Patent Application
20230357064
The present invention relates to petroleum hydrocarbons. In particular, the present invention relates to compositions and methods for the decontamination of petroleum hydrocarbons.
Between 0.47 to 8.4 million tonnes of oil are released to seas or oceans each year, depending on the frequency and severity of oil spills. Spilled oil reaches lakes, rivers, wetlands, and freshwater environments and can harm all organisms that live in, under, or around the water (direct and indirect effects on human health).
Over the past decade, various technologies such as chemical methods, physical treatments, thermal treatments, and bioremediation have been applied to groundwater, soils, and sediments clean-up. Bioremediation technologies offer the potential for significant cost savings and practical approaches compared to conventional remediation technologies, such as thermal treatment, excavation, and disposal in landfills, and pump & treat. Especially at remote sites in the north, conventional technologies are often not practical or feasible, for example requiring electricity provision, transport of chemicals, and their storage, among others.
Petroleum degrading bacteria can produce specific enzymes that can degrade petroleum hydrocarbons. Commonly, the best (optimized) scheme of biodegradation cannot be easily achieved since the biodegradation efficiency of contaminants is strongly influenced by the physicochemical characteristics of the pollutant, the contaminated matrices, and microbial growth condition. Hence, the biochemical remediation method based on using enzymes, rather than whole-cell degrading microorganisms, can be a promising method for cold-climate sites since the enzymatic method can specifically catalyze a series of reactions for pollutant removal. The enzyme cocktail can be particularly formulated for a contaminated site based on physicochemical characteristics of hydrocarbon contaminated soil/water matrices. It would also be desirable to provide an enzyme cocktail and method for the degradation of crude oil, diesel, and polycyclic aromatic hydrocarbons.
In addition, the development of devices/processes to target oil below the water surface has attracted the attention of researchers due to the complication that started to emerge recently. For example, the explosion in tar sands productions in Western Canada means increasing amounts of heavy crude oil making its way to the American Midwest via the Great Lakes.1 For these types of scenarios, the development of efficient remediation devices and/or processes is necessary to capture the plumes of the oil in the deep-water and limit the oil-impacted seafloor. In addition to extracting free phase hydrocarbon out of the water, the removal of aromatic hydrocarbons released into the water is important to avoid sediment contamination.
There remains a need for safe and efficient methods for managing contaminated sites.
In accordance with an aspect, there is provided a polyaromatic hydrocarbon (PAH)-degrading enzyme mixture obtained from a culture of PAH-utilizing microorganisms having been grown in presence of one or more enzyme inducers.
In an aspect, the culture is a mixed-culture of two or more PAH-utilizing microorganisms.
In an aspect, the PAH-utilizing microorganisms are bacteria that are capable of growing with a PAH-containing composition being their only source of carbon.
In an aspect, the PAH-utilizing microorganisms are indigenous to a PAH-contaminated soil sample.
In an aspect, the PAH-utilizing microorganisms comprise Pseudomonas sp. and/or Rhodococcus sp.
In an aspect, the PAH-utilizing microorganisms comprise Pseudomonas sp. URS-5, Pseudomonas sp. URS-6, Pseudomonas sp. URS-8, and/or Rhodococcus sp. URS-10.
In an aspect, the one or more enzyme inducers comprise a mixture of inducers.
In an aspect, the mixture of inducers is comprised in oil.
In an aspect, the one or more enzyme inducers comprise pure inducers.
In an aspect, the one or more enzyme inducers comprise naphthalene, anthracene, phenanthrene, pyrene, benzo[a]pyrene, Dilbit, and/or crude oil.
In an aspect, the PAH-degrading enzyme mixture comprises an optimum active enzyme temperature of less than about 30° C., such as less than about 25° C., such as about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., and any range therein between, such as from about 10° C. to about 15° C.
In an aspect, the PAH-degrading enzyme mixture comprises an optimum pH of from about 4 to about 9, such as from about 4, about 5, about 6, about 7, or about 8 to about 5, about 6, about 7, about 8, or about 9, such as about 4, about 5, about 6, about 7, about 8, or about 9, such as about 7.
In an aspect, the PAH-degrading enzyme mixture comprises less than about 10% salt or for use in an environment containing less than about 10% salt, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% salt.
In an aspect, the enzymes are adsorbed on soil particles.
In an aspect, the PAH-degrading enzyme mixture further comprises a dispersing aid, such as a surfactant or biosurfactant, such as a rhamnolipid.
In an aspect, the enzymes comprise naphthalene dioxygenase, naphthalene cis-dihydridiol dehydrogenase, dihydrodiol dehydrogenase, catechol 1,2 dioxygenase, catechol 2,3 dioxygenase, salicylaldehyde dehydrogenase, 1-hydroxy-2-naphthoate hydroxylase, salicylate hydroxylase, trans-2-carboxybenzalpyruvate hydratase-aldolase, lipase, toluene monooxygenase, lignin peroxidase, manganese peroxidase, esterase, and/or laccase.
In an aspect, the PAH-degrading enzyme mixture further comprises one or more species of live PAH-utilizing organisms.
In accordance with an aspect, there is provided Pseudomonas sp. URS-5, Pseudomonas sp. URS-6, Pseudomonas sp. URS-8, and/or Rhodococcus sp. URS-10.
In accordance with an aspect, there is provided a method for tailoring a polyaromatic hydrocarbon (PAH)-degrading enzyme mixture for a selected contaminated site for bioremediation, the method comprising:
isolating indigenous PAH-utilizing microorganisms from the contaminated site,
culturing the microorganisms in the presence of one or more enzyme inducers,
extracting enzymes from the culture, and
applying the enzymes to the contaminated site.
In an aspect, the method comprises culturing a multi-culture of two or more PAH-utilizing microorganisms.
In an aspect, the PAH-utilizing microorganisms are bacteria that are capable of growing with a PAH-containing composition being their only source of carbon.
In an aspect, the contaminated site comprises soil. In an aspect, the PAH-utilizing microorganisms comprise Pseudomonas sp. and/or Rhodococcus sp.
In an aspect, the PAH-utilizing microorganisms comprise Pseudomonas sp. URS-5, Pseudomonas sp. URS-6, Pseudomonas sp. URS-8, and/or Rhodococcus sp. URS-10.
In an aspect, the one or more enzyme inducers comprise a mixture of inducers.
In an aspect, the mixture of inducers is comprised in oil. In an aspect, the one or more enzyme inducers comprise pure inducers.
In an aspect, the one or more enzyme inducers comprise naphthalene, anthracene, phenanthrene, pyrene, benzo[a]pyrene, Dilbit, and/or crude oil.
In an aspect, the PAH-degrading enzyme mixture comprises an optimum active enzyme temperature of less than about 30° C., such as less than about 25° C., such as about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., and any range therein between, such as from about 10° C. to about 15° C.
In an aspect, the PAH-degrading enzyme mixture comprises an optimum pH of from about 4 to about 9, such as from about 4, about 5, about 6, about 7, or about 8 to about 5, about 6, about 7, about 8, or about 9, such as about 4, about 5, about 6, about 7, about 8, or about 9, such as about 7.
In an aspect, the contaminated site comprises less than about 10% salt, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% salt.
In an aspect, the enzymes are adsorbed on soil particles.
In an aspect, the PAH-degrading enzyme mixture comprises a dispersing aid, such as a surfactant or biosurfactant, such as a rhamnolipid.
In an aspect, the enzymes comprise naphthalene dioxygenase, naphthalene cis-dihydridiol dehydrogenase, dihydrodiol dehydrogenase, catechol 1,2 dioxygenase, catechol 2,3 dioxygenase, salicylaldehyde dehydrogenase, salicylate hydroxylase, trans-2-carboxybenzalpyruvate hydratase-aldolase, lipase, toluene monooxygenase, lignin peroxidase, manganese peroxidase, esterase, and/or laccase.
