WO2013138899A1

WO2013138899A1 - Euglena biofilter

Info

Publication number: WO2013138899A1
Application number: PCT/CA2013/000204
Authority: WO
Inventors: Adam J. NOBLE
Original assignee: Noble Adam J
Priority date: 2012-03-23
Filing date: 2013-03-07
Publication date: 2013-09-26

Abstract

A system and method for removing metal values and other contaminate materials from water using Euglena algae is disclosed. The Euglena algae is grown, concentrated and exposed to water having metal values and other contaminate materials therein. The metal values and other contaminate materials are sequestered by the Euglena algae so to yield treated water. The Euglena algae having metal values and other contaminate materials sequestered therein are separated from the treated water and the metal values and other contamination materials may be recovered from the Euglena algae. In particular nanosilver is sequestered and recovered from the Euglena algae.

Description

EUGLENA BIOFILTER

CROSS-REFERENCE TO RELATED APPLICATIONS This application is related to and claims benefit of priority to United States Provisional Patent Application Serial Number 61/615,027 entitled "EUGLENA BIOFILTER/^" tiled March 23, 2012 and United States Provisional Patent Application Serial Number 61/726.162, entitled "EUGLENA BIOFILTER,^" filed November 14.2012. the disclosures of which are herein fully incorporated by reference.

FIELD OF THE INVENTION

The present disclosure relates to use of Euglena algae as a biofilter for filtering nanosilver, other nano value metals, nanoparticles and other materials from water.

BACKGROUND

At the present time, Nanosilver (particles of silver, 1 to 100 nm in diameter), a compound that inhibits the growth of bacteria and fungi, is being used in an ever-increasing number of commercial, industrial and agricultural processes and products. Consequently, humans may be exposed to high levels of nanosilver in their daily lives. However, the detrimental effect(s) of nanosilver on living organisms remains controversial and largely untested. Studies demonstrating specific effects of nanosilver on living organisms are limited. It has been shown that Euglena, a photosynthetic protist in aquatic ecosystems, actively absorbs and concentrates nanosilver, causing structural and functional damage to Euglena cells and increasing the mortality rate of this organism. Since nanosilver particles are encapsulated and resist breaking down, it is reasonable to assume that there will be a progressive accumulation of nanosilver in the terrestrial environment from biosol id disposal and in the aquatic environment from wastewater effluent. Prel im inary research has indicated that nanosi lver is released in wastewater effluent in an urban environment.

Prel im inary studies also suggest that nanosi lver poses a potential chal lenge to the sustainability of our freshwater ecosystems and may also pose a serious health risk to humans. Therefore a method needs to be found whereby freshwater treatment faci lities can successfully remove this compound from wastewater effluents. This position provides the basis for the development of a Euglena biofi lter that is capable of removing nanosilver from wastewater. Therefore, it would be desirable to develop a Euglena Biofi lter for functioning as a remediation device for the removal of environmental nanosilver in wastewater effluent.

SUMMARY OF TH E DI SCLOS U RE

The following presents a simplified summary of the general inventive concept herein to provide a basic understanding of some aspects of the d isclosure. Th is summary is not an extensive overview of the disclosure. It is not intended to restrict key or critical elements of the disclosure or to delineate the scope of the disclosure beyond that explicitly or implicitly described by the following description and claims.

In one aspect, there is provided a system for filtering metal-value and other material contaminated water, the system comprising:

- a stock culture tank for growing a Euglena algae culture at a substantially constant density;

- a Euglena stock culture concentrator adapted to receive a substantially constant flow of Euglena algae from the stock culture tank and concentrate the Euglena algae stock culture so as to produce a concentrated Euglena culture therefrom;

- the Euglena stock culture concentrator in fluid communication with a metal- value contam inate water flowthrough system so as to receive the concentrated Euglena culture via a first in let;

- the metal-value and other material contam inate water flowthrough system including a second inlet for receiving the metal-value and other material contaminated water;

- the metal-value and other material contaminate water flowthrough system having a predeterm ined path along wh ich the concentrated Euglena culture and the metal-value and other material contaminated water co-travel so as to provide a residence time wherein the metal values and/or other contaminate materials are sequestered by the concentrated Euglena culture and exits via an outlet along w ith treated water.

In preferred embodiments, the metal-values and other materials present in the

contaminated water include nanosi lver, other nano value metals, nano particles, metal ions, phosphorous-based ions, nuclear waste and other materials.

In some embodiments, the system further comprises a filter system in fluid communication the outlet. The filter system has a fi lter system inlet for receiving the concentrated Euglena culture having the metal values and other contaminate materials sequestered therein and water. The filter system is provided for substantially separating the concentrated Euglena culture having the metal values and other contaminate materials sequestered therein and water.

In a preferred embodiment, the filter system includes at least one filter element that is eatable in a microwave digester for separating metal values from the concentrated Euglena culture. In a more preferred embodiment, the at least one filter element is a glass fibre filter having a pore size of from about 1 .0 μΜ to about 3.0 μΜ for entrapping therein the concentrated Euglena culture having the metal value and other contaminate materials sequestered therein. In some embodiments, the pore size is about 2.7 μΜ. The filter system also includes an outlet for collection of the treated water.

In some embodiments, the stock culture tank for growing a Euglena algae culture maintains the density of the Euglena culture at greater than about 4.2X 10⁶ celis/mL.

In preferred embodiments, the substantially constant flow of Euglena algae released to the Euglena stock culture concentrator so as to produce the concentrated Euglena stock culture therefrom is performed at a rate such that the density of the Euglena culture remaining in the stock culture tank remains substantially constant.

In preferred embodiments, the Euglena stock culture concentrator concentrates the Euglena algae to a concentration of at least about 6.8X 10⁶cells/mL.

In some embodiments, the stock culture tank further includes lengths of perforated black tubing as a growth substrate.

