WO2012151673A1 - Utilisation des algues dans des procédés de dépollution - Google Patents

Utilisation des algues dans des procédés de dépollution Download PDF

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Publication number
WO2012151673A1
WO2012151673A1 PCT/CA2012/000432 CA2012000432W WO2012151673A1 WO 2012151673 A1 WO2012151673 A1 WO 2012151673A1 CA 2012000432 W CA2012000432 W CA 2012000432W WO 2012151673 A1 WO2012151673 A1 WO 2012151673A1
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WO
WIPO (PCT)
Prior art keywords
euglena
nanosilver
constituent
sample
silver
Prior art date
Application number
PCT/CA2012/000432
Other languages
English (en)
Inventor
Adam J. NOBLE
Shayla R. LARSON
Original Assignee
Noble Adam J
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Noble Adam J filed Critical Noble Adam J
Publication of WO2012151673A1 publication Critical patent/WO2012151673A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/32—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae
    • C02F3/322—Biological treatment of water, waste water, or sewage characterised by the animals or plants used, e.g. algae use of algae
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/10—Inorganic compounds
    • C02F2101/20—Heavy metals or heavy metal compounds
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2209/00—Controlling or monitoring parameters in water treatment
    • C02F2209/003—Downstream control, i.e. outlet monitoring, e.g. to check the treating agents, such as halogens or ozone, leaving the process
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2303/00—Specific treatment goals
    • C02F2303/18—Removal of treatment agents after treatment
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2520/00—Use of whole organisms as detectors of pollution