In an aspect, the method further comprises applying one or more species of live PAH-utilizing organisms to the contaminated site.
In accordance with an aspect, there is provide a PAH-degrading enzyme mixture made by the method described herein.
In accordance with an aspect, there is provided a jellyfish-like device for bioremediation of a contaminated water site, the device comprising:
In an aspect, the PAH-degrading enzymes comprise the enzyme mixture described herein.
In an aspect, the hollow fiber module comprises hydrophilic modified polyethersulfone (mPES) hollow fibers.
In an aspect, the hollow fiber module comprises from about 1 to about 100 fibers, such as from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 fibers, such as about 10 fibers.
In an aspect, each fiber independently has an internal diameter of from about 0.1 mm to about 10 mm, such as from about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, or about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, or about 9.5 to about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, or about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10 mm, such as about 1.0 mm.
In an aspect, each fiber independently has a length of from about 10 to about 100 cm, such as from about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 to about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 cm, such as about 45 cm.
In an aspect, each fiber independently has a wall thickness of from about 0.01 mm to about 1 mm, such as from about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.50, or about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95 to about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.50, or about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0 mm, such as about 0.1 mm.
In an aspect, each fiber independently has a total surface area of from about 10 cm2 to about 100 cm2, such as from about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 to about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or 100 cm2, such as about 58 cm2.
In an aspect, the device further comprises a recycling pump, wherein the recycling pump optionally operates at a speed of from about 1 ml/min to about 100 ml/min, such as from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, ml/min, such as about 50 ml/min.
In accordance with an aspect, there is provided a mobile production plant for producing the PAH-degrading enzymes described herein, the production plant comprising a fermenter for culturing microorganisms, a bioreactor for enzyme production, and an ultrasonic device for producing cell extracts.
In an aspect, the fermenter is a stirred tank fermenter.
In an aspect, the fermenter cultures the microorganisms at room temperature. In an aspect, the fermenter comprises pH, temperature, and/or dissolved oxygen (DO) probes.
In an aspect, the bioreactor operates in continuous mode.
In an aspect, the plant further comprises a power generator.
In an aspect, the plant further comprises an oxygen source such as an oxygen cylinder.
In an aspect, the mobile production plant is provided on a trailer.
The novel features of the present invention will become apparent to those of skill in the art upon examination of the following detailed description of the invention. It should be understood, however, that the detailed description of the invention and the specific examples presented, while indicating certain aspects of the present invention, are provided for illustration purposes only because various changes and modifications within the spirit and scope of the invention will become apparent to those of skill in the art from the detailed description of the invention and claims that follow.
The present invention will be further understood from the following description with reference to the Figure, in which:
Indigenous microbial communities are constantly adjusting in response to environmental conditions. As described herein, using indigenous microorganisms can solve most of the challenges associated with environmental factors (temperature, pH, and salinity). The indigenous bacteria can produce enzymes to degrade high molecular weight hydrocarbons and result in less toxic metabolites that are more bioavailable for other bacterial strains in the community.
Using enzymes overcomes limitations of the slow rate of bioremediation by the whole-cell microorganisms. Enzymes can specifically catalyze a series of reactions in a minute timescale for pollutant removal.
Described herein are oxidoreductase enzymes from indigenous bacteria which are formulated for application for biodegradation of PAHs in soil and water. Also described is formulating the stabilized enzymes for a specified application such as for the breakdown of other petroleum hydrocarbons in specific conditions.
In aspects, enzyme cocktails were designed for effective biodegradation of at least one compound of polyaromatic hydrocarbons such as naphthalene, anthracene, phenanthrene and pyrene.
In aspects, a degradation method comprises bacterial enzymes for treating one of the above-mentioned materials in water and soil matrices.
Formulated bacterial enzymes from indigenous microorganisms in the environmental setting for degradation of poly-aromatic hydrocarbons may be of interest to remediation companies. In addition, the enzyme mixture can be formulated for a specific petroleum hydrocarbon in a contaminated site. This enzyme mixture can be combined with commercialized remediation products such as adsorbents to increase the remediation efficiency. This enzymatic technology can speed up the biodegradation of harmful chemicals by lowering the toxicity of target contaminants and increasing their bioavailability for other bacterial strains in the environment. By reducing the toxicity of contaminants in the system, intrinsic bioremediation can be reduced from years to weeks.
Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the typical materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.
It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Many patent applications, patents, and publications may be referred to herein to assist in understanding the aspects described. Each of these references is incorporated herein by reference in its entirety.
In understanding the scope of the present application, the articles “a”, “an”, “the”, and “said” are intended to mean that there are one or more of the elements. Additionally, the term “comprising” and its derivatives, as used herein, is intended to be open-ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives.
It will be understood that any aspects described as “comprising” certain components may also “consist of” or “consist essentially of,” wherein “consisting of” has a closed-ended or restrictive meaning and “consisting essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components, and components added for a purpose other than achieving the technical effect of the invention. For example, a composition defined using the phrase “consisting essentially of” encompasses any known acceptable additive, excipient, diluent, carrier, and the like. Typically, a composition consisting essentially of a set of components will comprise less than 5% by weight or volume, typically less than 3% by weight, more typically less than 1%, and even more typically less than 0.1% by weight of non-specified component(s).
It will be understood that any component defined herein as being included may be explicitly excluded from the claimed invention by way of proviso or negative limitation.
In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.
Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.
Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The abbreviation, “e.g.,” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.,” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.
“Active” or “activity” for the purposes herein refers to a biological activity of the compositions described herein, wherein “biological” activity refers to a biological function (either inhibitory or stimulatory) caused by the compositions.
The Enzyme Booster Technology (EnBooT) described herein was developed as a novel and green bioremediation method based on the combination of specifically formulated enzymes and highly efficient biosurfactants which can be easily applied for in-situ biodegradation in cold-climate regions. This technology can be adapted for other sites with harsh or mild conditions and products can be specifically tailored based on need.
Thus, in aspects, described herein is a polyaromatic hydrocarbon (PAH)-degrading enzyme mixture obtained from a culture of PAH-utilizing microorganisms having been grown in presence of one or more enzymes inducers.
In some aspects, the culture is a monoculture and in other aspects, the culture is a mixed one.
Also described herein are methods of use of the PAH-degrading enzymes and methods method for tailoring a PAH-degrading enzyme mixture for a selected contaminated site for bioremediation. The tailoring method comprises, for example, isolating indigenous PAH-utilizing microorganisms from the contaminated site, culturing the microorganisms in the presence of one or more enzyme inducers, extracting enzymes from the culture, and applying the enzymes to the contaminated site.
The method may comprise culturing a monoculture or a mixed-culture of two or more PAH-utilizing microorganisms. Typically, the PAH-utilizing microorganisms are bacteria that are capable of growing with a PAH-containing composition being their only source of carbon. The contaminated site typically comprises soil.
Any species of PAH-utilizing microorganisms are within the scope described herein. Typically, the PAH-utilizing microorganisms comprise Pseudomonas sp. and/or Rhodococcus sp., such as, for example, Pseudomonas sp. URS-5, Pseudomonas sp. URS-6, Pseudomonas sp. URS-8, and/or Rhodococcus sp. URS-10.
Typically, one or more enzyme inducers comprise a mixture of inducers and, typically, the mixture of inducers is comprised of oil. One or more enzyme inducers comprise pure inducers. Examples of enzyme induce include, for example, naphthalene, anthracene, phenanthrene, pyrene, benzo[a]pyrene, Dilbit, and/or crude oil.