In some embodiments, the stock culture tank contains a growth medium for growing the Euglena algae culture and is preferably provided at a temperature of from about 5°C to about 30°C. Furthermore, in preferred embodiments the growth medium is subjected to at least some light. The medium, in some embodiments, also includes yeast extract at a concentration of from about 1 g/L to about 25 g/L and/or plant hormones provide at from about 1 0⁶ moles per l iter to about 1 0⁹ moles per l itre. In some em bodiments, the growth med ium is maintained at a pH from about 6.5 to about 8.5 and preferably at a pH of about 7.2.

In a preferred embodiment, the growth med ium includes yeast extract ( l .Og/L), beef extract ( l .Og/L) and plant hormones: Tz( 1 0^~7mol/L, ABA( 10^"9mol/L) and the growth medium is subjected to full light for 1 6 hours a day and maintained at a temperature of about at 20°C. Furthermore, in preferred embodiments, the metabolic rate of the Euglena algae of the stock cu lture is maxim ised to increase its absorption potential.

In some embodiments, the metal value and other material contaminated water, in the water flowthrough system, inc ludes Euglena cells at a concentration of 20x 1 0 ' cel ls/m L to 1 .5x l 0⁶ cells/mL.

In some embodiments, the metal value and other material contam inated water, in the water flowthrough system, is held therein as the residence time for a time period of from about 30 minutes to about 5 hours.

In some embodiments, the metal value and other material contaminated water, in the water flowthrough system, is aerated at a rate of from about 1 00 mL to about 1 80 mL per minute.

In some embodiments, the metal value and other material contaminated water, in the water flowthrough system, is aerated with a nozzle wherein the nozzle has a diameter within a size range from about 1 .0 mm to about 1 0 mm.

In some embodiments, the pH of the metal value and other material contaminated water is adjusted so as to target the filtration of a desired contaminant.

In some embodiments, the metal value and other material contaminated water, in the water flowthrough system, is maintained at a pH of from about 3 to about 1 0.

I n a preferred embodiment, the metal value and other material contaminated water, in the water flowthrough system, includes the Euglena cells having a concentration of about 5.0X 1 0⁴ cells/mL, reaching a maxim um level at 1 hour in a culture and being aerated at 1 60mL/minute using a 1 0.0mm nozzle and being maintained at a pH of 8.5.

In another aspect, there is prov ided a method for removing metal values and other materials from a metal-value and other material contaminated water supply, the method including growing a Euglena algae culture in a stock culture tank at a substantial ly constant density; the stock cu lture tank including at least one black physical substrate having a plurality of perforations; adding to the Euglena algae culture one or more constituents for promoting the prol iferation of the Euglena algae culture; concentrating the Euglena algae culture so as to produce a concentrated Euglena algae culture; subjecting the concentrated Euglena algae culture to the metal-value and other material contaminated water for a predeterm ined period of time so as to al low the concentrated Euglena algae culture to sequester substantial ly al l the metal values and other contaminate materials and produce water substantial ly devoid of metal values and other contaminate materials;

separating the concentrated Euglena algae culture having substantially all the metal values and other contaminate materials sequestered therein from the water substantially devoid of metal values and other materials.

In some embodiments, the Euglena algae culture has Euglena algae concentration of about 4.2x 1 0⁶ cells/mL.

In some embodiments, the concentrated Euglena algae culture has a Euglena algae concentration of about 6.8x 1 0⁶cells/mL. In some embodiments, the constituents include yeast extract ( I .Og/L), beef extract ( I .Og/L ) and plant hormones: Tz( I O^"7mol/L, A BA( 1 0^~9mol/L).

In some embodiments, the Euglena algae culture is maintained at about 5°C to about 30°C. I n some embod iment, the Euglena algae cu lture having substantial ly al l the metal values and other materials sequestered therein is subjected to microwave digestion so as to retrieve the metal values and other contaminate materials.

In some embodiments, the pH of the metal value and other material contam inated water supply having added thereto Euglena algae culture is adjusted so as to target the fi ltration of a desired contam inant. I n preferred embodiments, the pH is adjusted to a desired level of from about 3 to about 1 0.

BRI EF DESC RI PTION OF THE DRAWINGS

Figure 1 is a histogram of the results of Experiment I, Part A, showing the diameter of Growth surface and growth of Euglena;

Figure 2a is a histogram of the results of Experiment I I, Part A, showing the effect of colour of the growth surface and growth rate of Euglena;

Figure 2b is a histogram of the results of Experiment I I I, Part A, showing the effect of surface shape and the growth surface and growth rate of Euglena;

Figure 3 is a histogram of the results of Experiment IV, Part A, showing the effect of light on the growth rate of Euglena;

Figure 4 is a histogram of the results of Experiment V, Part A, showing the effect of temperature and growth rate of Euglena;

Figure 5a is a histogram of the results of Experiment Via, Part A, showing the effect of plant hormones and growth rate of Euglena;

Figure 5b is a h istogram of the resu lts of Experiment Vlb, Part A. showing the effect of plant hormone combinations and growth rate of Euglena;

Figure 6 is a h istogram of the results of Experiment VI I, Part A, showing the effect of nutrients and growth rate of Euglena;

Figure 7 is a histogram of the results of Experiment VI I I, Part A, showing the effect of aeration and the growth rate of Euglena;

Figure 8 is a l ine graph of the results of Experiment 1, Part C, showing the effect of combination of growth factors on the growth rate of Euglena over time;

Figure 9 contains pictures of Euglena cells as various levels of nanosi lver partic le exposure of Experiment 1 , Part E;

Figure 1 0 contains pictures of Euglena cel ls as various levels of nanosi lver partic le exposure over time of Experiment I I, Part E;

Figure 1 1 contains confocal m icroscope images of Euglena cells as various levels of nanosilver particle exposure over time of Experiment I I I, Part E:

Figure 12 is a l ine graph showing Euglena stock culture density over time of Part F; and Figure 1 3 is a schematic diagram of exemplary Nanosilver Filtration System in accordance with an exemplary embodiment.

DESCRI PTION OF THE EXEMPLARY EMBODI M ENTS

It should be understood that the invention is not limited in its application to the details of construction and the arrangement of the step set forth in the fol lowing description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and term inology used herein is for the purpose of description and shou ld not be regarded as l im iting. The use of "inc luding." "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equ iva lents thereof as wel l as add itional items.