Definitions

  • Patent Application 61/484,327 filed May 10, 201 1 entitled “USE OF ALGAE IN RECOVERY", the entire subject matter of whichjs incorporated herein by reference.
  • the present disclosure relates to use of algae, for example, Euglena in recovery and isolation of nanosilver, other nano value metals, nano particles and other materials as described hereinbelow, capable of being absorbed or otherwise consumed thereby.
  • Nanosilver is a compound used in commercial and industrial processes to inhibit the growth of bacteria and fungi in such diverse applications as acne treatments, dressings, feminine hygiene products, babies' bottles, socks, undergarments, food preparation equipment, air-filtration devices, surface disinfection, and food containers.
  • the poultry, livestock, and aquatics industries use it to prevent disease and enhance weight gain.
  • Catheters and dialysis devices with nanosilver coatings are now available, and it is anticipated that artificial joints, pacemakers, and artificial heart valves will soon be manufactured using these coatings as well. Consequently, humans may be exposed to high levels of nanosilver due to its widespread use but its potentially detrimental effects on living organisms make its use controversial.
  • Nanosilver particles are encapsulated in gluconic acid, do not break down, and may be released into the water system in wastewater effluent. These particles range between 1 and 100 nm in diameter, which may allow them to pass through the cell membrane of living organisms. If nanosilver is harmful to living organisms, then plants and animals in aquatic ecosystems are at risk. If nanosilver does not break down, there must be an accumulation of its levels in the environment and there are serious implications as to whether wastewater treatment facilities are capable of removing nanosilver from drinking water. While some initial studies have maintained that treatment facilities are, indeed, capable of removing the necessary silver from effluent water, more recent studies indicate that this may not be accurate.
  • nanosilver particles represent a source of silver ions, which are reported to be toxic.
  • nanosilver levels in biosolids exported from wastewater treatment facilities for use as fertilizer on agricultural lands There is evidence that nanosilver has a toxic effect on the denitrification process that is essential in the breakdown of organic matter to form soil. In fact, one study showed nanosilver to be even more toxic to denitrifying bacteria than ionic silver.
  • Algae are often used as indicators to evaluate the existence of water pollution in bodies of water.
  • Euglena is a single-celled organism that is a primary producer in freshwater ecosystems. This form of algae has both plant and animal characteristics and is, therefore, representative of most organisms in the water system.
  • the Euglena is operable as an indicator of the impact of this chemical on living organisms in freshwater.
  • the present disclosure provides a method of recovering pollutants from a water sample, comprising adding to the sample a quantity of algae, isolating the algae from the sample and determining a level of pollution in the sample according to an identified amount of the pollutant absorbed by the algae.
  • the algae includes Euglena.
  • the pollutant includes metals.
  • the metals may include value metals, such as compounded silver.
  • the pollutants include capped nanoparticles.
  • a filter element comprising a substrate carrying Euglena, in sufficient quantity to absorb pollutants from freshwater passing therethrough.
  • a method of filtering fresh water comprising the step of providing the filter element of claim 7, passing a sample of freshwater therethrough, isolating the Euglena from the substrate and recovering pollutants from the Euglena.
  • a method for remediation of a sample of fresh water comprising depositing in the sample a sufficient amount of Euglena to absorb waterborne pollutant materials capable of passing the membrane of Euglena and removing the Euglena from the sample.
  • the method further comprises recovering the pollutant materials from the Euglena.
  • the pollutant includes capped nano metals or nano non- metals.
  • the capped nano metals includes gold, silver, platinum, titanium and lead.
  • a method of sequestering a constituent from a water sample comprising adding to the sample a quantity of Euglena so as to sequester the constituent therein.
  • the constituent is a transition metal component.
