WO2016209721A1

WO2016209721A1 - Biomolecular zonal compositions and methods for in-situ biological exfoliation of contaminated surfaces

Info

Publication number: WO2016209721A1
Application number: PCT/US2016/037993
Authority: WO
Inventors: Bryan Sims; Ray EAST
Original assignee: Bryan Sims; East Ray
Priority date: 2015-06-22
Filing date: 2016-06-17
Publication date: 2016-12-29
Also published as: EP3310505A1; AU2016283997A1; EP3310505A4; CA2990164A1

Abstract

A microbial mat is provided, including a contact layer; a core layer; a matrix of cellulose fibers coated with a liquid nutrient mix; and a carrying sheet; wherein the core layer comprises the matrix of cellulose fibers coated with a liquid nutrient mix, attached to the carrying sheet and coated with a dry nutrient mix; wherein the liquid nutrient mix and dry nutrient mix are composed of ingredients from the group consisting of Sodium Citrate, Ammonium Sulphate, Monosodium phosphate, Dipotassium Phosphate, Magnesium Sulphate, Eprom Salt, and Ferrous Sulphate; and a top layer, wherein the top layer is attached to the entire surface of the core layer.

Description

BIOMOLECULAR ZONAL COMPOSITIONS AND METHODS FOR

IN-SITU BIOLOGICAL EXFOLIATION OF CONTAMINATED SURFACES

FIELD OF THE INVENTION

This invention relates to compositions and methods for bioremediation of horizontal, sloping and vertical surfaces. The compositions comprise Biospheres and Miomats or Microbial Mats (if preferred) that combine to allow for contaminated surfaces to be cleaned in-situ by biological exfoliation. BACKGROUND

Bioremediation has emerged as a promising technology for the treatment of soil and groundwater contamination. Some conventional bioremediation approaches require the soil to be excavated for treatment either off site or ex-situ. Disadvantages of these approaches include disruption of the natural field and the need to transport large quantities of contaminated soil.

It would be beneficial to establish a predictable, cost effective bioremediation system in-situ in the field without the need for transporting the contaminated soil or water. It would also be beneficial to establish a bioremediation system in-situ in the field capable of cleaning contaminated surfaces without the use of harmful chemicals or the high-cost necessity to shut down operating systems during the treatment process. Methods for bioremediation in the field can use certain bacteria which digest and neutralize contaminants. Often, these bacteria are provided as a liquid culture and, thus, can be characterised as planktonic and freely existing in bulk solution. In these methods, water is used as a carrier to deliver bacteria and/or nutrients to the treatment area in the field. However, utilizing water as a medium to deliver and distribute bacteria is associated with various problems. Bacteria require moisture. However, simple liquid or water cultures in-situ cannot maintain adequate levels of moisture because water tends to evaporate and migrate. This causes massive losses in potential microbial activity. Hence, establishing and sustaining sufficiently large microbial populations at the contamination site becomes problematic. Bacteria obtain from their environment all nutrient materials necessary for their metabolic processes and cell reproduction. The food must be in solution and must pass into the cell. This is especially difficult when treating surface contamination in- situ due to high levels of toxicity being present at the start of a treatment and the lack of food that is inevitable towards the end of the process. Further, aerobes need oxygen for respiration and cannot grow unless oxygen is provided. Additionally, bacteria have a pH range within which growth is possible. Although the optimum pH value differs between species, an environment that is maintained to a neutral pH will best sustain most bacterial species utilized for in-situ bioremediation.

Successful bioremediation requires optimizing biomass in-situ, as this represents the total amount of suitable bacteria present in a given area or volume that will have the potential to metabolize and break down the contamination in order to remediate the targeted area of pollution.

The fate of in-situ bioremediation is generally considered to be uncertain when utilizing water as a medium to distribute bacteria because water is fluid and, thus, it is difficult to localize the distribution and delivery to one targeted area. The process may become wasteful and massive amounts of bacterial inoculant may be lost through natural migration. Moreover, much of the bacteria often misses the targeted pollution entirely, as the liquid culture passes across the surface too quickly to allow the formation of molecular bonds that are essential to both establishing and sustaining an effective process of biodegradation. Thus, there remains the need in the field for compositions and methods of delivering bacteria and other microorganisms in-situ for bioremediation of land, water, horizontal, sloping and vertical surfaces.

SUMMARY

At least some of these needs are addressed by remediation compositions and methods provided in this disclosure and are suitable for treatments in-situ. One embodiment provides compositions and methods for natural biodegradation of organic waste. Further embodiments provide compositions and methods for decontamination of soil, hard surfaces and construction materials such as bricks, concrete, gravel and stone masonry in the field.

Other embodiments provide compositions and methods for decontamination of water in the field, such as for example, ocean water, ground water and rivers.

Some embodiments provide compositions and methods for the bioremediation of horizontal, sloping and vertical surfaces. Further embodiments provide compositions and methods for contaminated surfaces to be cleaned in-situ by biological exfoliation.

One of the advantages of these compositions and methods is the reduction in number of in-situ applications needed in comparison to conventional compositions and methods.