I n accordance w ith the research presented herein it wou ld be desirable to develop a Euglena Biofi lter for functioning as a remed iation device for the removal of env ironmental nanosi lver and other contam inate materials in wastewater effluent. A Euglena biofi lter for such purposes has been developed so as to remove nanosilver, other nano value metals, nano partic les and other materials from water.

Adjusting the pH of the metal value and other material contam inated w ater hav ing added thereto Euglena cel ls causes a change in the protein channels of the Euglena cells and al lows the Euglena cel ls to take up various different pol lutants and therefore sequester therein. Such pol lutants may be, for example, but not l im ited to, heavy metal ions and nano particles thereof, nuclear waste, phosphorous-based ions and other water-borne pol lutants.

Briefly, the system 1 00 is schematically described w ith reference to Figure 1 3 and more details regarding the system are provided below with reference to Part F; Designing the Prototype Nanosilver Filter as wel l as other details relating to the system and the materials also being described below. Turning now to Figure 13, an exemplary system 1 00 for fi ltering metal-value, in particular nanosilver, and other material contaminants from water using a concentrated culture of Euglena algae is discussed.

The system, general ly shown at 1 00, includes a Euglena stock culture tank 1 1 0 for growing a Euglena algae culture 1 1 0a at a substantial ly constant density in a su itable med ium 1 08 for growing Euglena algae cultures. The Euglena algae I 1 0a is then fed into a Euglena stock culture concentrator 1 1 2 adapted to receive a substantial ly constant flow of 1 1 0a Euglena algae from the stock culture tank 1 1 0 and concentrate the Euglena algae stock cu lture 1 1 0a so as to produce a concentrated Euglena cu lture 1 1 2a.

With in the Euglena stock culture tank 1 10 a growth substrate is prov ided (not shown). I n general, the growth substrate is provided as a surface to aid in the growth, and

multipl ication of the Euglena algae 1 10a. For example, the growth substrate may be provided as section of clear or black plastic tubing. In preferred embodiments, sections of black plastic tubing are used. Furthermore, the d iameter of the tubing may be provided ranging from about 0.25 cm to about 3.0 cm. I n preferred em bod iments, the tubing is provided as black plastic tubing having a diameter of about 2.5 cm and in lengths of about 2.0 cm. I n certain embodiments, more than one section of tubing may be provided.

Additional ly, the sections of tubing may also be provided as flatten plastic pieces as the tube section noted above (i.e. a sheet) and/or perforated tube sections such a perforated cyl inders. In preferred embodiments, the tubing is provided as perforated cyl inders.

From the Euglena stock culture concentrator 1 1 2, the concentrated Euglena culture 1 1 2a, provided as slurry, is placed into contact with a metal-value contam inated other contaminate containing water 120. Briefly, with reference to Figure 1 3, for example, the concentrated Euglena algae culture 1 12a is moved into a flowthrough system 1 1 6, also termed herein as a Nanosilver Absorption Flow Through System, via tubing 1 14 in fluid communication with the flowthrough system 1 16. The Euglena stock culture tank 1 10 is provided to continually produce a culture of Euglena algae 1 10a which can be moved into the Euglena stock culture concentrator I 1 2 at a desired rate so as to provide the flowthrough I 1 6 with a required and substantial ly constant flow of concentrated Euglena cu lture 1 1 2a.

The flowthrough system 1 1 6 receives the concentrated Euglena culture 1 12 via a first in let 1 1 8a and the metal-value and other material contam inated water 1 20 is received into the flowthrough system via a second inlet 1 20a. The flowthrough system 1 16 is prov ided with a predetermi ned path, as show n schematically in Figure 1 3, for example, along which the concentrated Euglena culture 1 1 2a and the metal-value and other material contaminated water 1 20 co-travel so as to provide a residence time where the concentrated Euglena algae cu lture 1 12a is in contact with the metal-value and other material contaminated water 120 such that at least a portion of the metal values and other contam inate materials are sequestered by the concentrated Euglena culture. The water and the concentrated Euglena cu lture now having metal values and other materials sequestered therein then exit the flowthrough system 1 1 6 via an outlet 1 22.

From the outlet 122, the water and the concentrated Euglena culture having metal values and other contaminate materials sequestered therein enter a multiple stacking filter system 1 24 designed to filter the outflow of water from the flowthrough fi ltration system 1 1 6 and thus separate the concentrated Euglena culture having metal values and other materials sequestered therein from the water. Briefly, in preferred embodiments the multiple stacking filter system 124 is provided with a Nanosi lver Re-claiming Filter having glass filters 126 comprising glass fibres for entrapping the concentrated Euglena culture having metal values and other contaminate materials sequestered therein. In some embodiments the glass fiber filter 126 has a pore size of from about 1 .0 μΜ to about 3.0 μΜ and in preferred embodiments, the glass fiber filter has a pore size of about 2.7 μΜ.

In some embodiments the concentrated Euglena culture materials may sequester nanosilver, other nano value metals, nano particles and other materials. Furthermore, the concentrated Euglena culture may also sequester phosphorous-based ions, nuclear waste and other materials.

From the multiple stacking filter system 124 effluent water which is now substantially devoid of at least nanosilver, and in some embodiments, other contaminants is released as treated water 128.

The concentrated Euglena culture having metal values and other materials sequestered therein entrapped by the glass filter fibers 1 26 of multiple stacking filter system 124 are subjected, in some preferred embodiments to microwave digestion at 130. Of course, contaminates sequestered in the Euglena algae may also be recovered by other methods as desired. In the case of nanosilver contamination, the microwave digestion 130 of the Euglena algae renders the sequestered nanosilver as shown at 1 32, for example.

Turning now to experimental methods and data, the Euglena algae culturing and sequestering of contaminates associated with above-described system are discussed, below.