  • a method of indicating water quality comprising providing a non-polluted water sample, providing a first quantity of Euglena, adding the first quantity to the non-polluted water sample, providing a polluted water sample, providing a second quantity of Euglena, adding the second quantity of Euglena to the polluted water sample, culturing the first quantity of Euglena and the second quantity of Euglena for a given time period, and comparing the growth or an attribute of the first quantity of Euglena to the second quantity of euglena, so as to determine in the respective growth rates.
  • the attribute is the shape.
  • the Euglena forms a ball shaped configuration in the polluted water sample.
  • the second quantity forms a ball shaped configuration in the polluted water sample, while the first quantity adopts an oval shaped configuration in the non-polluted sample.
  • a method of indicating impact of a contaminant on organisms in a water sample comprising providing an uncontaminated water sample, providing a first quantity of Euglena, adding the first quantity of Euglena to the uncontaminated water sample, providing a contaminated water sample, providing a second quantity of Euglena, adding the second quantity of Euglena to the contaminated water sample, culturing the first quantity of Euglena and the second quantity of Euglena for a given time period, and comparing differences in population or attribute of the first quantity of Euglena to the second quantity of euglena, so as to determine respective growth rates thereof.
  • a method of recovering a constituent from a water sample comprising providing a water sample with the constituent, adding a quantity of Euglena to the water sample, and after a given time period, recovering the Euglena from the water sample, and isolating the constituent from the Euglena.
  • the isolating includes isolating the constituent from a vacual cell structure of the Euglena.
  • the constituent is a quantity of capped nanoparticles.
  • a method for isolating capped silver nanoparticles from a silver solution containing a capped silver nanoparticle constituent and one or more of an elemental silver constituent and an ionic silver constituent in the silver solution comprising filtering substantially all of the elemental silver constituent from the silver solution, and/or adding to the silver solution an ionic constituent in sufficient concentration to react with the ionic silver constituent to remove substantially all of the ionic silver constituent from solution.
  • the ionic constituent includes bromide, carbonate, chloride, oxide, phosphate, sulphate and/or dichromate.
  • Figures 1 to 16 are representations of certain observations related to the exemplary embodiments.
  • nanosilver particles provide a greater relative surface area, allowing for maximal exposure of silver within the body. This means that greater absorption of nanosilver could occur at lower
  • nanosilver can diffuse through the cell membrane and release silver ions directly inside the cell; that nanoparticles can be absorbed through the skin, eyes, and nose; and that they can cross the blood-brain barrier. In terms of human health consequences, it has been hypothesized that nanosilver could cause drug to drug interactions within the liver.
  • nanosilver technology has gone from billions of dollars to an estimated trillion in the last five years.
  • millions of tons of nanosilver are produced yearly and it has been estimated that, in Europe, up to 15% of the total silver added to the environment each year may come from textiles and biocidal plastics treated with nanosilver. It has been estimated that over 400 tons of silver are released into wash water in Europe alone annually from nanosilver embedded fabrics such as socks.
  • nanosilver embedded fabrics such as socks.
  • nanosilver levels in our freshwater ecosystems are not being monitored currently and may already be approaching a lethal threshold which could challenge the sustainability of these ecosystems.
  • Water resources and ecosystem management concerns would dictate that further study needs to be done on this source of water pollution.
  • the possible effect of nanosilver on water quality suggests that there could be potential for negative repercussions related to the health of all living organisms.
  • Nanosilver will be shown to negatively affect structure and function of
  • EXPERIMENT #1 DETERMINATION OF THE LC50 FOR EUGLENA CULTURED IN NANOSILVER
  • a concentrated Euglena culture was grown in algae optimum growth media, and nanosilver solutions of: 0, 5, 10, 40, 75, and 100 ⁇ g/L concentrations were added.
  • Euglena cultures were grown in 10 ⁇ g/L and 50 ⁇ g/L nanosilver solutions. Chlorophyll concentration and cell counts were determined at 0, 1 , 2, 3.5, 5.5, 24, 48, 72, and 96 hours.