Further embodiments provide Microbial Mats and methods for treating the surfaces in-situ when conditions are predicted to be hot. In these applications, the top layer would be a perforated heat reflective membrane to aid cooling across the surface being treated. The cellulosic nutrient rich core or middle layer would range between 100 gsm and 300 gsm and have a water absorbent capacity of between 4 to 20 times the materials own weight to aid moisture levels and the stabilizing bottom 'contact' sheet would be phobic to aid attachment as microorganisms have been found to attach more rapidly to hydrophobic and non-polar surfaces than hydrophilic surfaces. The production process, combined with the fibres selected, provide 'spaces and pathways' to further aid microbial attachment as the degree of colonization of the Microbial Mat's contact surfaces have been found to increase with surface roughness. This is due to the physical 'valleys' present that can allow the microbes to occupy protected areas with reduced shear forces whilst the surface roughness also increases the surface area available for bacterial attachment.

Further embodiments provide Microbial Mats and methods for treating the surfaces in-situ when conditions are predicted to be cold. In these applications, the top layer would be a black thermal insulating membrane to reduce heat loss across the surface being treated. The cellulosic nutrient rich core or middle layer would range between 300 gsm and 400 gsm to provide additional insulation and have a water absorbent capacity of between 4 to 20 times the materials own weight to control moisture levels. The stabilizing bottom 'contact' sheet would be phobic to aid attachment as microorganisms have been found to attach more rapidly to hydrophobic and non-polar surfaces than hydrophilic surfaces. The production process, combined with the fibres selected, provide 'spaces and pathways' to further aid microbial attachment as the degree of colonization of the Microbial Mat's contact surfaces have been found to increase with surface roughness. This is due to the physical 'valleys' present that can allow the microbes to occupy protected areas with reduced shear forces whilst the surface roughness also increases the surface area available for bacterial attachment.

In one embodiment, a microbial mat is provided, including a contact layer; a core layer; a matrix of cellulose fibers coated with a liquid nutrient mix; and a carrying sheet; wherein the core layer comprises the matrix of cellulose fibers coated with a liquid nutrient mix, attached to the carrying sheet and coated with a dry nutrient mix; wherein the liquid nutrient mix and dry nutrient mix are composed of ingredients from the group consisting of Sodium Citrate, Ammonium Sulphate, Monosodium phosphate, Dipotassium Phosphate, Magnesium Sulphate, Eprom Salt, and Ferrous Sulphate; and a top layer, wherein the top layer is attached to the entire surface of the core layer.

In an embodiment, the top layer is a polymer sheet between 100 and 500 gsm. It is contemplated that the core layer is between 100 and 400 gsm, with a water absorbent capacity between 4 to 20 times the materials weight. In an embodiment, the contact layer is hydrophobic material. In an embodiment, the contact layer is hydrophilic material. In an embodiment, the top layer is a perforated, heat reflective membrane. In an embodiment, the top layer is a thermal insulating membrane. A method is disclosed for in-situ biological exfoliation of contaminated surfaces using a microbial mat, including: applying a biosphere blend to the contaminated surface; waiting for a predetermined time; and covering the contaminated surface with a microbial mat, wherein the microbial mat comprises a contact layer, a core layer, and a top layer. In an embodiment, the method further contemplates waiting a predetermined time; removing the microbial mat; washing the contaminated surface with a recharging biological blend; and assessing treatment of contaminated surface. In an embodiment, the method further includes waiting a predetermined time;

removing the microbial mat; washing the contaminated surface with a recharging biological blend; reapplying biosphere blend; and covering the contaminated surface with a microbial mat. In an embodiment, the method further includes waiting a predetermined time; removing the microbial mat; washing the contaminated surface with a recharging biological blend; drying the contaminated surface for a

predetermined time; reapplying the biosphere blend; and covering the contaminated surface with a microbial mat.

A composition is provided including comprising biospheres and bacteria, wherein the biospheres are cellulosic biopolymers and sized in the range from 500 nanometers to 80 microns and have a minimum free swell absorption capacity of 400 times by weight and a maximum free swell absorption capacity of 1200 times by weight.

In an embodiment, the biospheres gradually release moisture. In an embodiment, the viscosity of the liquid composition is from 6 centipoise (cps) to 217 centipoise (cps). In an embodiment, the viscosity of the liquid composition is from 217 centipoise (cps) to 1 , 159 centipoise (cps). In an embodiment, the viscosity of the liquid composition is from 1 ,236 centipoise (cps) to 5,021 centipoise. In an embodiment, the viscosity of the liquid composition is from 5,021 centipoise (cps) to 47,31 1 centipoise (cps).

A method is provided for bioremediation in situ, the method comprising: preparing a blend of liquid bacterial culture with biospheres, wherein the biospheres are cellulosic biopolymers and sized in the range from 500 nanometers to 80 microns; and have a minimum free swell absorption capacity of 400 times by weight and a maximum free swell absorption capacity of 1200 times by weight; applying the blend at a site in need of bioremediation; and letting the blend to form a gelatinous matrix.

A method is provided for delivering and/or hosting biological and/or chemical reagents in a gelatinous matrix, the method comprising: obtaining biospheres which are cellulosic biopolymers and sized in the range from 500 nanometers to 80 microns and have a minimum free swell absorption capacity of 400 times by weight and a maximum free swell absorption capacity of 1200 times by weight; mixing the biospheres with a bacterial and/or chemical; and letting the mixture to form a gelatinous matrix.