The PAH-degrading enzyme mixture typically comprises an optimum active enzyme temperature of less than about 30° C., such as less than about 25° C., such as about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., about 10° C., about 11° C., about 12° C., about 13° C., about 14° C., about 15° C., about 16° C., about 17° C., about 18° C., about 19° C., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C., and any range therein between, such as from about 10° C. to about 15° C. and an optimum pH of from about 4 to about 9, such as from about 4, about 5, about 6, about 7, or about 8 to about 5, about 6, about 7, about 8, or about 9, such as about 4, about 5, about 6, about 7, about 8, or about 9, such as about 7.
Typically, the contaminated site comprises less than about 10% salt, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% salt.
In other aspects, the enzymes are adsorbed on soil particles.
In aspects, the PAH-degrading enzyme mixture comprises a dispersing aid, such as a surfactant or biosurfactant, such as a rhamnolipid.
Exemplary enzymes include naphthalene dioxygenase, pyrene dioxygenase, dihydrodiol dehydrogenase, 1-hydroxy-2-naphthoate hydroxylase, catechol 1,2 dioxygenase, catechol 2,3 dioxygenase, salicylate hydroxylase, lipase, toluene monooxygenase, lignin peroxidase, manganese peroxidase, esterase, and/or laccase.
In aspects, the method further comprises applying one or more species of live PAH-utilizing organisms to the contaminated site.
Also contemplated herein is a PAH-degrading enzyme mixture made by the method described herein.
The following examples do not include detailed descriptions of conventional methods. Such methods are well known to those of ordinary skill in the art and are described in numerous publications including Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, which is incorporated by reference herein.
Further described herein is a jellyfish-like device for bioremediation of a contaminated water site. The device comprises an enzyme reservoir containing PAH-degrading enzymes, and a hollow fiber module in fluid communication with the enzyme reservoir, wherein the hollow fiber module is permeable to PAHs but not to the PAH-degrading enzymes. In typical aspects, the PAH-degrading enzymes comprise the enzyme mixture described herein (
In aspects, the hollow fiber module comprises hydrophilic modified polyether sulfone (mPES) hollow fibers. The hollow fiber module typically comprises from about 1 to about 100 fibers, such as from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, or about 95 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, or about 100 fibers, such as about 10 fibers.
Typically, each fiber independently has an internal diameter of from about 0.1 mm to about 10 mm, such as from about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, or about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, or about 9.5 to about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.5, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, about 5.0, or about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10 mm, such as about 1.0 mm.
Typically, each fiber independently has a length of from about 10 to about 100 cm, such as from about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 to about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 cm, such as about 45 cm.
Typically, each fiber independently has a wall thickness of from about 0.01 mm to about 1 mm, such as from about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.50, or about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, or about 0.95 to about 0.02, about 0.03, about 0.04, about 0.05, about 0.06, about 0.07, about 0.08, about 0.09, about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.50, or about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0 mm, such as about 0.1 mm.
Typically, each fiber independently has a total surface area of from about 10 cm2 to about 100 cm2, such as from about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 to about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or 100 cm2, such as about 58 cm2.
Typically, the device further comprises a recycling pump, wherein the recycling pump optionally operates at a speed of from about 1 ml/min to about 100 ml/min, such as from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, or about 90 to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100, ml/min, such as about 50 ml/min.
To improve production efficiency resulting in cost-effective enzymes, an unsterile and continuous fermentation process was developed by using a compact production plant for increased responsiveness and mobility.
The enzymatic remediation of petroleum-contaminated sites is a promising approach for site clean-up. However, the cost and efficiency of the enzyme cocktails that are employed in these processes are still a significant bottleneck. In order to address issues regarding the clean-up of contaminated sites in cold regions, described herein are a mobile product plant and method for on-site production of enzyme solutions and biochemical products using a compact and mobile production plant in order to reduce the production cost of this technology. Typically, the mobile production plant comprises: (1) a 3 L customized stirred tank fermenter for culturing of strains at room temperature equipped with pH, temperature and DO probes; (2) a 15 L customize hybrid bioreactors applied for bench-scale enzyme production operating in continuous mode equipped 5-inch exhaust filter; and (3) an ultrasonic device used for pilot-scale production of cell extract. Piping would connect components. All these equipment as well as other additional equipment such as a power generator, oxygen cylinder, may be provided pre-packaged on a trailer, for increased mobility and responsiveness.
Based on the literature review2, the most representative enzymes involved in bioremediation belong to the family of oxidoreductases. Kadri et. al (2018) studied bench-scale production of the cocktail enzymes using 5 L stirred tank reactor (STR) experiments. They showed that a significant decrease in the dissolved oxygen (DO) values was detected during the first stage of fermentation. The decrease in DO observed is coincidental with the exponential growth phase of strains that required a higher oxygen uptake rate (OUR). Considering the fact that maximum biomass and enzyme activities can be achieved in presence of high dissolved oxygen, a higher agitation from 250 to 500 rpm (representing the large power domain in the process) and a higher aeration rate reaching 3.5 L min-1 were applied to maintain dissolved oxygen greater than 60%.3 The challenges to achieving effective and economical production become greater for larger volumes where utility costs generally dominate fermentation economics. The airlift bioreactor has the potential for use in the large-scale production of these enzymes due to its advantages over STR with respect to higher gas hold-up, higher mass transfer, lower shear stress, lower microbial contamination, lower energy consumption, installation, and operation costs. In addition, an airlift bioreactor can supply more oxygen, and efficiency of petroleum degrading enzymes is increased compared to stirred conventional reactors.4 Air-lift bioreactors have been widely used in a number of industrial processes, such as the production of amino acids, antibiotics, enzymes, vitamins, and organic acids. However, the continuous enzyme production in air-lift reactors was reported for several enzymes, to the best of our knowledge, there is no report available on the production of petroleum-hydrocarbon enzymes.
Petroleum hydrocarbons have a significant role in the production of degrading enzymes and using petroleum hydrocarbons as the sole source of carbon inhibits the growth of other non-degrading microorganisms. Due to the inhibitory and toxicity effect of such compounds, selective growth will be obtained with a specific C: N ratio of media for seed cultivation and enzyme production. Thus, a complicated facility operating in sterile conditions is not needed for the production of targeted enzymes because the specific conditions for the growth of specific bacteria are provided.
Thus, more generally, described herein is a mobile production plant. In aspects, the mobile production plant is for producing the PAH-degrading enzymes described herein. The production plant comprises a fermenter for culturing microorganisms, a bioreactor for enzyme production, and an ultrasonic device for producing cell extracts.
In certain aspects, the fermenter is a stirred tank fermenter and optionally cultures the microorganisms at room temperature. The fermenter typically comprises pH, temperature, and/or dissolved oxygen (DO) probes. Typically, the bioreactor operates in continuous mode.
In certain aspects, the mobile production plant further comprises a power generator and/or an oxygen source such as an oxygen cylinder. Typically, the mobile production plant is provided on a trailer.
Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions of the present invention and practice the claimed methods. The following working examples, therefore, specifically point out the typical aspects of the present invention and are not to be construed as limiting in any way in the remainder of the disclosure.
This study aims to investigate (1) the bench-scale production of aliphatic and PAHs degrading enzymes; (2) the feasibility study for soil application through bench-scale and soil column tests; and (3) the fabrication of jellyfish-like devices for marine spill clean-up. This device is inspired by the long, elegant appendages of jellyfish that use their sticky tentacles to catch the oils below the surface water during a passive process when they float into the piece of oil by an incident. Each device is composed of a recirculation reservoir (e.g., jellyfish bell), and hollow fiber membranes used for physical immobilization of PAH degrading enzymes.