Procedures and Results:

Part A: Testing Variables for Optimal Growth of Euglena

Part A Design of Euglena Stock Culture Tank

Methods and Materials - All Euglena test cultures for experiments: 1, II, III, IV, V, VI, VI I, and VIII were prepared in culturing flasks containing 25ml of Euglena stock culture in 25ml of Euglena growth media and incubated at 25 °C for 1 44 hours at pH 7.5 in the greenhouse, unless otherwise stated. A Vi-Cell counter was used to determine the initial cell concentration in 1 .0 mL of the Euglena test culture in each experiment. Five repetitions were performed for each experiment below.

Data Analysis - A Vi-Cell counter was used to determine cell counts for each Euglena test culture in experiment I, I I, I I I, IV. V. V], VI I, VI I I and compared with control cultures. ANOVA was performed on observed data at the 5% level of significance {p<0.05).

Experiment I - Diameter of Plastic Tubing to Serve as a Growth Surface

Five Euglena test cultures were prepared in culturing flasks, each containing sections of clear plastic tubing, 2 cm in length but with diameters of: 0.5 cm, 0.8 cm, 1 .2 cm, 1 .8 cm. 2.5 cm.

As shown in Figure 1 , a maximum Euglena cell concentration of 4.6x 10³ cells/mL was obtained when plastic tubing with a diameter of 2.5 cm was used as a growth surface in Euglena cultures.

Experiment II - Colour of Plastic Tubing to Support Growth of Euglena

Three Euglena test cultures were prepared in culturing flasks, each containing sections of black, clear, or white plastic tubing, each 2.5 cm in diameter and 2 cm in length.

As shown in Figure 2a, a maximum cell concentration of 1.9x1 ο³ cells/mL was obtained when black tubing was added as growth surface in Euglena cultures.

Experiment III - Shape of Growth Surface and Growth Rate of Euglena Three Eugiena test cultures were prepared in culturing flasks, each containing a d ifferent shape of black plastic; tube sections, flat plastic, and perforated cyl inders.

As shown in Figure 2b, a maxim um cell concentration of 2.7x 1 0⁵ cells/m L was obtained when perforated cylinders and flat plastic were added to separate cultures as a growth surface in Eugiena cu ltures.

Experiment IV- Light as a Stimulant for Growth of Eugiena

Two Eugiena test cultures were prepared in culturing flasks; one flask was covered in tinfoi l to block out all light, the other flask was left to let al l light in.

As shown in figure 3, a maximum cel l concentration of 2.6x l 0⁴ cells/mL was obtained in Eugiena cultures that were exposed to fu l l l ight.

Experiment V - Temperature and Growth of Eugiena

Four Eugiena test cultures were prepared in culturing flasks and incubated at four d ifferent temperatures: 1 0, 20, 30 and 40 °C.

As shown in Figure 4, a maximum cel l concentration of Ux l O' cells/mL was obtained when Eugiena cultures were grown at 20°C.

Experiment VI a) - Plant Hormones and Growth Rate of Eugiena

Thirty Eugiena test cu ltures were prepared in culturing flasks and grown in six different plant hormones: BAP, ABA, I AA, I P, Tz, GA₃, at five different concentrations: 1 0 \ 1 0 ^" ⁶, 10 ^"7, 10 ⁸ and 10^"9 mol/L.

As shown in Figure 5a, a maximum cell concentration of 4.0x 10³ cells/mL was observed using BAP at Ιθ ηοΙ/L, ABA at 10^"9 mol/L, IAA at !0^~9mol/L, IP at 10^"5mol/L. Tz at 10⁷ mol/L.

Experiment VI b) - Plant Hormone Combinations and Growth Rate of Euglena

Thirty-two test cultures of Euglena were grown in thirty-two different combinations of plant hormones consisting of I.2.3.4. and 5 different plant hormones at their optimum concentration for growth from Experiment VI a).

As shown in Figure 5b, a maximum cell concentration of 2.3x10^" cells/mL was observed using Tz ( 10 ⁷ mol/L) combined with ABA ( 10 ^~9 mol/L).

Experiment VII - Determination of the Nutrients for Growth of Euglena.

Ten Euglena test cultures were prepared in culturing flasks and grown in five

concentrations of yeast extract and beef extract (separately)(l .0, 10.0, 25.0, 50.0. and 100.0 g/L).

As shown in Figure 6, a maximum cell concentration of l.lxlO⁶ cells/mL was observed using 1.0g/L of yeast extract and 1.0g/L of beef extract in the Euglena growth media.

Experiment VIII - Aeration and Growth Rate of Euglena

Three Euglena test cultures were prepared in culturing flasks and aerated at 160mL/min. using a 1.0 mm and 10.0 mm diameter nozzle.

As shown in Figure 7, a maximum cell concentration of4.1xl0³ cells/mL was observed using the 1.0mm nozzle. Part B: Testing Factors Affecting the Absorption of Nanosilver and Ag+ By Euglena Methods and Materials - All Euglena test cultures for experiments: I. II. III. IV. V, VI. VII, VIII were prepared in culturing flasks containing 25ml of Euglena stock culture in 25ml of Euglena growth media and incubated at 25 °C for 12 hours at pH 7.5 in the greenhouse, unless otherwise stated. A Vi-Cell counter was used to determine the initial cell concentration in 1.0 niL of the Euglena test culture in each experiment. Three repetitions were performed for each experiment in Part B.

Data Analysis - Silver absorption levels for each experiment were determined using 1CP- MS (Inductively Coupled Plasma-Mass Spectrophotometer) and compared to control cultures. ANOVA was performed on observed data at the 5% level of significance (p<0.05).

Experiment I - Determination of Nanosilver and Ag-i- Absorption By Euglena

Ten test cultures of Euglena at a concentration of 5. OX 10⁴cells/mL were grown in nanosilver (nAg) and silver ion (Ag+) solutions of: 1.0, 10.0, 25.0, 50.0. 100.0 μg/L.

Results:

Table 1 . Average Nanosilver (nAg) and Ag+ Absorption by Euglena

Experiment ΙΓ - Variable Concentrations of Euglena Cells and Nanosilver

Absorption

A dilution series of twenty-five Euglena test cultures (5.0x l 0⁴, 2.0X 10% 6.0x 10% 10⁶, 1 .5x l 0⁶ cells/mL) were grown in the following nanosilver solutions (concentrations: 1 .0, 10.0, 25.0, 50.0. 100.0 μg/L).