BRIEF DESCRIPTION OF THE DRAWINGS FIGURE. 1 reports results from treatment with average hot spot concentrations (> 5,000 mg/kg);

FIGURE 2 reports volumetric projections; and

FIGURE 3 reports results of a CUP analysis after 14 and 28 weeks. FIGURE 4 reports results of CFU/CUP analysis over 90 days - Conventional Blend v Novel Blend with Biospheres & Microbial Mats.

FIGURE 5 is a schematic vertical cross-section of the present microbial mat. DETAILED DESCRIPTION

Referring now to FIGs. 1-5, suitable bacteria include, but are not limited to, naturally occurring bacteria and genetically engineered bacteria. Some suitable bacteria include those that produce at least one enzyme that can be used for biodegradation of organic waste. A person of skill will further appreciate that in addition to bacteria, other microorganisms, such as for example algae, can be suitable in certain embodiments.

One embodiment provides a composition that transforms water from being a simple carrier into a host environment where microbial activity can thrive. The composition creates an organic gelatinous matrix and is well suited for delivering, sustaining and containing microorganisms in situ at a contamination site. The network is easy to manage and localize to a contamination zone in part because it has a very slow migratory rate and will remain in place at the contamination site long enough for bacteria to digest and clean the organic waste. One embodiment provides a blend that transforms liquid microbial culture, which can be characterised as planktonic cells freely existing in bulk solution, into continuous gelatinous superstructures that can act as the bacteria's essential foundation for life as they store key elements such as carbon, hydrogen, oxygen and nitrogen. The resulting architecture being a highly complex matrix that forms the structure and acts like a biofilm to mediate the attachment of cells to different substrata and provide protection against various environmental stresses and dehydration. In some embodiments, the blend is mixed with absorbent cellulosic biopolymers that range in size from between 500 nanometers to 80 microns. In addition to forming a cellular, rather than crystalline matrix when hydrated, these tiny particles, which are referred to in this specification as biospheres, must have a minimum free swell absorption capacity of 400 times by weight and a maximum of 1200 times.

This range is critical in terms of achieving the correct carbon balance for each gelatinous mixture. This delicate balance is caused by the necessity to provide sufficient levels of carbon as a food source to sustain optimized levels of microbial activity, but not overloading the mixture with carbon to the point where it becomes possible for the genome, in the bacteria, to adapt towards favoring food that is easier to digest and, as a consequence, encouraging the microbial process to switch off from the food source being targeted, which, of course, is the contamination.

In some embodiments, the blend carries biospheres that act like tiny building blocks in the ground to supplement the soil's retentive processes and its ability to redistribute various essential elements. This optimizes the life support system that represents the host environment within the land or contaminated water source, and, thus, the blend can significantly increase a specific biomass and the potential for biodegradation wherever it is needed within the profile of the soil or contaminated water source.

The biospheres that form the basic molecular structure within the blend have the ability to release moisture. Primarily, this action facilitates a process of slow release for a range of critical life supporting constituents which are rapidly lost when applying conventional liquid cultures, and, significantly, the biospheres, that remain, can be recharged by either simple human intervention or, in a number of scenarios, remotely through nothing more than rehydration by capturing the rain. It should be noted that conventional synthetic super absorbent polymers are much less suitable for the production of these biological blends. They would form molecular structures that would be inimical, rather than optimizing to the microbial process. Further embodiments provide a method that creates an adaptable living gelatinous matrix by transforming water into a sustainable host microenvironment that optimizes the process of biodegradation and represents a major difference and advance over conventional bioremediation using liquid cultures. In further embodiments, the blend with biospheres can be used with water and a broad range of biological and chemical reagents with scope for application on any scale. The blend assists organic molecules to dissolve, mix and interact with bacteria to optimize the process of predictable in-situ bioremediation, notwithstanding that the number of treatments are minimized - even in scenarios where no potential for biological activity exists.

The blends with biospheres can be devised and engineered so that they suit specific applications. Hence, selecting the most appropriate particle size, when producing site specific blends with biospheres, represents an important part of this technology. Typically, the size of particles utilized in the blends fall into five main categories, as discussed in more detail below.

Some embodiments provide blends and methods for treating the soil surface in-situ. In these applications, the dynamic viscosity is level 1 and biospheres are greater in size than 500 nanometers to avoid excessive reactivity that, due to their very large surface area to volume ratio, can cause agglomeration in the soil, but, equally important, is that they are less than 5 microns to ensure the particles are not filtered out as the blend migrates through the soil. The viscosities of these blends are between Factors 3 and 6, dependent upon geology and contamination, wherein factor 3 equals 6 centipoise (cps) and factor 6 equals 217 cps.

Some other embodiments provide blends and methods for treatment of soil surfaces where the gelatinous matrix should have a higher viscosity which is valued as dynamic viscosity levels 1 and 2. In these blends and methods, biospheres are in the range between 10 and 30 microns to avoid deep penetration into the soil. The viscosity ranges of these blends are between Factor 6 & 14, dependent upon geology and contamination, wherein Factor 6 is equal to 217 cps and Factor 14 is equal to 1 , 159 cps.