Future work includes the application of formulated enzymes for the field scale tests (in-situ and ex-situ) and scale-up of enzyme production and combination of enzyme mixture with commercialized remediation products. In addition, the application of metagenomics will be used for the analysis of microbial community to understand the shift in microbial population because of detoxification of the contaminant in soil and degradation of polyaromatic hydrocarbons. This date will be used for the optimization of enzyme concentrations.
Before inoculation for enzyme production, each bacterium was cultured in a nutrition broth (NB) at 15±1° C. and 150 rpm. A correlation was obtained between cell concentration and the number of viable bacteria by measuring optical density at 600 nm and counting colony-forming units (CFU).
The enzyme production based on monoculture of newly isolated was carried out in 250 mL serum bottles containing 50 mL of NB supplemented with 100, 250, and 500 ppm of oil. The target enzymes were also produced based on the mixed-culture of newly isolated Pseudomonas URS-5, URS-6, URS-8, Rhodococcus URS-10. In this regard, the enzyme production was carried out in 250 mL serum bottles containing 50 mL of NB supplemented with 250 mg/L of oil as a mixture of enzyme inducers. After 24h of incubation, the culture was centrifuged at 3810×g for 20 min. Cell extracts containing PAH degrading enzymes were prepared from 100 ml of each cultured Pseudomonas sp. URS-5, Pseudomonas sp. URS-6, Pseudomonas sp. URS-8, and Rhodococcus sp. URS-10 grown on oil. When growth reached the late exponential phase, cells were harvested from the media after culturing by centrifugation (16,000 rpm for 4 min at 4° C.). The pellets with the biomass were resuspended in phosphate buffer, pH 8, and then sonicated on ice using an Ultrasonifier (Branson Ultrasonics Corporation, Danbury, CT, USA) at 22 kHz and 30 kHz frequencies of ultrasounds for 10 min. Bradford assay was performed using Pierce TM BCA Protein Assay Kit (Thermo Fisher Scientific, Canada) and bovine serum albumin (BSA) standard curve.
Naphthalene dioxygenase activity is assayed based on the formation of indigo at 500 nm per time unit. The enzyme reaction was carried out using 5 μl of indole 100 mM as a substrate in DMF to the cellular lysates. The absorbance of the reaction mixture at 500 nm was recorded against a blank containing all ingredients except the substrate. The specific enzyme activity was defined as the initial rate of indigo formation by plotting the increase in absorbance at the first 2 h of reaction normalized to the protein content of the sample. The maximal absorbance of indigo was determined by mass spectrum at different wavelengths using a standard indigo solution dissolved in N,N′-Dimethyl-Formamide (DMF)5.
Naphthalene cis-dihydrodiol dehydrogenase activity was assayed by spectrophotometric measurement of the reduction of NAD at 340 nm. Routine assays were performed at 30° C. in a 1-ml quartz cuvette maintained under argon. The assay mixture (0.6 ml) contained 1 mM NAD and 0.1 mM dihydrodiol substrate in 0.1 M potassium phosphate, pH 7.0. The reaction was initiated by an appropriate amount of enzyme, and the absorption at 340 nm was recorded at 0.1-s intervals over 1 min with an HP8452 spectrophotometer equipped with a thermostated cuvette holder (Agilent Technologies, Les Ulis, France). The enzyme activity was calculated from the initial linear portion of the time course using an absorption coefficient of 6,220 M1·cm1 for nicotinamide adenine dinucleotide (NADH). One enzyme unit was defined as the amount that catalyzed the formation of one micromole of NADH per minute6.
Catechol 2, 3-dioxygenase and catechol 1, 2-dioxygenase activities are assayed by monitoring the production of 2-hydroxymuconic semialdehyde and muconic acid at 375 nm and 260 nm, respectively. The reaction mixture contained 1 mM of catechol, 2.0 mL of phosphate buffer (pH=7.0), and cellular lysates. One unit of enzyme activity is defined as the amount of enzyme that produced 1 μmol of cis, cis-muconic acid, and 2-hydroxy muconic semialdehyde per time unit.7
1-hydroxy-2-naphthoate and salicylate hydroxylase activities were measured at 340 nm by an assay described by Balashova et al.8 The reaction mixture (1 ml) contained 20 mM KH2PO4 (pH7.5). 100 μl cell extract, 100 μM NADH, and 50 μM 1-hydroxy-2-naphthoate or salicylate, respectively. Molar reaction coefficient at 340 nm of 5.08 and 6.22 mM−1cm−1 were used to determine the reaction of 1-hydroxy-2 naphthoate and salicylate. The rate of change in light absorption was measured at 340 nm. One unit of activity is the amount of enzyme that is needed to catalyze the conversion of one mol of NADH per minute into oxygen.9
Pre-selected isolates were tested for the key enzymes involved in the degradation of target substrates. The involved enzymes identification is studied by carrying out LC-MS/MS to confirm the presence of target enzyme in enzyme mixture obtained from mixed-culture. To provide a reference data set for peptide spectrum match (PSM) analysis for target enzymes, an enzymes cocktail was prepared using the suspension trapping (S-Trap) method as described in detail in our previous study (Miri et al., 2021a). Resulted peptides were subjected to mass determination analysis using a mass spectrometer coupled to an EASY-nLC 1000 system (Thermo Fisher Scientific, USA) and mass spectrometry (MS)-MS sequence analysis using Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, USA). All raw data were processed using the software Proteome Discoverer (version 2.2, Thermo Fisher Scientific, USA), then aligned with the protein database of Universal Protein Resource, UniProt (www.uniprot.org) and BLASTP (https://blast.ncbi.nlm.nih.gov).10, 11.
Example of Process for Isolation of Bacteria for Producing Enzymes
A series of pyrene biodegradation tests were conducted using SSM supplemented with three PAHs (naphthalene, phenanthrene, pyrene); and among all the isolated obtained (28 strains), URS-5, URS-6, URS-8, and URS-10 were selected based on their ability to grow on pyrene as a sole carbon and energy source. The constant freely dissolved concentrations of PAHs were provided using O-ring silicone as described in the experimentation section. Positive biodegradation ability was confirmed by measuring the percent depletion of PAHs in the silicone O-ring after 37 and 47 days for the biodegradation experiments (
A comparison of the degradation of pyrene by strains under a single and mixture substrate system was conducted in order to determine if the presence of other substrate would affect (either suppressing or enhancing) the degradation of the primary substrate. As can be seen in
Pseudomonas URS-5, URS-6 could grow in the range of temperature between 4±1° C. and 25±1° C., but not as effectively at 37±1° C. as previously reported for P. synxantha and P. mandelii strains.7 The presence of these characteristics indicates that the strains do not pose any potential pathogenic risk to humans or animals. The partial 16S rRNA sequencing showed that URS-6 was close to the type of strain of Pseudomonas putida strain NR 043434.1T with 99% identity. The sequence identity of URS-5 and URS-8 were 99% with a type of strain of Pseudomonas mandelii strain Cl P 105273T and Pseudomonas syringae ATCC 19310T respectively. In the sequence analysis of URS-5, the sequence identity was 98% with a type of strain of Rhodococcus erythropolis JCM 3201T.