Results:

Table 2. Average Nanosilver (nAg) Absorption in Different Concentrations of Euglena

Cells

*Abs= Absorbed Con= Concentration

Maximum nanosilver absorption of 100% was observed at all Euglena cell concentrations and a nanosilver concentration of 1 .C^g/L. 99.9% of the nanosilver was absorbed by Euglena cel ls at all concentrations and nanosilver concentrations of: 1 0.0. 25.0, and

99.0% of the nanosilver was absorbed by Euglena cells at all concentrations and a nanosi lver concentration of 100^ /L.

Experiment III -Nanosilver Absorption By Euglena Over Time

Eighteen Euglena test cultures at a concentration of 5.0X I 0⁴ cells /niL were grow n in the follow ing nanosilver solutions (concentration: 1 .0, 25.0, 50.0 μg/L). Nanosilver absorption was determined at 1. 5, 12, 24 and 48 hours.

Results:

Table 3. Average Nanosilver Absorption Over Time

Abs= Absorbed Con= Concentration

Experiment IV - Temperature and Nanosilver Absorption By Euglena

Nine Euglena test cultures, at a concentration of 5. OX 1 0⁴ cells/L were grown in the following nanosilver solutions (concentration: 1 .0, 25.0, 50.0 μg/L) and incubated at I 0°C, 20^°C, and 30°C. Results:

Abs= Absorbed Con= Concentration

Experiment V - Aeration and Nanosilver Absorption By Euglena

Nine Euglena test cultures, at a concentration of 5.0X 10⁴ cells/mL were grown in the following nanosilver solutions (concentration: 1 .0, 25.0, 50^g/L) and aerated at l 60mL/L using a 1 .0mm and 10.0mm nozzle.

Results:

Table 5. Avera e Nanosilver Absor tion at Different Sized Aeration Nozzles

*Abs= Absorbed Con= Concentration

Experiment VI - pH and Nanosilver Absorption By Euglena

Nine Euglena test cultures, at a concentration of 5.0X 10⁴ cells/ were grown in the following nanosilver solutions (concentration: 1 .0, 25.0, 50^g/L) and incubated at pH 6.5, 7.5, and 8.5. Results:

Table 6. Average Nanosilver Absorption at Different pH Values

*Abs= Absorbed Con= Concentration

Experiment VII - Culture Age and Nanosilver Absorption By Euglena

A new Euglena stock culture was started. 50 mL of this culture was added to nanosilver solutions (concentrations: 1 .0, 10.0, 50.0 and 100^g/L) on week I of the stock culture. The same steps were repeated in weeks 3. 6 and 12 using the same Euglena stock culture Results:

Table 7. Average Nanosilver Absorption at Different Culture Ages

*Abs= Absorbed Con= Concentration

Part C: Combining the Variables to Maximize Euglena Growth and Nanosilver Absorption

Part C of the experiment was conducted to determine the maximum nanosilver absorbance at the lowest concentration of Euglena in the shortest time interval. Methods and Materials - All Euglena test cultures for Experiments: I and II were prepared in culturing flasks containing 1.2 L of Euglena stock culture in 1.2 L Euglena growth media and incubated at 25 °C. A Vi-Cell counter was used to determine the initial cell concentration in 1.0 mL of the Euglena test culture in each experiment. Five repetitions were performed for each experiment in Part C.

Data Analysis - Cell counts in Euglena test cultures were determined daily using a Vi- Cell counter. Silver absorption levels were determined using ICP-MS. ANOVA was performed on observed data at the 5% level of significance {])<().05).

Experiment I - Maximizing Cell Density of Euglena Culture

A Euglena test culture was grown under conditions for optimum growth identified for each variable tested in Part A (Experiments I- VIII).

As shown in Figure 8, a maximum cell concentration of 4.2X10⁶cells/mL was observed at day 7 after which the cultures entered a death phase.

Experiment II - Maximizing Nanosilver Absorption by Euglena

A Euglena test culture was grown under conditions for optimum absorption identified for each variable tested in Part B (Experiments 11-VI). and exposed to the following nanosilver solutions (concentration: 1.0, 10.0, 25.0, 50.0, I00^g/L).

Results:

Table 8. Avera e Maximum Nanosi lver Absor tion B Eu lena

Part D: Determination of Nanosilver Concentration in Water Samples

Materials and Methods - Each water sample went through a si lver separation process. A glass fibre membrane filter was used to remove the elemental silver. N itric acid was added to the remaining solution to form si lver nitrate w ith the ionic si lver. Hydrochloric acid was added to this solution to react w ith si lver n itrate and form a si lver chloride precipitate. The suspension of si lver chloride precipitate was centrifuged and the si lver chloride precipitate was filtered from the solution. The addition of hydrochloric acid and fi ltration of the precipitate was repeated until the reaction reached the endpoint and a precipitate was not produced. The supernatant was digested to elim inate organic matter from the sample for nanosilver.

Data Analysis - The resulting supernatant from Part D: Experiment I and I I was microwave digested and analyzed for total si lver using ICP-MS.

Experiment I - Testing Nanosilver Concentration in Influent and Effluent Water from the Peterborough Wastewater Treatment Plant (WWTP)

Influent and effluent samples were collected at the Peterborough wastewater treatment facility and the nanosilver in the samples was isolated using the method outlined above. Res u Its:

Table 9. Average Nanosi lver Concentration in Peterborough Wastewater Treatment Plant

Experiment II - Testing Nanosilver Concentration in Freshwater

Water samples collected from : Percy Lake. Clear Lake, Otonabee River North

Peterborough, Otonabee River above and below the Peterborough Waste ater Treatment Plant, Trent Severn Water Way (TSWW) (Trenton), Lake Ontario (Trenton), Lake Ontario (Harbourtront), and Humber River were tested using the method outlined above to isolate the nanosilver.