Further blends and methods include those suitable for in-situ treatment of porous hard surfaces. In these blends, the dynamic viscosity levels are 1 and 2 and biospheres are in the range between 5 & 40 microns to achieve sufficient penetration and provide an adequate coating across the surface being treated to sustain an optimized level of microbial activity. The viscosity ranges of these biospheres are equal to Factors of between 6 and 18, dependent upon surface material and contamination and wherein Factor 6 is equal to 217 cps and Factor 18 is equal to 1 ,236 cps.

Further blends and methods are suitable for treatment of non-porous hard surfaces with dynamic viscosity Levels 2 and 3. In these blends, biospheres are in the range between 30 and 80 microns to provide an adequate coating across the surface being treated to sustain an optimized level of microbial activity. The viscosity ranges of these biospheres are equal to a Factor between 18 and 36, dependent upon surface material and contamination, wherein Factor 18 is equal to 1 ,236 cps and Factor 36 is equal to 5,021 cps. Dynamic Viscosity Level 3 is particularly suitable for treating heavy contamination where surfaces require high levels of moisture retention due to little or no on-site attendance.

Further embodiments provide blends and methods for in situ treatment of vertical surfaces with dynamic viscosity levels between 3 and 4. In these blends, biospheres are in the range between 40 and 80 microns to provide an adequate coating and attachment across the surface being treated to sustain an optimized level of microbial activity. The viscosity ranges of these biospheres are equal to a Factor of between 36 and 72, dependent upon surface material and contamination, wherein Factor 36 is equal to 5,021 cps and Factor 72 is equal to 47,31 1 cps. In some embodiments, the blends are applied to provide an optimizing microbial wrap or coating that interacts, in-situ, with surfaces that are saturated by a pretreatment utilizing either a symbiotic low viscosity blend with a Factor of 3 or liquid culture. Further embodiments provide blends and methods for in situ treatment of horizontal, sloping and vertical surfaces. These compositions comprise blends with biospheres and nutrient rich Microbial Mats (MMs) that combine to allow for contaminated surfaces to be cleaned rapidly in-situ by the novel process of biological exfoliation. These treatment categories demonstrate one of the advances presented by this technology over using simple liquid cultures: the ability of the blend with biospheres to adapt as an optimizing host environment to distribute selected bacteria in a form devised to suit specific treatment requirements based upon the type of geology and surfaces to be remediated, whilst also accounting for the weather and accessibility to the location requiring treatment. Even in highly problematic cases, where it would be impossible to treat using conventional liquid cultures, the blends with biospheres can be adapted to provide an optimized biological solution e.g. when pollution is located on vertical surfaces, such as brick walls, which can be affected by contamination, through subsurface migration, in an underground tunnel.

Referring now to FIGURE 5, another significant advance is the combined use of these dynamic viscosity blends with Microbial Mats, which are applied to the entire surface area immediately after being treated with the appropriate blend. This provides an additional nutrient rich top cover that further augments the biomass and, thus, ability of the resultant biofilm to naturally disperse, emulsify, metabolise and clean, in-situ, horizontal, sloping and vertical contaminated surfaces by the novel process of biological exfoliation. The nutrient rich Microbial Mats, like the blends with biospheres, can be devised and engineered so that they suit specific applications and treatment conditions. Hence, for the production of site specific Microbial Mats, the most appropriate fibers, top and bottom covers and nutrient content must be selected. Also, the level of hydrophobicity and the contact surface profile are important to the successful functionality of this technology. Typically, specifications to produce the Microbial Mats fall into four main categories, as they are modified to suit prevailing weather conditions during the treatment process. In some embodiments, the mat, generally designated 10, has a polymer top sheet 12, a core 14 and a carrying sheet 16. The core 14 of the Microbial Mat is a manufactured matrix of fine 0.5 to 8.0 denier and cellulose fibers with a fiber length ranging between 5mm to 60mm. These fibres are individualised before being coated with a liquid nutrient mix based upon the ingredients listed in Table 2. The treated fibres are then air laid to form a consistent web of between 100 gsm to 400 gsm (grams per square metre).

The fibrous web is then needled onto the polymer carrying sheet 16. The carrying sheet 16 preferably ranges between 12 gsm and 25 gsm. A dry nutrient mixture (See Table 2) is then mechanically distributed across the surface of the fibrous web.

Finally, dependant upon the application, the polymer top sheet 12 of between 100 gsm and 500 gsm is glued over the entire surface of the fibrous web 14 to seal and secure the dry nutrient mix to make this element an integral part of the Microbial Mat 10.

Width of the Microbial Mats can range between 10cm and 250cm and the length between 15cm and 40 metres dependent upon application. Some embodiments provide Microbial Mats and methods for treating surfaces in-situ when conditions are predicted to be dry. In these applications, the top layer would be hydrophilic to allow hydration across the surface without disturbing the treatment process. The cellulosic nutrient rich core or middle layer would range between 200 gsm and 400gsm and have a water absorbent capacity of between 4 to 20 times the materials own weight to aid moisture levels and the stabilizing bottom 'contact' sheet would be phobic to assist attachment as microorganisms have been found to attach more rapidly to hydrophobic and non-polar surfaces than hydrophilic surfaces. The production process, combined with the fibres selected, provide 'spaces and pathways' to further aid microbial attachment as the degree of colonization of the Microbial Mat's contact surfaces have been found to increase with surface roughness. This is due to the physical 'valleys' present that can allow the microbes to occupy protected areas with reduced shear forces whilst the surface roughness also increases the surface area available for bacterial attachment.