The sequences for URS-5, URS-6, URS-8, and URS-10 have been deposited in the NCBI Genbank database and were assigned the accession of MZ144068.1, MZ144069.1, MZ144070.1 and MZ959374, respectively. Phylogenetic analysis also grouped URS-5, URS-6, URS-8 and URS-10 with the strain of Pseudomonas putida, Pseudomonas mandelii (P. mandelii), Pseudomonas synxantha, an Rhodococcus erythropolis with 98% bootstrap support (
Pseudomonas spp. and Rhodococcus spp. remarkably degrade various xenobiotics and often are isolated from hydrocarbon-contaminated sites.12 Wald et. al 2015 indicated that the Pseudomonas syringae group (namely Pseudomonas cichorii LMG 2162T) was one of the most dominant Pseudomonads in the sequestration and of carbon from naphthalene in the samples and potential degradation of other PAHs upon aeration of the sediment at both 20 (7%) and 10° C.13 Margesin et al., (2008) isolated cold-tolerant strains from contaminated soil which can degrade BTEX at 10° C.14
Regarding the URS-5, general sequental pattern of degradation was observed in which naphthalne was transformed first, followed by phenanthrene, and pyrene (
Regarding the URS-8, the same pattern of degradation for pyrene was observed. This strain did not show extensive degradation of pyrene in the PAHs mixture; however, the lack of accumulated intermediate can be considered positive effect that might be attributed to the enzyme system presented in liquid medium. The lack of salicylate hydroxylase activity in cell free lysate suggesting a pathway for three PAHs that does not pass through salicylate. However, protocatechuic acid 3,4-dioxygenase enzyme assay suggesting the pathway for the catabolism of phthalate to central metabolites via intradiol cleavage of protochatechuic acid (Table.1). Other studies reported the relationship between degradation rate of PAHs and naphthalene dioxygenase activity.17
Compared to URS-5 and URS-8 strains, URS-6 showed higher degradation rate that might be attributed to increasing the bioavailability of pyrene as well as high activity of naphthalene dioxygenase enzyme (
For URS-10, differet pattern of degradation was observe in which this strain degraded small amount of pyrene in the PAHs mixture, and degraded considerably more pyrene when it was present as the sole subtrate (
As shown in
The above-mentioned bacterial strains, including but not limited to, Pseudomonas, and Rhodococcus, can be mixed culturing for enzyme production. Two types of fermentation media (minimum salt media (MSM) and nutrition broth (NB)) are compared for enzyme production. The NB media contains all the elements for growth and is not selective and the MSM media can provide inorganic nutrients for the growth of bacteria. Both media are supplemented with 50-500 mg/L of crude oil as a mixture of enzyme inducers. Using standard media can support the high-cell density of cultures and can provide all needed nutrition of mixed-culture of bacterial strains. For example, produced biomass in NB is around 5-fold higher than MSM, and enzymes are produced 10-fold higher in NB compared to MSM. Using pure inducers (such as naphthalene, anthracene, phenanthrene, pyrene, and benzo[a]pyrene) results in the induction of certain enzymes NOT all the target enzymes. For example, naphthalene can induce the production of naphthalene dioxygenase and catechol 2,3 dioxygenase in Pseudomonas strains NOT lipase and toluene monooxygenase.
Crude oil assimilating resting cells including Pseudomonas sp. URS-5, Pseudomonas sp. URS-6, Pseudomonas sp. URS-8, and Rhodococcus sp. URS-10 was applied for the production of PAH degrading enzymes. The time-course experiments showed that 37 hours of incubation might be the best time when the production of enzymes is close to maximum when growth reaches its stationary phase. Ultrasonic dispersion is a bioavailability improvement method used prior to incubation to reach favorable biodegradation efficiency by PAH-utilizing microorganisms. Table 1 showed the results of cell lysates from cultured bacteria in NB supplemented with 100, 250 and 500 mg l−1 of crude oil as inducer. As seen in this table, the concentration of the supplement (inducers) is one of the determining factors in enzyme expression7. The cell lysate from cultured Pseudomonas sp. URS-6 in the presence of 250 mg l−1 of crude oil showed high naphthalene dioxygenase and catechol 2,3 dioxygenase activity, while cell extract from Pseudomonas sp. URS-8 and URS-5 showed high Protocatechuic acid 3,4-dioxygenase, and 1-hydroxy-2-naphthoate hydroxylase activity, respectively. Rhodococcus sp. URS-10 showed high activity of pyrene dioxygenase. Moreover, the extract cells from the cultured bacteria in the presence of 100, 250 and 500 mg l−1 of crude oil showed a significant difference in activity of the enzyme in order of 500>250>100 (p<0.05), and 250 mg l−1 was the optimal concentration for enzyme production.
Pseudomonas sp. URS-8, and Rhodococcus sp. URS-10. (T = 10° C., pH = 7, Salinity = 10%)
Pseudomonas sp. URS-5
Pseudomonas sp. URS-6
Pseudomonas sp. URS-8
Rhodococcus sp. URS-10
As discussed previously, removal of more than 50% is achieved for targeted PAHs in the multi-culture of bacteria than the monoculture of isolated strain (
The production of target enzymes was carried out in 250 ml serum bottles containing 50 ml of NB supplemented with 250 mg l−1 of crude oil. As can be seen in
Large-scale production of EnBooT revealed that the yield coefficients (overall specific cell yield on substrate) and total productivity cannot reach their highest theoratical values with the cultivation at current stirred tank fermenters in spite of controlling oxygen transfer through pressurization of the reactor, agitation, air inlet and feed rate. Stired-tank reactor is the most ccommonly used gas-liquid contactor that often cinsists of flat-blade turbine for gas dispersion and agitation. Traditionally, rushton turbine (RT) impeller used for the process of gas-liquid stirred reactor resulted in poor gas dispersibility in the regions far away from impeller due to limited gas handling capacity of RT impeller and drastic pressure. Thus, a custom- design bioreactor was needed to balance the culture's oxygen demand over the full biomass range thereby cells consume oxygen at such rate high rate that the maximum possible oxygen utilization value was approached. The multi-impeller stirred tank with draft tube design configuration consisting of an improved Rushton turbine combined with a pitched-blade turbine offers the advantage of enhanced media circulation, mixing and gas-liquid mass transfer coefficient for suspending solids (biomass). This fermentation has design criteria similar to mechanically stirred hybrid airlift reactors fermenters. The upper impeller is a down-pumping axial flow impeller (e.g., six-pitched blade disc turbine impeller). In order to achieve relatively high local gas holdup and long bubble residence time, the circulation of the fluid below the upper paddle is downward-directed, opposite to the mainstream of the bubble. The gas introduced from the gas sparger can be effectively dispersed along the radial direction above the bottom impeller (e.g. Punched rigid-flexible impeller) and rises upward in the annular region between the tank wall and the draft tube. Punched rigid-flexible impeller was proposed to improve the gas-liquid dispersion performance in the reactor.
To improve production efficiency resulting in cheaper enzymes, a fermentation process was developed by using a compact production plant for maximum responsiveness and mobility (
To confirm the presence of target enzymes in the crude cocktail obtained from mixed-culture of PAH-degrading bacteria, the liquid chromatography-Tandem mass spectrometry (LC-MS/MS) was carried out. This method can provide substantially more information in the identification of enzymes in a mixture compared to conventional biochemical assays10. The identified proteins with more than 1% false discovery rate (FDR) are listed in Table 2. Five PAH-degrading enzymes were identified: naphthalene dioxygenase, naphthalene 1,2-dioxygenase, dihydrodiol dehydrogenase, catechol-2,3-dioxygenase, salicylaldehyde dehydrogenase. The sequence of the crude cocktail showed the presence of other proteins (such as membrane proteins, stress-response proteins, ATP-binding proteins, etc.), however, they did not show strict identity to the sequences of any proteins of known PAH-degrading function in the mixture.
1Molecular mass was calculated from the respective UniprotKB entry (Theoretical Mass).
2Molecular mass was obtained from LC-MS/MS (Experimental Mass).
Several proteins that are associated with oxidative stress, which prevent the unfolding of proteins were detected. High levels of reactive oxygen or chlorine species (ROS/RCS) cause protein unfolding and aggregation in the cytosol. To cope with this stress, bacterial cells employ ATP-independent chaperones such as the DnaK system and the GroEL system, which bind unfolded or aggregated proteins and maintain their solubility19. It is reported that the oxidation of PAH trans-dihydrodiols to catechols generates reactive ROS.20. The presence of chaperones in the crude cocktail obtained from mixed-culture of PAH-degrading bacteria indicated that growth on these compounds induces a stress response. Similarly, Tomas-Gallardo et al., (2006) reported that Rhodococcus sp. strain TFB produced 60-kDa Chaperonins in the presence of phthalate. They identified phthalate-induced proteins using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS/MS)21.
