claims 1 to 18, wherein the at least one strain of bacteria produces enzymes for desulphurization of sulfur containing contaminants in synthetic rubber, wherein the sulfur containing content comprises Dibenzothiophene.

20. The method of any one of claims 1 to 19, wherein the growth medium comprises Modified Saline Medium.

21. The method of any one of claims 1 to 20, wherein the at least one strain of bacteria comprises Rhodococcus erythropolis.

22. The method of any one of claims 1 to 21, wherein the at least one strain of bacteria comprises Cupriavidus basilensis.

23. The method of any one of claims 1 to 22, further comprising catalyzing the enzymatic reactions with nanoparticles prepared using cultures of Chlorella.

24. The method of any one of claims 1 to 23, further comprising culturing in a humidity controlled environment for more than about one month, alternatively more than about two months, or alternatively more than about three months.

25. The method of any one of claims 1 to 24, further comprising a sonication pretreatment.

26. A bioremediating composition comprising: a consortial culture comprising at least one fungal culture and at least one bacterium culture; and a growth medium comprising a waste material, wherein at least a portion of the waste material comprises synthetic rubber.

27. The composition of claim 26 wherein the portion of the waste material comprising synthetic rubber comprises rubber crumb.

28. The composition of claims 26 or 27 wherein mycelium growth penetrates the synthetic rubber, forms aggregates with synthetic rubber, and / or adheres to the synthetic rubber.

29. The composition of any one of claims 26 to 28, wherein the growth medium consists essentially of synthetic rubber.

30. The composition of any one of claims 26 to 29, wherein the growth medium does not compromise soil.

31. The composition of any one of claims 26 to 30, wherein the fungi and bacteria of the composition exhibit high enzyme activity and growth together.

32. The composition of any one of claims 26 to 31, wherein the at least one fungal culture comprises:Pleurotus ostreatus, Purpureocillium lilacinum, Rhodotorula mucilaginosa yeast, Meripilus giganteus, Trametes hirsute, Hypholoma fasciculare, Chaetomium cuniculare, Dactylonectria macrodidyma, Ganoderma sinense, Cladosporium clodosporioides, Rhodotorula mucilaginosa, Acremonium or Pseudomonas putida,' and the at least one bacterium culture comprises:Cupriavidus basilensis or Rhodococcus erythropolis.

33. The composition of any one of claims 26 to 32, wherein the composition is configured for degradation of sulfur compounds and / or breakdowns sulfur cross-linkages when the growth medium comprises a source of sulfur.

34. The composition of any one of claims 26 to 33, wherein the bacterium culture metabolizes polycyclic aromatic hydrocarbons during cultivation with the rubber crumb.

35. The composition of any one of claims 26 to 34, wherein the composition accelerates biological degradation of the waste material.

36. The composition of any one of claims 26 to 35, wherein the composition renders the waste material less toxic.

37. The composition of any one of claims 26 to 36, wherein the composition renders the waste material biodegradable.

38. The composition of any one of claims 26 to 37, wherein the composition wherein the portion of the waste material comprising synthetic rubber is pretreated by sonication.

39. The composition of any one of claims 26 to 38, wherein the composition further comprises Modified Saline Medium.

40. The composition of any one of claims 26 to 39, wherein the growth media has a pH range from about 4 to about 10, alternatively a pH range from about 5 to about 9, alternatively a pH range from about 6 to about 8, or alternatively a pH range from about 7 to about 7.5.

41. The composition of any one of claims 26 to 40, wherein the composition stored in a controlled environment wherein the temperature is held between about 20°C to about 35°C.

42. The composition of any one of claims 26 to 41, wherein the composition produces polyhydroxy alkanes.

43. A biodegradable byproduct produced from a method of bioremediation comprising: providing a growth medium, wherein at least a portion of the growth medium comprises a waste material, wherein the waste material comprises rubber crumb; inoculating the growth medium with a bioremediating composition comprising at least one strain of fungi, wherein the at least one strain of fungi adheres or penetrates the rubber crumb; and at least one strain of bacteria, wherein the at least one strain of bacteria produces a biofilm; and, the at least one strain of fungi and the at least one strain of bacteria form a co-culture.

44. A method of mitigating climate change by reducing and / or preventing additional greenhouse gas emissions and / or monitoring, tracking, and / or verifying greenhouse gas emission reductions, and / or reducing water contamination and reuse of waste materials, the method comprising:providing a media, wherein at least a portion of the media comprises a waste material, wherein the waste material comprises rubber crumb; inoculating the media with at least one strain of fungi; and, inoculating the media with at least one strain of bacteria.

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