Results:

Table 10. Avera e Nanosilver Concentration in Ontario Water Sam les

Part E: Absorption of Nanosilver by Living Organisms

Experiment 1 - Transmission Electron Microscopic Examination of Euglena Cultured in

Nanosilver and

Materials and Methods - Euglena, incubated in 50μg/L nanosilver for 24 hours, was fixed in 7.5% gluteraldehyde. centrifuged, rinsed with sodium phosphate buffer, and a 1 % tetra osmium oxide was added to the resulting Euglena pellet. The pel let was immersed in agar, cut into blocks and dehydrated with ethanol. embedded in epoxy and hardened overnight. One-micron thick sections were produced using a diamond blade. Sections were stained for TEM analysis using a Joel 1 00 CX. Pure nanosilver was dried on formvar coated copper grids and placed in the TEM for characterization. Results are shown in Figure 9.

Experiment II - Light Microscope Examination of Euglena in Nanosilver

Materials and Methods - Euglena samples were exposed to different concentrations of nanosilver ( 1 .0, 10.0. 50.0, 100.0 μg/L) for specific time periods ( 1 min, 30 min. 5 hours. 24 hours, 48 hours) and then examined under the light microscope for morphological changes. Unexposed Euglena were also examined under the light microscope and then exposed to nanosilver to observe the initial reaction to the nanosilver. Results are shown in Figure 10.

Experiment III - Confocal Microscope Examination of Euglena in Nanosilver Materials and Methods - Euglena samples were exposed to different concentrations of nanosilver ( 1 .0, 1 0.0, 50.0, 100.0 μg/L) for specific time periods ( 1 min, 30 min, 5 hours, 24 hours, 48 hours) and then examined under the confocal microscope for morphological changes. Unexposed Euglena were also examined under the confocal microscope and then exposed to nanosilver to observe initial reaction to nanosilver. Results are shown Figure 1 1. Experiment IV - LC50 for Euglena Cultured in Nanosilver and Ag+ Materials and Method- A Euglena culture ( 1 .3x l 0⁴ cells/mL) was grown in Euglena growth media and in nanosi lver solutions (concentrations: 1 .0, 5.0, 10.0, 40.0, 75.0. and 100.0 μg/L). These cultures were incubated for 24 hours in a greenhouse. This experiment was repeated with Euglena growing in Euglena growth media and Ag+ solutions

(concentrations: 1 .0. 5.0, 10.0, 40.0, 75.0, and 100^g/L). The Euglena and Ag+ cultures were incubated for 48 hours in a greenhouse.

Data Analysis - Using a Vi-Cell counter, cell counts were determined in test samples and compared to a control to determine the concentration of nanosilver and of Ag+ causing 50% mortality in the Euglena population (LC50).

Results: The LC50 for Euglena in nanosilver was determined to be 5^g/L and determined to be 46^g/L for Euglena in Ag+.

Table 1 1 . Average Euglena Death In Nanosilver and Ag+ Solutions

Experiment V - LC50 for Lake Trout Embryos Cultured in Nanosilver and Ag+ Materials and Method - Freshwater Lake Trout (inland species) were collected from Seneca Lake (strain of broodstock), Sault Saint Marie, Ontario, Canada. Eggs and milt were gently placed in a dry bowl, cold water added and the mixture was stirred for two minutes with a feather. Eggs were rinsed and incubated at 7 °C for I hour to water-harden. Dead eggs were removed and the viable fertilized eggs were separated into I I groups of 1 00. These 1 1 groups of freshwater trout embryos were incubated at 7-8°C, in trays of nanosilver and Ag+ in the following concentrations: 0.1. 1 .0, 10.0. 50.0. 1 00.0 μg/L. Data Analysis - Dead embryos were removed and counted daily and compared to a control, to determine the LC50 for each concentration of nanosi lver and of Ag+.

Results: The LC50 for lake trout embryos cultured for 10 days was determined to be 37^g/L. The LC50 for lake trout embryos cultured for 15 days was determined to be 23^g/L. Trout embryos cultured in ionic silver did not exhibit a 50% decl ine in population.

Table 12. Average Trout Embryo Death In Nanosilver and Ag+ Solution

* Con= Concentration

Part F: Designing the Prototype Nanosilver Filter

Step 1

Based on the optimization experiments for growth, in Part A, and the optimization studies, in Part C, a Euglena Stock Culture Tank 1 10 was designed to produce an on-going supply of Euglena I 1 0a to regenerate the Nanosilver Absorption F low Through (NAFT) System as needed.

Method: The Euglena Stock Culture Tank 1 10 consisted of a 2.4L funnel-shaped titration vessel connected to the Euglena concentrator tank I 12 by plastic tubing I 14. To maintain a constant maximum cell density of 4.2X 10⁶ cells/mL, 2% of the total culture volume (50mL) was gravity drained (at a constant rate of 50mL/hr.) and replaced with Euglena growth media, via pump, after the culture attained a cel l density of 4.2X 10⁶cell/mL. As shown in Figure 12, a cell concentration of 4.2X 1 0⁶ cells/mL was maintained for twenty days with 4.2X I 0⁶ Euglena cells/mL/day harvested.

Step 2

A Euglena Concentrator 1 12 was designed to provide a means of hyper-concentrating Euglena 1 12a for a regulated direct transfer to the Nanosi lver Absorption Flow Through System (NAFT-system).

Method: A second, 2.4L funnel-shaped titration vessel was incorporated to utilize gravity to concentrate Euglena cells.

Results: Maximum concentration of 6.8X 10⁶cells/mL was observed at the bottom of the Euglena concentrator vessel.

Step 3

A study was conducted to determine cost effective materials for construction of a

Nanosilver Absorption Flow Through (NAFT) System 1 16. In determining the construction materials and dimensions of this NAFT System, the following criteria were taken into account a) Existing methods for water treatment currently applied at the Peterborough Water Treatment Facility;

b) The optimum conditions of temperature and time for maximum nanosilver absorption; and

c) Any pertinent conditions to support absorption of nanosilver by Euglena that emerge from the Part A, Part B or Part C.

Step 4

Nanosilver Re-claiming Filter was designed to capture Euglena and therefore absorbed nanosi lver in outflow water from the NAFT System.