Six different temperatures including 10, 15, 20, 30, 40, and 50° C. and seven different pH values including 3, 4, 5, 6, 7, 8, and 9 were tested for enzyme activity assays. For the salinity test, five solutions of enzyme mixture were mixed with different NaCl concentrations ranging from 2 to 10% (w/v). The preferred temperature and pH values for the enzyme mixture are 20° C. and 7 respectively. Salt concentrations up to 5% have no effect on enzyme activities.
This procedure is generally outlined in
Briefly, 0.5 g of the impacted sediments was transferred in a crimp sealed serum bottle (150 mL) containing 25 mL of MSM supplemented with 200 mg/L of oil as the sole carbon source and incubated for a month at 4° C. After 1 month of incubation of the final enrichment culture on the mixture of oils, aliquots (100 μl) of 10−10 dilutions, in MSM were spread on agar plates (M3 and NB agar) through the conventional spread-plate technique and then incubated at 15° C. for 4 days. After that, single colonies with different morphology were selected and each of them inoculated to the freshly prepared MSM or NB medium (50 mL) and used as inoculum for degradation and enzyme production screening7,22.
To investigate the ability of individual strains to degrade PAHs at 15±1° C., 6×109 CFU/mL (exponential phase bacteria) of each bacterial cell were spiked to each sample. All tests were performed in MSM and PAHs (naphthalene, anthracene, phenanthrene, and pyrene) as the sole carbon sources. To control the freely dissolved concentration of PAHs rather than the total concentration, the PAHs were loaded on the silicone O-rings before culturing the bacteria as described in the next section. The mixture was then incubated at 15±1° C. on a rotary shaker at 150 rpm. These conditions were selected based on optimum growth and aerobic PAH biodegradation using the above-mentioned genera 23. The biodegradation efficiency was calculated by the following Eq. (1)
where, Co and Cn are the concentration of test and abiotic control, respectively. Among a total of 18 isolates obtained, four isolates were selected based on their ability to grow on PAHs in separate and mixture substrate tests at 15±1° C. Bacterial growth was measured using optical density (OD) with a spectrophotometer at A600 nm (OD600 nm˜1-1.2 that corresponded to 1.1×109 cells/ml).
Finally, the selected colonies were identified with 16S ribosomal RNA gene, the gene fragment was amplified using the following universal primers for PCR: 27F (5′-AGAGTTTGATCCTGGCTCAG-3′), 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplified products were sequenced and then the 16S rRNA fragment sequences were compared with the reference sequences using the BLASTn program) in the National Center for Biotechnology Information (NCBI) database24. Thereafter, the phylogenetic analysis was developed using the neighbor-joining algorithm of MEGA 7.0 software with 1000 bootstraps25.
For soil application, enzymatic biodegradation in soil involves key factors that should be verified in batch tests and soil columns. Key factors are enzyme stability, degradation kinetics, dispersion of enzymes, and availability of contaminants. The adsorption of enzymes/proteins on the soil solid phase is known as a quasi-reversible phenomenon with great significance. Adsorption can stabilize enzymes by increasing their resistance toward denaturation. However, the adsorption of contaminants can reduce the bioavailability of the substrate for the enzymatic reaction. Dispersing aids such as surfactants, biosurfactants can be added to the enzyme mixture.
The soil samples used for column test experiments have been collected from a contaminated site in Quebec, Canada. The initial content of metals is reported in Table 6 which is applicable norms in Quebec for commercial and industrial uses. The soil samples were originally contaminated with PAHs (Table 3) and have been artificially contaminated until an initial concentration of 1000 μg/kg for each target PAHs (1:1:1:1) was obtained (average concentration in site). Sieving analysis was carried out according to the ASTM D422-63 procedure (ASTM 2007). Grain size distribution and characteristics of the soil are described in Table 4. Particle-size analysis of the soil is shown in
Basic characteristics of the soil, such as zeta potential (
The soil columns used were made from a stainless-steel cylinder (wall thickness of 2 mm, 3.5 cm in diameter, and 14.5 cm in length (140 mL)). Each set had two replicates. Tubes were clamped between Teflon end reservoirs and stainless-steel endplates. To avoid particle losses, one stainless-steel mesh was inserted at both the inlet and outlet ends of the column, and two Vitton© O-rings were used to seal the cylinder with the endplates. Column packing and water saturation were carried out according to Martel and Gelinas (27. Briefly, dry packing was carried out by pouring discrete soil portions (with a spoon) into the column and then mechanically compacting them with a plunger. The soil portions were deposited in layers of 1 cm and each layer was artificially contaminated with selected polyaromatic hydrocarbons using a micropipette of 100 μL to a final detected concentration in soil. To ensure hydraulic connectivity between the layers, lightly scarifying the soil surface (0.5 cm of each layer) was carried out after compaction and before the addition of another layer. This procedure was repeated several times until the top of the soil column was reached (Gilbert et al. 2014). The bulk density of the soil after packing was 1.8±0.02 g/cm3. The columns were saturated with water overnight, following the injection of CO2 at a pressure of 4 psi to remove entrapped air for complete water saturation. As CO2 is a water-soluble gas, its bubbles can quickly dissolve in water. The soil columns were allowed to saturate with deaerated water (approximate 5 pore volume) using a peristaltic pump (2 mL/min) from bottom to top to eliminate CO2. Polyethylene tubes were used for the inflow and outflow of the aqueous solutions.
For laminar flow through the porous column bed, the hydraulic conductivity K of each column was calculated using Darcy's law (Eq. (2)) following a permeability test which was conducted in duplicates. Three hydraulic gradients were imposed for each column at 5, 10, and 15 cm/cm (
This range of hydraulic gradients might be appropriate for the packed column because it did not alter soil fabric, structure, and the rearrangement of the soil particles and it did not result in channelization in the cracks in the soil samples (Mohamed and Paleologos 2017).
Where K is hydraulic conductivity (cm/s), Q is the flux (cm3/s), A is cross-sectional area (cm2) H2 and H1 are the hydraulic heads and L is the length of column (cm). Hydraulic conductivity, indicating permeability of porous media, measurement is necessary to study the ability of fluid (enzyme mixture in aqueous solution) to flow through the porous media and evaluate the enzyme accessibility to contaminated soils. The pore volume of each column was measured according to Eq. (3):
PV=m
1
−m
2
−m
3 (3)
Where, m1, m2 and m3 are the saturated soil column mass, the mass of water in both ends reservoir (including the attached tubing sections at the time of water injection) and the mass of the dry column respectively. The conversion of the mass of water into volume was assumed at room temperature to calculate the pore volume. The pore volume and the porosity (the media's own internal porosity (pore volume/total volume of the soil column) of each packed column were evaluated28, 29.