Some other embodiments provide Microbial Mats and methods for treating surfaces in-situ when conditions are predicted to be wet. In these applications, the top layer would be hydrophobic to act as a diffuser and, thus, prevent the mat from becoming saturated and inimical to the process. The cellulosic nutrient rich core or middle layer would range between 100 gsm and 300 gsm and have a water absorbent capacity of between 4 to 20 times the materials own weight to control moisture levels and the stabilizing bottom 'contact' sheet would be phobic to aid attachment as microorganisms have been found to attach more rapidly to hydrophobic and non- polar surfaces than hydrophilic surfaces. The production process, combined with the fibres selected, provide 'spaces and pathways' to further aid microbial attachment as the degree of colonization of the Microbial Mat's contact surfaces have been found to increase with surface roughness. This is due to the physical 'valleys' present that can allow the microbes to occupy protected areas with reduced shear forces, whilst the surface roughness also increases the surface area available for bacterial attachment.

In all these novel applications and treatment applications, both field and bench-scale studies, have demonstrated the ability of the blends with biospheres, when combined with the nutrient rich Microbial Mats, to enhance the process of biodegradation and allow for contaminated surfaces to be cleaned rapidly in-situ by biological exfoliation. As a result, many of the active properties that combine to produce these highly productive outcomes also distinguish the in-situ technology with biospheres and nutrient rich Microbial Mats from conventional bioremediation.

One particularly important distinction is the capacity to establish and sustain microbial activity at levels where potent micellar bio-surfactant solutions naturally occur and assist organic molecules to dissolve, mix and interact with the selected bacteria to simplify application and optimize the process of biodegradation.

These optimized, naturally occurring, environmentally safe biological catalysts disrupt the complex molecular chains of hydrocarbon based contaminants and this process, occurring within the gelatinous matrix created by the blend with biospheres, produces more easily digestible molecules that become encapsulated, together with the bacteria, in cores of nano-sized micelles that sustain favorable contact with the water that surrounds them and, thus, provide an ideal microenvironment for optimizing the reaction kinetics associated with successful in-situ biodegradation.

Therefore, the blend with biospheres, combined with nutrient rich Microbial Mats, provides an optimized biological cleaning process in which nano-sized micelles are created, while, typically, no micelles are usually created in a conventional bioremediation process with a conventional liquid bacterial culture. This distinction is important because of the following significant advantages, which become apparent, when comparing the key characteristics of using the blend with biospheres combined with nutrient rich Microbial Mats rather than a conventional liquid culture to perform in-situ biological treatments to clean surfaces.

Water's natural ability to freely migrate through the soil and flow across surfaces, is as essential for life as it is wasteful and impractical when being used as the primary vehicle for distribution of specific treatments to remediate the soil or biologically clean contaminated surfaces. Therefore, with no alternative to water for the distribution of specialist biodegrading bacteria, other than the way it comes out of a tap, in-situ bioremediation remains a highly unpredictable and intensive procedure. In complete contrast, the blend with biospheres augments water, and while it remains fluid and continuous, it also becomes a controllable life optimizing gelatinous host, which is organic with a cellular matrix to optimize the potential for biological life cycles to thrive.

Despite being a much less intensive process, the gelatinous matrix, which is rechargeable in-situ, is also a far more predictable inoculant than conventional liquid cultures. The blend with biospheres combines absorption, rapid capture and controlled release, within a microbial inoculant, which transforms the water element into an optimizing super carrier that can act as a biodegradable subterranean sink, to sustain moisture levels. The gelatinous matrix makes maintaining sufficient moisture and, consequently, in-situ remediation, much more efficient.

Further and in complete contrast to conventional liquid cultures, the blend with biospheres establishes a natural nutritious reservoir in the ground or across any surface when it is applied. This presents a major advance as the novel properties, within this gelatinous reservoir of rechargeable super absorption, rapid capture and slow release combine to help even out the unpredictability associated with microbial survival. The most critical stages being at the start of a project, due to contamination causing high levels of toxicity, and towards the end of the process, when a lack of food occurs as a result of the land or surface becoming clean again. Another significant advance, is the Microbial Mat, which is specifically designed to simulate the best possible strata from the bacteria to facilitate adhesion to this additional protective layer that also provides a nutrient rich surface. The Microbial Mat's novel properties augment the biomass and capacity of the resultant biofilm to disperse, emulsify, metabolise and ultimately, clean the contaminated surface by a rapid process of biological exfoliation.

Moreover, this groundbreaking invention, devised specifically for use with Biomolecular Zonal Compositions (BZC), utilises the extracellular polymeric substances, which embody the molecular architecture within the complex matrix of blends made with biospheres, to bring the three separate layers, which form the initial profile of the treatment area, together through encapsulation and molecular bonding to, ultimately, act as one and, thus, allow for contaminated surfaces to be cleaned in-situ by the novel process of biological exfoliation. The three separate layers being i) the contamination, ii) a coating of an appropriate Biomolecular Zonal Composition (BZC) i.e. a blend with biospheres formulated to a Dynamic Viscosity of between 2 and 3 and, iii) the protective and conditioning nutrient rich Microbial Mat. A significant advantage is that the novel process of in-situ biological exfoliation typically occurs within a period of seven to fourteen days after the completion of just one to three applications in-situ, dependent upon type and level of contamination. In some embodiments the Microbial Mats (MMs) are devised to protect the treatment from dry, wet, hot and cold conditions. However, these MMs also act as a conditioning top layer, providing anchorage and additional nutrients that augment growth of the microbial community. This is particularly successful as the highly complex matrix, which forms the molecular structure of all BZCs, acts like a biofilm rather than a conventional liquid culture and, therefore, the physical appendages of bacteria stimulate chemical reactions such as oxidation and hydration as cells become irreversibly adsorbed and bond with the additional surface area provided by the MMs.