  • a dilution series of Euglena cultures (0.25, 0.5, 1.0, 3.75, and lOmL of the stock culture) was grown in nanosilver solutions of: 0, 1 , and 10 ⁇ g/L nanosilver. Chlorophyll concentration and cell counts were then determined.
  • EXPERIMENT #5 EXAMINATION OF EUGLENA CELLS USING A COMPOUND MICROSCOPE
  • a pipette was used to remove gluteraldehyde from centrifuged Euglena pellets.
  • EXPERIMENT #7 SEPARATION OF NANOSILVER FROM OTHER FORMS SILVER IN SOLUTION Elemental silver, ionic silver, and nanosilver, each in known concentration, were added to de-ionized water to create a solution. A glass fibre membrane filter was then used to remove the elemental silver. Nitric acid was added to the remaining solution to form silver nitrate with the ionic silver. Hydrochloric acid was then added to this solution to react with the silver nitrate and form a silver chloride precipitate. This suspension was centrifuged to separate the silver chloride precipitate from solution. The supernatant was then analysed and compared with the original concentration of nanosilver added to the solution.
  • Nanosilver was shown to be harmful to Euglena with the LC50 for Euglena, grown in the presence of nanosilver, being calculated as 25.0 by determining the decrease in total chlorophyll concentration and then confirmed by cell count (see Figures 1 and 2).
  • an inverse relationship was found between nanosilver concentration and chlorophyll concentration.
  • chlorophyll concentration decreased proportionally (see Figure 1).
  • a similar pattern was found with Euglena cell count.
  • the number of typical, fully formed Euglena cells decreased proportionally (see Figure 2).
  • nanosilver concentration in influent and effluent water collected from a waste water facility were also used to determine nanosilver concentration in influent and effluent water collected from a waste water facility (see Table 1).
  • the nanosilver concentration in influent water was determined to be 7 ⁇ g/L while the nanosilver concentration in effluent water was determined to be 67.0 ⁇ /L.
  • Nanosilver particles were found to pass through the cell membrane and were identified in the cytoplasm and the vacuoles of these Euglena cells (see Figure 12). Table 1. Nanosilver Concentration at Different Locations Along the Trent Severn Waterway
  • the LC50 was discovered to be 25 ⁇ ⁇ of nanosilver for concentrated solutions of Euglena and 0 ⁇ g/L of nanosilver for dilute solutions of Euglena.
  • nanosilver may have a lethal effect on primary producers in our fresh water ecosystems.
  • nanosilver also being used in the poultry, livestock, and aquatic industries to prevent disease and enhance weight gain in animals, we predict that the level of nanosilver contamination in fresh water ecosystems will continue to increase, creating an increased potential for bioaccumulation of nanosilver in fresh water ecosystems and we maintain that the potential for humans to be exposed to detrimental levels of nanosilver on a daily basis similarly will continue to increase.
  • nanosilver' s toxic effect was determined to be dose-time dependent. Cultures with higher concentrations of Euglena absorbed proportionally more nanosilver than cultures with lower concentrations of Euglena. In addition, at 96 hours all the nanosilver had been absorbed in a 50 ⁇ g L nanosilver solution. This result suggests that Euglena has the potential to be a new, previously unrecognized means of removing nanosilver from wastewater. Therefore, we propose that Euglena may serve as a bioremediation measure for nanosilver contamination in freshwater.
  • Euglena is an effective indicator species of water pollution at one or more predetermined sites, based on some knowledge of the sites, where the possibility of pollution may exist where Euglena does not grow effectively. Further, Euglena is believed to be useful in detecting, by changes in its growth rate, negative changes in a fresh water ecosystem, particularly to pollutants, such as nanosilver and other constituents that are capable of passing the membrane of the Euglena. In this case, microscopic examination may identify very small concentrations of nanoparticles, such as nonosilver particles.
  • the LC50 for nanosilver and Euglena in vitro was determined to be 25 ⁇ g/L.
  • nanosilver present in the 1.0 g/L nanosilver solutions At 72 hours, Euglena absorbed 100% of the nanosilver particles present in the 10 ⁇ g/L nanosilver solutions. After 96 hours of exposure to nanosilver, Euglena was found to absorb 99% of the nanosilver present in the 50.( g/L nanosilver solutions.
  • a method was determined and successfully used to isolate nanosilver from solutions containing elemental silver, ionic silver, and nanosilver.
  • Euglena was used to successfully remove nanosilver from contaminated water samples.