The dynamic behavior of water flowing into the packed soil columns was described by measuring the axial dispersion coefficient to evaluate the assumption of dispersed plug flow. The axial dispersion coefficient (DL) in the packed column was measured by injecting a conservative tracer (Br−). This ion is not initially present in soil and in enzyme solution, is not affected by soil adsorption, and has no effect on enzymes activity. The concentration of Br− collected in the effluent samples over time was measured using an ion-selective electrode detector. Dispersity by using the one-dimensional Ogata-Banks (1-D) dispersion according to Eq. (4):
Where, C is the concentration in column outflow, C0 is the initial Br concentration (1000 μg/L in this study), erfc is the complementary error function, x is the depth (position in the column), v is the pore water velocity, and t is the time. Dispersion coefficient measurement is needed to evaluate if the column is packed properly and if a diffusion-dominated flow may exist. The volume of Br solution injected at C/C0=0.5 gives the transport pore volume of the column. The ratio of the volume of enzyme solution injected at C/C0=0.5 to the one of Br gives the retardation factor (Renzyme) or the adsorption coefficient (KD) of the enzyme in the soil according to Eq (5) and (6). This is very important information to calculate how much enzyme solution is needed to flood completely the porous media while achieving an enzyme activity that is equivalent to the solution being injected. Fetter et al. (1999) showed that the Freundlich sorption isotherm coefficient (Kf) can be calculated using the retardation factor as follow
Where R is the retardation factor, θ the porosity, ρb the bulk density and n Freundlich isotherm constant. (Table 5).
(1) Soil density was selected based on site soil density ranging 1.7-1.9 g/cm3.
To design the best-formulated enzymes for biodegradation of specific contaminates, site characterizations such as pH, annual temperature, contaminant concentration, salinity, metal concentration are needed. For example, the contaminated soils used in this study are collected from a contaminated site in Quebec, Canada. This site is selected because of its intense oil contamination which contained ˜1,000 mg/kg of crude oil in the soil. The initial characterizations of the samples are presented in Table 6.
Risk assessment can help to determine goals of remedial action at a site. The maximum level of residual risks that remain at the site after clean-up needs to be specified. The concentration value of the target contaminant is referred to as a preliminary remediation goal. The preliminary remediation goal is the average concentration of target chemicals in an exposure area that will yield the specified target risk in an individual who is exposed at random within the exposure area. The average concentration after remediation is required to be reduced to the preliminary remediation goal or below. Since the contaminated site is located in the province of Quebec (Canada), total PAHs 0.1 mg/kg of soil is considered as the local soil threshold and a benchmark for evaluating the efficiency of treatment30.
The contaminated soil and groundwater were provided from a petroleum site with intense contamination of oil. The mineralogy of soils varies in different parts of the site but, in addition to quartz and feldspar, clay minerals and carbonates were also found. The results of groundwater characterization also confirmed the presence of carbonates (hard water) with a pH of 7.2±0.4. The soil sample comprised 59% of particles with a size range between 1-5 mm, 38% with a size range of 250 μm-500 μm and 3% of very fine particles (less than 250 pm in size). The physical characteristics of the soil were determined as follows: moisture content of 24.17±0.5%, pH 7.1±0.2, total solids: 75.36±0.91%. Batch tests indicated that 85-90% of PAHs were degraded after 4 weeks (Table 7).
The packed soil columns include six treatments are shown in Table 8. The enzyme mixture contained around 30 U/mg of naphthalene dioxygenase, 40 U/mg of dihydrodiol dehydrogenase, and 40 U/mg of catechol-2,3-dioxygenase, 10 U/mg of salicylaldehyde dehydrogenase, 10U/mg of salicylate hydroxylase and 5 U/mg of Trans-2-carboxybenzalpyruvate hydratase-aldolase was injected into the soil column as influents until saturation. The effluents were collected for each 5 mL at regular intervals to determine the total protein concentration and Br concentration (
As mentioned in Table 9, the soil column tests were carried out over 8 weeks at 15±1° C. (average annual temperature of the targeted site). The enzyme injections were repeated after 4 weeks for the columns that were planned to open after 8 weeks. The time intervals for injection were selected based on the enzyme stability results in batch tests.
To determine the PAHs concentration in soil samples, about 0.1 g of soil were taken from the head, middle and bottom part of the soil column (depth of 2, 7, and 12 cm) in triplicate for each part. Each sample was homogenized, and a 5 g subsample was placed in a 43 ml vial where it was mixed with 5 mL of hexane as mentioned in PAH concentration analysis. The results showed that 50-70% of PAHs were degraded after 4 weeks (Table 9).
a head (depth of 1-2 cm),
b middle *dept of ((depth of 7-8 cm) and
c bottom (depth of 12-13 cm) of soil column.
Laboratory Experiments Approach for Preparation and Characterization of the Jellyfish-Like Device
Spectrum® modified poly(ether sulfone) mPES hollow fibers with a molecular weight of (MWCO) of 10,000 Da (D02-E010-05-N, Spectrum Labs, USA) were used for multiple enzyme and cofactor immobilization. Since the molecular weight of PAH substrates is about 169 and the molecular weight of the studied enzyme is more than 10,000 Da, Spectrum® mPES membrane appeared appropriate for this study. Moreover, thin-skinned hydrophilic modified polyether sulfone (mPES) hollow fibers are void-free and anisotropic in a structure that provides high relative hydraulic permeability and flux rates at a maximum pressure of 30 psi. Hydrophilic neutrally charged Spectrum® mPES hollow fiber minimized non-specific protein binding as well as fouling by hydrophobic molecules31. The hollow fiber ultrafiltration module consisted of 10 fibers with an internal diameter of 1.0 mm, a length of 45 cm, a wall thickness of 0.1 mm, and the total surface area was 58 cm2.
After selecting the membrane, the second step was to ensure that these membrane modules were intact. To do so, the diffusion test method was applied to evaluate its integrity.
The second step was the systematic exploration of membrane hollow-fibers characteristics including the permeation of substrate and the retainment ratio of the co-enzymes. The permeation equipment, shown schematically in
Recycling-pump speed was fixed at 50 ml min−1 to circulate enzyme solution or co-enzyme (NADH) in 20 mM Tris-HCl buffer pH=9. The retention of 1 mM enzyme or co-enzyme (NADH) inside the lumen of hollow fiber membrane was studied by calculating the retainment ratio as follow
Where V, V0 C, and C0 are the volumes of the inner solution before and after the permeation experiments and the solute (i.e., coenzyme) concretions at time t, and t0. The concentration of co-enzymes (NAD+) retained in pre-selected ultrafiltration with 10 kDa were measured via Biowave UV-vis spectrophotometer at 260 nm, respectively.
To determine the permeation coefficient for substrates, single fiber reactor consisting of a gas-liquid chromatograph was applied. Experimental data were analyzed according to the mass exchanger equation (Eq. (8)):33
Where K0, Ki, a, a+b, Di2 and Ki2 are mass transfer coefficients for the external flowing stream, mass transfer coefficient for the internal flowing stream, the radius of the hollow region in fiber (I.D), the diffusion coefficient of permeating solute in fiber wall and solubility of permeating solute in fiber wall respectively. The high flow rate of the internal flowing stream (100 ml/min) and vigorous agitation at 500 in a 1L flask were adjusted so that the first and second terms in the denominator of the quantity inside the brackets were small (the absence of internal and external diffusional limitation). By assuming the high mass-transfer coefficient for the external and internal flowing streams, the permeation coefficient of the substrate can be calculated (Pi2=ki2Di2).
To study the benefit of using hollow fiber modules for enzyme-catalyzed reactions, the performance of a beaker-type hollow fiber system was evaluated theoretically based on simple mathematical models and then examined experimentally in the degradation of the model substrate. Ultrafiltration oscillation is induced in the beaker side of the reactor by introducing a pulsatile flow to the hollow fiber lumen as well as by maintaining outflow from the lumen at the average value of the pulsatile inflow rate. The phenomenological transport equation may be written according to Kedem and Katachalsky34
J
s
=P
m
ΔC+σJ
w
C
b (9)
Where σ, Cb, ΔC, Js, and Jw refer to sieving coefficient, solute concertation in the bulk solution phase, concentration difference between the two compartments, substrate, and water flux, respectively. The sieving coefficient
in the absence of concentration polarization can be 135 Mass balance for the substrate in compartments I and II, respectively, are
Where A and Q are mass transfer areas of hollow fiber membrane (cm2) and volumetric flowrate (cm3). If we assume square or sine wave ultrafiltration swing, in the absence of concentration polarization, water flux varies with time as
Le d and d are the difference between the outlet and the inlet flowrates in the lumen appears as ultrafiltration and the fraction of average outlet velocity. For sine-wave ultrafiltration swing, Eq. (10) and (11) can be transformed into dimensionless forms by using the following dimensionless variables
F and W are dimensionless frequency for square and sine wave pulsations. Eq. (12) and (13) were solved numerically using the Runge-Kutta scheme to study reactor performance with ultrafiltration swing.