In further embodiments, a rapid increase in microbial population can be observed during an 'exponential growth phase' and, as a consequence, biosurfactant production begins. Fortunately, as this leads to enhanced bioavailability and biodegradation, the extremely high biomass density that develops can be sustained throughout the process as available nutrients, pH conditions, redox potential and hydration levels are optimised in the vicinity of the cells within the architecture that is uniquely formed so rapidly by combining the BZCs with MMs.

Another novel advantage of in-situ biological exfoliation is that, ultimately, the pollution becomes completely encapsulated within the extracellular polymeric substances (EPS) created by the BZC and the natural excretion of the bacteria. Moreover, the intercellular adhesion that occurs between the entire gelatinous mass and the MMs facilitates the exfoliation of contamination from the surface by simply rolling up the MMs. This novel process is manifested by the treatment's ability to release the contamination from the surface being cleaned through the production of potent micellar surfactant solutions whilst, simultaneously, encapsulating it in a gelatinous biological structure that becomes fixed to the contact surface of the MMs. Consequently, the innovative and novel process of biological exfoliation is made possible and occurs rapidly in comparison with conventional in-situ bioremediation methods and treatments. In other embodiments, treatments for in-situ biological exfoliation are so predictable they can be set out as prescriptive treatment modules that predict both the number of treatments and time required to complete a specific cleaning process. (See example in Table 1 )

In at least some embodiments, blends with biospheres utilised for in-situ biological exfoliation are formulated to act as a self-buffering system to independently maintain the correct pH value across the surface throughout the treatment process. Moreover, the exceptionally high retention characteristic, within blends with biospheres, results in a far greater proportion of its pH buffering components remaining in position for much longer than would be possible when using a conventional liquid culture and, thus, the blend with biospheres optimizes the potential for these components to act as a continuous bacteria specific pH buffering stimulant. To prevail over the many limitations facing conventional in-situ bioremediation, the blend with biospheres, utilised for in-situ biological exfoliation, transforms water to intensify targeting and interaction with contaminants, while still sustaining a healthy microenvironment that optimizes the potential for biological life cycles to flourish. This transformation massively increases the surface area that is made available for the bacteria to grow up on and, thus, the biomass that results is also increased exponentially.

These advances are realized as organic micro-particles (biospheres) are meticulously blended with a water based liquid culture and optionally with other natural synergistic ingredients to develop both site and application specific embodiments.

In some embodiments, the blend with biospheres and other optional components discussed below in Table 2, is obtained by using a vacuum induction system so that the biospheres are mixed with the water under intense sheer energy. This is essential as it increases the specific surface of the available liquid by several hundred thousand times and, thus, as the biospheres are separated momentarily, they become wetted and dispersed completely without forming any lumps through agglomeration.

Finally, the blend can be further refined by low to medium rotation before being left to rest and bottling.

In some embodiments, the blend with biospheres can be further formulated with at least one component selected from Table 2. Table 2. Components for Heterotrophic Bacterium Growth.

Important Note: Use only food-grade chemicals

The blend with biospheres can be used with any microorganisms. At least in some embodiments, the microorganisms utilized are indigenous to the soil and the ocean, they are not genetically altered and fall within nonpathogenic homology groups. Such microorganisms may include any of the following: a) Pseudomonas putida - A gram negative rod that was isolated from fuel oil contaminated soil. This aerobic Pseudomonas falls within the non-pathogenic P. flourescens homology group; b) Acinetobacter johnsonii/genospecies 7 - A non-spore forming gram negative rod that was isolated from an Atlantic Ocean estuary off the coast of Hampton, New Hampshire. These bacteria were selected for their ability to degrade crude oil and other petroleum hydrocarbons in marine environments; c) Alcaligenes faecallis Type II - A gram negative rod that was isolated from fuel oil contaminated soil. Alcaligenes faecallis Type II. These bacteria are not gram positive and, thus, they are not Staphylococci sp., Bacillus sp., or Streptococci sp. Biolog analyses also excluded Salmonella, fecal coliform and Shigella; d) Pseudomonas-unidentified fluorescent - A gram negative rod that was isolated from fuel oil contaminated soil. This aerobic Pseudomonas falls within the nonpathogenic P. flourescens homology group.

A person of skill would further appreciate that the blends with biospheres can be used in in-situ methods where precision delivery is needed.

In complete contrast to conventional liquid cultures, where planktonic cells freely exist in bulk solution, the gelatinous matrix with biospheres produces controlled migration through dynamic viscosity management so bacteria can remain in-situ with negligible losses throughout the cleaning process.