Method: Glass filters 126 with different pore sizes were tested for effectiveness in trapping Euglena with absorbed nanosilver. A fluorometer was used to assess the concentration of Euglena in entry water to the fi lter and in the filtrate. Results: A glass fibre filter with a pore size of 2.7μιη was the most effective filter for trapping the Euglena. The filter trapped 99.99% of Euglena.

Step 5

A Multiple Filter Stacking System 124 was designed to support the continual filtration of outflow water from the NAFT System for application in a Water Treatment Facility. Method: A 7.2L rectangular, clear plexiglass tank was designed incorporating plexiglass dividers that control water flow rate. An inlet and an outlet allowed water to enter and exit the system.

Step 6

A prototype Nanosilver Filtration System was built which incorporates all identified operational factors (those tested in steps 1 to 5 and those that emerged during the design or construction phase).

Method: The Nanosilver Filtration System was tested for effectiveness first, using water to which a known concentration of nanosilver had been added, and second, using influent and effluent water collected at the Peterborough Water Treatment Facility, the metal-value and other contaminate containing water 1 20. The nanosi lver concentration in this influent and effluent water was determined prior to its use in the Nanosilver Filtration System. This data was used to determine the percent efficiency of the Nanosilver Filtration System prototype. Results:

Table 13: Average Absor tion of Nanosilver b Protot e Nanosilver Filtration S stem

In the above discussed experiments, it has been surprisingly discovered the Euglena cell growth is maximized when a Euglena growth medium that incorporates the following components, is added to a Euglena stock culture: a) yeast extract ( 1 .0g/L), and beef extract ( 1 .0g/L);

b) the plant hormones: Tz( 10^~7mol/L, ABA( 10^"9mol/L);

c) black, perforated cylinders or flat, black plastic; and

d) is grown in full light at 20°C.

Furthermore, nanosilver absorption by Euglena cells at a concentration of 5.0X 10⁴ cells/mL reaches a maximum level at 1 hour in a culture aerated at 160 mL/minute using a 1 0.0 mm nozzle at pH 8.5.

As sol id waste influent is d igested and broken dow n, nanosi lver is released. An average of 8.75g/day of nanosi lver is released in the Peterborough Wastewater Treatment Plant effluent water.

Nanosilver concentrations increase from north to south along the Trent Severn Waterway with a decline in nanosi lver concentrations being found in Lake Ontario and the h ighest concentration nanosilver in the H umber River.

The outer membrane of Euglena cel ls attracts and absorbs nanosilver particles.

Subsequently, Euglena algae absorbs nanosi lver particles thereby, concentrating nanosi lver. Absorption of nanosi lver particles causes Euglena cells to cease movement and contract into spheres. At 48 hours. Euglena cel ls in h igh concentrations of nanosilver lyse. While the LC50 for Euglena in nanosi lver is 5.0μ«/Ε, the LC50 for Euglena in ionic si lver is greatly increased (46^g/L).

The LC50 for lake trout embryos in nanosilver is determ ined to be 23 ^ig/L.

A stock culture of Euglena algae at a concentration of 4.2X 10⁶ was maintainable for 20 days when all conditions identified in optimization studies for Euglena growth were incorporated in the experimental sy stem.

Surprisingly, Euglena cells absorbed about 99.9% of nanosilver from wastewater effluent. Moreover, utilizing the abovementioned system, about 99.9% of the Euglena cells containing nanosi lver can be reclaimed from the NAFT System (Nanosilver Absorption Flow-Through System).

Given above observations, a biofilter that can effectively and efficiently remove nanosilver from wastewater has been developed which is both environmentally and economical ly beneficial. Such as nanosilver poses a potential risk to ecosystems and human health, the abil ity to remove th is compound from freshwater and wastewater is an innovation that represents sustai nabi l ity to l iv ing organ isms. Given that 8.75g/day of nanosilver is released in unfiltered wastewater, the annual deposition into the Otonabee River is 3.2kg/year from the Peterborough Wastewater Treatment Plant. Rec laiming 99.9% of this compound using an econom ical biofi lter represents $4.2 m illion dol lars annual ly. It is estimated that, in Europe, over 400 tons of nanosilver is released annual ly into the environment in wastewater effluent. Th is represents 53 trill ion dol lars of nanosi lver that cou ld be reclaimed.

Those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations of the materials, components, process and steps noted herein. Wh ile a system and method for fi ltering metal-value, and other material contam inated water and a method for removing metal values and other contaminants from a metal-value and other material contaminated water supply is provided for what are presently considered preferred and exemplary embod iments, the inv ention is not so limited. To the contrary, the invention is intended to cover various modifications and equivalent materials included within the spirit and scope of the appended claims. The scope of the following claims is to be accorded the broadest interpretation so as to encompass all such modifications and equivalent materials and functions thereof

Claims

is claimed is:

A system for fi ltering metal-value, and other material contam inated water, the system comprising:

- a stock culture tank for growing a Euglena algae culture at a substantial ly constant density;

- a Euglena stock culture concentrator adapted to receive a substantially constant flow of Euglena algae from the stock culture tank and concentrate the Euglena algae stock cu lture so as to produce a concentrated Euglena culture therefrom :

- the Euglena stock culture concentrator in fluid communication with a metal- value contaminate water flowthrough system so as to receive the

concentrated Euglena culture via a first inlet;

- the metal-value and other material contam inated water flowthrough system including a second inlet for receiving the metal-value and other material contaminated water;

- the metal-value and other material contam inate water flowthrough system having a predetermined path along which the concentrated Euglena culture and the metal-value and other material contaminated water co-travel so as to provide a residence time wherein the metal values and/or other contaminate materials are sequestered by the concentrated Euglena culture and exits via an outlet along with treated water.

2. The system as defined in claim I . wherein the metal -value and other material contaminated water includes nanosilver, other nano value metals, nano particles and other materials.

3. The system as defined in c laim 1 , wherein the metal value and other material

contaminated water incl udes phosphorous-based ions, nuc lear w aste and other materials.