The release of PAHs to water following the oil spill and its biodegradation in the presence of pre-selected strains and obtained enzymes solutions were confirmed by GC-MS (Agilent model 6890 GC, 5973 MSD) analyses which were carried out at 0, 30, 47 days of incubation.36 Biotic and abiotic controls were used in the same conditions. The preparation of samples for GC/MS analysis was described elsewhere in detail.36 The fermentative medium (20 mL) containing the remaining PAHs and metabolites and surrogate standards (Acenaphthene D10, Chrysene D12, Anthracene D10 each—0.7 μg·mL−1, Pyrene D10—1.0 μg·mL−1; purchased from Chromatographic Specialities Inc., Canada) were added to glass tubes (50 mL with covers). After mixing, 20 mL of Dichloromethane (HoneyWell™, Fisher Scientific, Canada) was added to each sample and mixed thoroughly for 30 minutes. 5 g of silica (SiliCycle, Canada) was added, and the tubes were shaken on an oscillating shaker for 10 minutes. 5 mL of supernatant were withdrawn and transferred in a calibrated glass tube, reduced to 1 mL (35° C. with nitrogen gas), and then analyzed by GC/MS (1 mL/min, 300° C., 35 minutes).36.
It is very important to control the freely dissolved PAH concentration and keep it unchanged throughout the experiment by equilibrium partitioning from the silicone. Silicone O-rings (1 g each) were used as passive dosing format to provide constant freely dissolved concentrations and avoid spiking with cosolvents as described elsewhere.37 In brief, the rings were loaded by equilibrium partitioning i.e., pushing PAHs (log Kow,3.33-4.88) from a methanol suspension of naphthalene, anthracene, phenanthrene, pyrene into the polymer, followed by methanol removal with water.38 The performance of passive dosing was determined by studying O-ring loading, substrate release into the medium and equilibrium concentration in the medium. During the loading of PAHs, individual PAHs were dissolved in methanol (loading solvent) to saturation level. To create the PAHs mixture, the concentration in the loading solution was calculated using partitioning coefficients for each PAH from Gilbert et. al. (2015).39 Then, cleaned and dried O-rings were added to 50 mL volumes of loading solution kept at 15±1° C. A criterion for successful application of the passive dosing system was ensuring the mass of PAHs dissolved into methanol was much greater than that partitioned into the silicon O-ring which in turn was greater than that partitioned into the MSM37. After 24, 72 and 128 h, O-ring was removed from each loading batch, and they were transferred into MSM to measure O-ring (Crest) and freely dissolved concentration of each PAH (Cfree) at 0.33, 0.66, 1.33, 2.66, 5.33, 12, 24,48, and 72 h. The partition coefficient between different phases (KMethanol:Oring and KOring:MSM) were obtained using the measured Ctest,initial and Cfree, initial. The obtained partitioning coefficient (KOring:MSM) was applied to calculate the required O-ring volume to avoid depletion (<5%) using the following Equation:
With a 5 mL MSM and partitioning coefficients from Table 10, the O-ring volume needed for each PAH and PAH mixture were determined. The initial concentrations of freely dissolved PAHs (Cfree initial) and O-ring concentrations (Ctest) in medium samples were calculated via the approach published by Butler et. al (2013).37 Finally, for the determination of the losses, freely dissolved concentrations of PAHs were directly measured in medium samples to compare with calculated Cfree initial.
Two controls were included to study biodegradation versus dissolved concentrations. The first control contained 5 mL of strain inoculating suspension in passive doing vials with O-rings to account for losses through volatilization. The second control contained 5 mL of MSM without any strain in passive dosing vials in the presence of O-ring loaded with PAHs to account for losses through biodegradation.
Table 10 shows the partition coefficients obtained from measured PAH concentrations in loading solution, MSM, and O-ring. The losses of naphthalene, anthracene, phenanthrene, and pyrene through volatilization for control 1 were 19, 4, 0 and 0%, respectively. To buffer the losses, a 5-higher volume of O-ring was used for naphthalene to avoid depletion of more than 5%.
As mentioned previously, jellyfish may have a thing to teach us.
The test to measure diffusion air flow rate was carried out as described elsewhere.41 Briefly, ultrafiltration (UF) membranes were wetted and pressurized with air to about 30 psi. The shell side is isolated (Valve 2 [V2] is closed and V3 open to measure the volume of displaced air or liquid), and the volume of air flowing through the membrane is measured and compared with values for diffusive airflow for the intact membrane.
The measured diffusion specification less than 2 SCCM/0.1 m2 area showed that the selected module was intact.
The ratio of permeation coefficient for anthracene used as model substrate (2x 10-3 cm2s−1) to ultrafiltration velocity (54×10−3-75×10−3 cm 2s−1) obtained in this section will be applied to determine the conversion rate of the substrate at different experimental conditions. The other important permeability characteristic of the selected membrane under experimental condition are coenzyme retention that affects the performance of hollow fiber immobilized enzymes. The membrane reactor system selected benefits a high recycling speed (range of pumping rate from 0.2 ml/min to 200 ml/min) and it was assumed that in this reactor there is no steric problem for interactions between enzymes and coenzymes, and there is a force convection transport through the membrane of the substrate.
As shown in
For kinetic analysis of such vertical reactor, enzyme and substrate solution recycle was modeled as a two-compartment membrane reactor separated by an ultrafiltration membrane. The solutions contained in the enzyme compartment (II) and the substrate compartment (I) are assumed to be fully mixed. Reaction rates at various substrate concentrations were measured in free solution and at a recirculating flow rate of 200 ml/min. The Lineweaver-Burk equation was applied to calculate Km of soluble enzyme and hollow fiber immobilized enzyme through the linear fitting. The observed values for the anthracene relate to a multienzyme system of target enzymes. Biotic and abiotic controls are used in the same conditions to distinguish between adsorption and biodegradation for anthracene removal. Two parallel control experiments with inactivated enzyme and without solution circulation were also run. As shown in
To investigate the effect of electrospun nanofibers on the performance of the reactor system, it has been applied in a recycled hollow fiber enzyme reactor containing oil-water emulsion (e.g., water, anthracene, and lubricant oil) in compartment I (
A great deal of effort has been made to decrease the tendency of membrane fouling through the application of different techniques to improve membrane properties (e.g., pore size, hydrophilicity, etc.) or pre-treatment of feed stream (e.g., flocculation and microfiltration (MF)). Thus, it is important to take care of the particle loading before the feed gets to membrane units42.
Irwin, R. J., Mouwerik, M. V., Stevens, L., Seese, M. D., & Basham, W. (1997). Environmental contaminants encyclopedia, naphthalene entry. National Park Service, 1-80.
Minnesota Department of Health. (2019). Anthracene and Groundwater. https://www.health.state.mn.us/communities/environment/risk/docs/guidance/gw/anthrac eneinfo.pdf
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The above disclosure generally describes the present invention. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
All publications, patents and patent applications cited above are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
This application claims priority from a provisional application U.S. application No. 63/339,357 filed May 6, 2022, which is hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| 63339357 | May 2022 | US |