This is important as this additional control maximizes the opportunity for bacteria to attach themselves to the target food source, which is the contaminant to be removed from the surface. Moreover, whilst reducing costs by minimizing both the amount of inoculant that is wasted and time spent on performing unproductive treatments, this novel method also creates an organic optimising host environment capable of establishing billions more Colony Utilising Populations (CUPs) far more quickly to make the whole procedure faster and much more predictable.

In further embodiments, fluorescence can be added to a biosphere so that the migratory patterns and stability of a gelatinous matrix can be tracked in the field and observed. In these embodiments, samples can be analyzed under UV light and/or by UV microscopy.

Further embodiments include kits which comprise a blend with biospheres and MMs. Such blends can be stored as a dry powder and mixed with water and a bacterial culture of choice prior to being used in the field. The blend with biospheres and the gelatinous matrix it creates, has the potential to improve many commercial practices in the areas of water conservation, diffuse pollution management, land remediation, restoration of soils and the maintenance remediation of construction materials.

This invention will be further described by the way of the following non-limiting examples.

Example 1.

Distinction in performance between conventional liquid culture and a blend with biospheres were demonstrated by the results from a large field trial that was conducted utilizing a conventional liquid bacterial culture in Phase 1 as shown in Fig. 1 . See Cells 1 and 4 - before Phase 2 treatment. In Phase 2, both cells were treated with a blend comprising biospheres and a positive outcome was achieved. See Figure 1. (Cells 2 & 3 not treated during Phase 1 ).

Moreover, the graph in Fig. 1 also delineates a second set of identical patterns of TPH biodegradation. These results are also significant, as they occurred in the treatment areas designated as the control locations during the study and, therefore, neither area had received any treatment whatsoever before Phase 2.

Ultimately, in these four heterogeneous cases, analysis had demonstrated identical patterns of biodegradation. Thus, the indicated change in TPH concentrations was due to biodegradation that resulted from the blend with biospheres that was utilized for the in-situ remediation procedure carried out in the second phase of this environmental study.

See Fig 1 . (Cells 2 & 3 not treated during Phase 1 )

Additional studies were conducted and demonstrate the overall reduction in contaminant mass that was achieved after the in-situ remediation procedure, with a blend comprising biospheres, that was completed in the second phase of the same environmental study. These results are reported in Fig. 2 alongside instructive comparative data taken from the conventional in-situ bioremediation treatments that were performed in Phase 1 of the study. These results also verify the only significant reduction in pollution that had occurred on the site, over a period of eight years of scientific monitoring, was due to the biodegradation that resulted from utilizing the blend with biospheres.

Example 2.

Distinction in performance between conventional liquid culture and a blend with biospheres can also be observed in shelf-life studies where random examples are taken at a specific point of final production and stored in separate twenty liter containers so that periodic samples can be taken for analysis to assess microbial viability over various periods of time. As shown in Fig. 3, a variety of microbial blends with biospheres remained viable after 28 weeks in storage. Overall, after fourteen weeks, these laboratory results demonstrated three blends with biospheres had sustained strong viability, one blend with biospheres had sustained moderate viability and the liquid culture, used as the control, had sustained only a low level of viability. After 28 weeks, the results demonstrated all four blends with biospheres had sustained strong viability juxtaposed with the liquid culture that demonstrated no activity. See Fig. 3.

The results from this study are particularly instructive because various blends with biospheres tested and the control were all produced from the same batch of liquid culture.

Example 3.

Further distinctions in performance between conventional liquid culture and a blend with biospheres combined with Microbial Mats (MMs) are demonstrated by the results of a scientific study that was structured specifically to observe and compare the potential of a proven high performance biological degrader to utilize high molecular weight mineral oil as a sole carbon source when; a) it is applied to surface contamination as a conventional liquid and; b) when the same biological degrader is applied to the surface contamination in a blend with biospheres combined with Microbial Mats (MMs) to perform in-situ biological exfoliation of surfaces heavily contaminated with high molecular weight mineral oil. This study was carried out over a period of ninety days with scientific analysis of Colony Forming Units (CFUs) and Colony Utilising Populations, for high molecular weight mineral oil, performed on the first, thirtieth, sixtieth and ninetieth day.

On the first day, the conventional liquid culture and the blend with biospheres combined with MMs both demonstrated exceptionally strong viability, although, scientific analysis did indicate the conventional liquid culture to be the most active. (CUP Result: Convention Liquid = >10¹³ per gram v Novel Blend with Biospheres and Microbial Mats = >10¹⁰ per gram - See Fig. 4) After thirty days, laboratory results demonstrated a significant reduction in the viability of the conventional liquid culture as levels of CUPs decreased. However, in complete contrast to these poor results, viability of the blend with biospheres combined with MMs had remained exceptionally strong with levels of CUPs increasing significantly.