4. The system as defined in claim 1 . further comprising a filter system in fluid

commun ication the outlet, the filter system having a filter system in let for receiv ing the concentrated Euglena cu lture having the metal values and other contam inate materials sequestered therein and water, the fi lter system provided for substantial ly separating the concentrated Euglena culture having the metal values and other contam inate materials sequestered therein and water.

5. The system as defined in claim 4, wherein the filter system includes at least one filter element wh ich is locatable in a microwave digester for separating metal values and other contam inate materials from the concentrated Euglena culture, the filter system including an outlet for col lection of the treated water.

6. The system as defined in c laim 5, wherein the at least one filter element is a glass fibre filter.

7. The system as defined in claim 6, wherein the glass fibre fi lter has a pore size of from about 1 .0 μΜ to about 3.0 μΜ .

8. The system as defined in c laim 6, wherein the glass fibre fi lter has a pore size of about 2.7 μΜ .

9. The system as defined in claim 1 , wherein the stock culture tank for growing a

Euglena algae culture maintains the density of the Euglena culture greater than about 4.2x l 0 cel ls/m L.

1 0. The system as defined in claim 9, wherein the substantial ly constant flow of Euglena algae released to the Euglena stock culture concentrator so as to produce the concentrated Euglena stock culture therefrom is performed at a rate such that the density of the Euglena culture remaining in the stock culture tank remains substantially constant.

1 1 . The system as defined in claim 1 0, wherein the Euglena stock culture concentrator concentrates the Euglena algae to a concentration of at least about 6.8x l O^ft cel ls/mL.

12. The system as defined in claim 1 , wherein the stock cu lture tank further includes lengths of perforated black tubing as a growth substrate.

1 3. The system as defined in claim 1 , wherein the stock cu lture tank contains a growth medium for growing the Euglena algae culture.

14. The system as defined in claim 13, the growth medium being at a temperature of from about 5°C to 30°C.

15. The system as defined in claim 13, the growth medium being subjected to at least some light.

16. The system as defined in claim 13, the growth medium including yeast extract, at a concentration ranging from about 1 g/L to about 25 g/L.

17. The system as defined in claim 13, the growth medium including plant hormones provided at about 10⁶ moles per liter to about 10⁹ moles per litre.

18. The system as defined in claim 13, the growth medium is maintained at a pH from about 6.5 to about 8.5.

1 . The system as defined in claim 13, the growth medium is maintained at a pH of about 7.2.

20. The system a defined in claim 13, wherein the growth medium includes yeast extract (l.Og/L), beef extract (l.Og/L) and plant hormones: Tz(10^~7mol/L, ABA(10^"9mol/L) and the growth medium is subjected to full light for 16 hours a day and maintained at a temperature of about at 20°C.

2 1 . The system as defined in claim 1 3. wherein the metabol ic rate of the Euglena algae of the stock cu lture is max im ised to increase its absorption potential .

22. The system as defined in claim 1 , wherein the metal value and other material

contaminated water, in the water flowthrough system, inc ludes Euglena cel ls at a concentration of 20.0x 10 ' cells/m L to 1 .5 l 0⁶ cel ls/m L.

23. The system as defined in claim 1 , wherein the metal value and other material

contaminated water, in the water flowthrough sy stem, is held therein as the residence time for time period of from about 30 minutes to about 5 hours.

24. The system as defined in claim 1 , wherein the metal value and other material

contaminated water, in the water flowthrough system, is aerated at a rate of about 1 00 mL to about 1 80 mL per minute.

25. The system as defined in claim 1 , wherein the metal value and other material

contaminated water, in the water flowthrough system, is aerated with a nozzle wherein the nozzle has a diameter within a size range from about 1 .0 mm to about 10 mm.

26. The system as defined in claim 1 , wherein the pH of the metal value and other

material contaminated water is adjusted so as to target the fi ltration of a desired contaminant.

27. The system as defined in claim 1 , wherein the metal value and other material contam inated water, in the water flowthrough system, is maintained at a pH of from about 3 to about 10.

28. The system as defined in claim 1 , wherein the metal value and other material

contam inated water, in the water flowthrough system, includes the Euglena cel ls having a concentration of about 5.0x 1 0 cel ls/mL. reaching a maximum level at I hour in a culture and being aerated at 1 60mL/m^'mute using a 1 0.0mm nozzle and being maintained at a pH of 8.5.

29. A method for removing metal values and other contam inants from a metal-value and other material contam inated water supply, the method including growing a Euglena algae culture in a stock culture tank at a substantial ly constant density; the stock culture tank including at least one black physical substrate having a plural ity of perforations; adding to the Euglena algae culture one or more constituents for promoting the prol iferation of the Euglena algae culture; concentrating the Euglena algae culture so as to produce a concentrated Euglena algae culture; subjecting the concentrated Euglena algae culture to the metal-value and other material contaminated water for a predetermined period of time so as to allow the concentrated Euglena algae culture to sequester substantially all the metal values and other contaminate materials and produce water substantially devoid of metal values; separating the concentrated Euglena algae culture having substantially al l the metal values and other contaminate materials sequestered therein from the water substantially devoid of metal values and other contaminate materials.

30. The method as defined in claim 29. wherein the Euglena algae culture has Euglena algae concentration of about 4.2x 1 0⁶ cel ls/m L.

3 1 . The method as defined in claim 29. wherein the concentrated Euglena algae culture has a Euglena algae concentration of about 6.8x 1 0⁶cel ls/mL.

32. The method as defined in claim 29, wherein the constituents include yeast extract ( l .Og/L), beef extract ( l .Og/L) and plant hormones: Tz( 1 0^"7mol/L. A BA( 1 0^"9mol/L).

33. The method as defined in claim 29. wherein the Euglena algae culture is maintained at about 5°C to about 30°C.

34. The method as defined in claim 29, w herein the Euglena algae culture having

substantially all the metal values sequestered therein is subjected to microwave digestion so as to retrieve the metal values and other contaminate materials.

35. The method as defined in claim 29, wherein the pH of the metal value and other material contaminated water supply having added thereto Euglena algae culture is adjusted so as to target the filtration of a desired contaminant.

36. The method a defined in claim 35, wherein the pH is adjusted to a desired level of from about 3 to about 10.