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  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Microbiology (AREA)
  • Hydrology & Water Resources (AREA)
  • Environmental & Geological Engineering (AREA)
  • Water Supply & Treatment (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Water Treatment By Sorption (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

Cette invention concerne un procédé de dépollution d'un échantillon d'eau comprenant l'ajout audit l'échantillon d'une quantité d'Euglène pour isoler les algues et la détermination du degré de pollution de l'échantillon selon une quantité identifiée de polluant absorbée par les algues.
PCT/CA2012/000432 2011-05-10 2012-05-08 Utilisation des algues dans des procédés de dépollution WO2012151673A1 (fr)

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US201161484327P 2011-05-10 2011-05-10
US61/484,327 2011-05-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018184120A1 (fr) * 2017-04-07 2018-10-11 Winters Cameron Procédés et utilisations d'exsudats encapsulés et de biomasse d'euglena séchée pour lier un métal
US10773978B2 (en) 2016-08-19 2020-09-15 Noblegen, Inc. Methods and uses of dissolved organic material fractions for binding metal ions

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
AHMED H ET AL.: "Monitoring of Waste Water Samples Using the ECOTOX biosystem and the Flagellate Algae Euglena gracilis", WATER AIR SOIL POLLUT, vol. 216, 2011, pages 547 560 *
AHMED H ET AL.: "Short term Bioassay of Chlorophenol Compounds Using Euglena gracilis", SRX ECOLOGY, vol. 2010, 16 August 2012 (2012-08-16), Retrieved from the Internet <URL:http://www.hindawi.com/archive/2010/421950/ref> *
AZIZULLAH A ET AL.: "Ecotoxiological Evaluation of Wastewater Samples from Gadoon Amazai Industrial Estate (GAIE), Swabi, Pakistan", INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES, vol. 1, no. 5, February 2011 (2011-02-01), pages 959 976 *
BOWER E ET AL.: "Lakefield High School Students Presenting Nanosilver Research in Sweden this Month", PETERBOROUGH EXAMINER, 6 August 2011 (2011-08-06), Retrieved from the Internet <URL:http://www.thepeterboroughexaminer.com/2011/08/06/lakefield-high-school-students-presenting-nanosilver-research-in-sweden-later-this-month> [retrieved on 20120705] *
BRAYNER R ET AL.: "ZnO Nanaoparticles: Synthesis, Characterization, and Ecotoxicological Studies", LANGMUIR, vol. 26, no. 9, 2010, pages 6522 6528 *
DE BASHAN LE ET AL.: "Immobilized Microalgae for Removing Pollutants: review of Practical Aspects", BIORESOURCE TECHNOLOGY, vol. 101, 2010, pages 1611 1627 *
ISHII N ET AL.: "Removal of Rare Earth Elements by Algal Flagellate Euglena gracilis", J ALLOYS AND COMPOUNDS, vol. 408-412, 2006, pages 417 - 420 *
ISHII N ET AL.: "Removal of Technetium from Solution by Algal Flagellate Euglena gracilis", J EVNIRON. QUALL, vol. 35, 2006, pages 2017 - 2020 *
KENNEDY AJ ET AL.: "Fractionating Nanosilver: Importance for Determining Toxicity to Aquatic Test Organisms", ENVIRON. SCI. TECHNOL, vol. 44, 2010, pages 9571 9577 *
LARSON S ET AL.: "The Hazards of Nanosilver Uncovered", INTERNATIONAL WATER INSTITUTE, SIWI, DROTTINGGATAN, SWEDEN., 21 August 2011 (2011-08-21), STOCKHOLM, Retrieved from the Internet <URL:http://www.sivdi.org/documents/SJWPprojectsfinal/SJWPProjects2011/SJWP2011Canada.pdf> *
NAVARRO E ET AL.: "Toxicity of Silver Nanoparticles to Chlamydomonas reinhardtii", ENVIRON. SCI. TECHNOL, vol. 42, 2008, pages 8959 8964 *
REHMAN A: "Heavy Metals Uptake by Euglena proxima Isolated from Tannery Effluents and Its Potential Use on Wastewater Treatment", RUSSIAN JOURNAL OF ECOLOGY, vol. 42, no. 1, 22 March 2011 (2011-03-22), pages 44 - 49 *
STREB C ET AL.: "ECOTOX-Biomonitoring Based on Real Time Movement Analysis of Unicellular Organisms", PROCEEDINGS OF ''LIFE IN SPACE FOR LIFE ON EARTH'', 8TH EUROPEAN SYMPOSIUM ON LIFE SCIENCES RESEARCH IN SPACE, 23RD ANNUAL INTERNATIONAL GRAVITATIONAL PHYSIOLOGY MEETING, KOROLINKA INSTITUTET, September 2002 (2002-09-01), STOCKHOLM, SWEDEN,2-7 JUNE 2002 (ESA SP-501 *
TAHEDL H ET AL.: "Automated Biomonitoring Using Real Time Movement of Euglena gracilis", ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY, vol. 48, 2001, pages 161 169 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10773978B2 (en) 2016-08-19 2020-09-15 Noblegen, Inc. Methods and uses of dissolved organic material fractions for binding metal ions
US11440822B2 (en) 2016-08-19 2022-09-13 Noblegen, Inc. Methods and uses of dissolved organic material fractions for binding metal ions
WO2018184120A1 (fr) * 2017-04-07 2018-10-11 Winters Cameron Procédés et utilisations d'exsudats encapsulés et de biomasse d'euglena séchée pour lier un métal
JP2020516768A (ja) * 2017-04-07 2020-06-11 ノーブルジェン・インコーポレーテッド 金属を結合するための方法並びにカプセル化された滲出物及び乾燥ユーグレナ（ｅｕｇｌｅｎａ）バイオマスの使用
US11053139B2 (en) 2017-04-07 2021-07-06 Noblegen Inc. Methods and uses of encapsulated exudates and dried euglena biomass for binding metal

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