(CUP Result: Convention Liquid = >10⁴ per gram v Novel Blend with Biospheres and Microbial Mats = >10¹³ per gram - See Fig. 4)

After sixty days, the pattern continued with laboratory results demonstrating a further reduction in the viability of the conventional liquid culture as levels of CUPs continued decreasing. Again, in complete contrast to these poor results, viability of the blend with biospheres combined with MMs remained exceptionally strong with consistently high levels of CUPs. (CUP Result: Convention Liquid = <10¹ per gram v Novel Blend with Biospheres and Microbial Mats = >10¹³ per gram - See Fig. 4) After ninety days, laboratory results confirmed a clear distinction in performance between the conventional liquid culture and the blend with biospheres combined with Microbial Mats. As the level of CUPs continued to be low, the potential for utilisation of the contamination by the conventional liquid culture remained extremely poor. However, in complete contrast to these results, the viability of the blend with biospheres combined with MMs had remained exceptionally strong with scientific analysis continuing to demonstrate high levels of CUPs. (Final CUP Result: Convention Liquid = <10¹ per gram v Novel Blend with Biospheres and Microbial Mats = >10¹¹ per gram - See Fig. 4)

Once again, the results from this study are particularly instructive because the biological constituents, within the conventional liquid culture and the blend with biospheres combined with Microbial Mats, were identical. Thus, presenting iterative lines of indubitable evidence that the higher levels of performance, demonstrated during this study, were due entirely to the optimising effect upon bacterial activity that occurs within the complex matrix of the novel blend with biospheres combined with MMs. While a particular embodiment of the present microbial mat and biomolecular zonal compositions and methods for in-situ biological exfoliation of contaminated surfaces have been described herein, it will be appreciated by those skilled in the art that changes and modifications may be made thereto without departing from the invention in its broader aspects and as set forth in the following claims.

Claims

CLAIMS:

1 . A microbial mat comprising:

a contact layer;

a core layer;

a matrix of cellulose fibers coated with a liquid nutrient mix;

a carrying sheet;

wherein the core layer comprises the matrix of cellulose fibers coated with a liquid nutrient mix, attached to the carrying sheet and coated with a dry nutrient mix;

wherein the liquid nutrient mix and dry nutrient mix are composed of ingredients from the group consisting of Sodium Citrate, Ammonium Sulphate, Monosodium phosphate, Dipotassium Phosphate, Magnesium Sulphate, Eprom Salt, and Ferrous Sulphate; and

a top layer, wherein the top layer is attached to the entire surface of the core layer.

2. The microbial mat of claim 1 , wherein the top layer is a polymer sheet

between 100 and 500 gsm.

3. The microbial mat of claim 1 or 2, wherein the core layer is between 100 and 400 gsm, with a water absorbent capacity between 4 to 20 times the materials weight.

4. The microbial mat of any of the above claims, wherein the contact layer is hydrophobic material.

5. The microbial mat of any of claims 1 -3, wherein the contact layer is

hydrophilic material.

6. The microbial mat of any of the above claims, wherein the top layer is a

perforated, heat reflective membrane.

7. The microbial mat of any of claims 1 -5, wherein the top layer is a thermal insulating membrane.

8. A method for in-situ biological exfoliation of contaminated surfaces using a microbial mat, comprising:

applying a biosphere blend to the contaminated surface;

waiting for a predetermined time; and

covering the contaminated surface with a microbial mat, wherein the microbial mat comprises a contact layer, a core layer, and a top layer.

9. The method of claim 8 further comprising:

waiting a predetermined time;

removing the microbial mat;

washing the contaminated surface with a recharging biological blend; and assessing treatment of contaminated surface.

10. The method of claims 8 or 9 further comprising:

waiting a predetermined time;

removing the microbial mat;

washing the contaminated surface with a recharging biological blend;

reapplying the biosphere blend; and

covering the contaminated surface with a microbial mat.

11. The method of any of claims 8-10 further comprising:

waiting a predetermined time;

removing the microbial mat;

washing the contaminated surface with a recharging biological blend;

drying the contaminated surface for a predetermined time;

reapplying the biosphere blend; and

covering the contaminated surface with a microbial mat.

12. A composition comprising biospheres and bacteria, wherein the biospheres are cellulosic biopolymers and sized in the range from 500 nanometers to 80 microns and have a minimum free swell absorption capacity of 400 times by weight and a maximum free swell absorption capacity of 1200 times by weight.

13. The composition of claim 12, wherein the biospheres gradually release moisture.

14. The composition of claims 12 or 13, wherein the viscosity of the liquid

composition is from 6 centipoise (cps) to 217 centipoise (cps).

15. The composition of claim 12, wherein the viscosity of the liquid composition is from 217 centipoise (cps) to 1 ,159 centipoise (cps).

16. The composition of claim 12, wherein the viscosity of the liquid composition is from 1 ,236 centipoise (cps) to 5,021 centipoise

17. The composition of claim 12, wherein the viscosity of the liquid composition is from 5,021 centipoise (cps) to 47,31 1 centipoise (cps).

18. A method for bioremediation in situ, the method comprising:

preparing a blend of liquid bacterial culture with biospheres, wherein the biospheres are cellulosic biopolymers and sized in the range from 500 nanometers to 80 microns; and

have a minimum free swell absorption capacity of 400 times by weight and a maximum free swell absorption capacity of 1200 times by weight;

applying the blend at a site in need of bioremediation; and

letting the blend to form a gelatinous matrix.

19. A method for delivering and/or hosting biological and/or chemical reagents in a gelatinous matrix, the method comprising:

obtaining biospheres which are cellulosic biopolymers and sized in the range from 500 nanometers to 80 microns and have a minimum free swell absorption capacity of 400 times by weight and a maximum free swell absorption capacity of 1200 times by weight;

mixing the biospheres with a bacterial and/or chemical; and

letting the mixture to form a gelatinous matrix.