TECHNICAL FIELD THIS INVENTION relates to a biosorbent. More particularly, the invention relates to a biosorbent that may be particularly useful in the removal of an oxyanion and/or metal, cations from water and/or other aqueous solutions.

BACKGROUND

Selenium (Se) is an essential element for all vertebrates and some plants, where it forms a core component of selenoproteins that play a role in DNA synthesis and defense against oxidati ve stress (Hesketh, 2008; Wiseman, el al, 20 I I : Patching and Gardiner, 1999). There is, however, a narrow gap between the amounts of Se required for these essential functions and the concentrations at which Se becomes toxic to terrestrial and aquatic life (Chapman, el /., 2009; Sappington, 2002). Se is globally distributed within soil, coal and oil deposits from which it is slowly released through natural erosion. Human activities such as mining, combustion of fossil fuels and irrigation increase the rate of Se transport from these sources into aquatic habitats (Chapman, el al., 2009) and each of these practices have been explicitly linked to instances of relatively severe Se ecotoxicity, particularly in the United States (US)(Sappington, 2002).

The relative bioavailability and toxicity of Se is strongly linked to its oxidation state and subsequent chemical speciation. In aqueous environments, water soluble inorganic Se species with oxidation states of Se ^l and Se ^l dominate. In oxygenated surface waters with a pH typical of natural water bodies (~ range pH 6- 8) Se is primarily found as Se " ¹ in the form of the selenate (SeO^ ^' oxyanion (Sappington, 2002; Torres, et al., 201 1 ; Chapman, et al., 2009). Aquatic primary producers accumulate inorganic Se species and transform them into reduced organic forms such as selenomethionine (Se-Met) and selenocysteine (Se-Cys) (Sappington, 2002).

Once incorporated by primary producers, Se is readily transferred through aquatic food webs and higher vertebrates accumulate the majority of Se from dietary sources (Sappington, 2002; Chapman, et al., 2009). Se toxicity is linked to its tendency to displace sulfur in amino acids, thus altering the structure and function of 9 proteins in higher vertebrates (Amweg, et al, 2003; Perez-Corona, et al, 1997). This has critical implications for the management of seleniferous waste waters. A common management strategy for mining and mineral processing industries is to confine Se-contaminated ^' waters within long-term storage areas (such as fly ash dams, tailings ponds or irrigation ditches). These storage structures often support water birds and fish and thus represent significant sources of Se to local vertebrate populations despite their apparent confinement in artificially created aquatic habitats.

Given the environmental issues surrounding Se there is a clear need to develop methods that will improve management processes in industrial settings. While there are several existing treatment technologies for Se, most are unable to achieve Se concentrations that satisfy regulatory requirements, are too costly to be implemented, at the necessary scales, or are ineffective at treating dilute waste effluents (Amweg, Stuart, and Weston, 2003; Gonzalez-Acevedo, t a!., 2012). In addition, many are effective only against selenite, while the predominant form of selenium in surface waters is selenate.

It will, also be appreciated that selenium-containing contaminants such as selenite and selenate are not the only oxyanion contaminants that are environmentally damaging. There is also a need to remedy contamination b other oxyanions. such as nitrogen-, sulfur-, phosphorus-, arsenic-, chromium-, molybdenum- and boron-containing oxyanions. it may also be advantageous to remove metal cation contaminants from the environment.

SUMMARY

The invention is broadly directed to a biosorbent, and methods of producing and using the biosorbent for reducing or removing an oxyanion from water and/or another aqueous solution. In an additional or alternative form, there may be provided a method for removing metal cations from water and/or other aqueous solutions.

In a first aspect, the invention provides a biosorbent comprising;

algal based biomass coated with one or more metal cations, whereby the coated algal based biomass is capable of reducing or removing an oxyanion from water and/or another aqueous solution.

Suitably, the metal cation has a valence of > 2.

In one embodiment the metal cation is an iron cation. Preferably, the iron cation is ferric iron. In general embodiments, the oxyanion may be selenium-, nitrogen-, carbon-, iodine-, chlorine-, bromine-, vanadium-, manganese-, sulfur-, phosphorus-, arsenic-, chromium-, molybdenum- and/or boron-containing oxyanion.

In one embodiment the oxyanion is selected from the group consisting of selenite, selenate, sulfate, arsenite, arsenate, chromate, molybdate, borate, phosphate and nitrate. Preferably, the oxyanion is selenate and/or seienite.

In one embodiment the algal based biornass may comprise raacroalgae.

In a further embodiment the algae are selected from the group consisting of red (Rhodophyta), brown (Phaeophyta) and green (Chlorophyta) algae.

Preferably, the algae species are selected from one or more of the group consisting of Ui spp., Derbesi ^' a spp., Oed gonium spp., Cladophora. spp., Padina spp., Cystoseira spp., Hatymenia spp., Gracilaria spp,, Eucheuma spp. _. . Kappaphycvs spp., Saccharitta spp., Sargass m spp., Undaria spp. and Phaeophyceae spp .

More preferably, the algae species is a freshwater green algae species.

Even more preferably, the algae is of the species Cladophora spp. and/or of the species Oed.ogomum spp, (e.g., Oedogonium crispum).

In one embodiment, the algae species are selected from at least two of the group consisting of Ulva spp., iJerbesia spp., Oedogonium spp., Cladophora spp,, Padina spp., Cystoseira spp., Hatymenia spp., Gracilaria spp,, Eucheuma spp,. Kappaphyciis spp., Sacckarina spp., Sargassum spp. and Undaria spp.

In a certain embodiment, the algal based biomass comprises one or more algae species derived, obtained from, grown and/or cultivated m waste water.

In one embodiment, the algal based biomass comprises waste residues remaining after extraction of phycocolloids from algae.

Preferably, the algal based biomass comprises waste residues remaining after extraction of phycocolloids from one or more of the group consisting of Gracilaria spp, Eucheuma spp., Kappaphyciis spp., Saccharina spp,, Sargassum spp. and Undaria spp.

More preferably, the algal based biomass comprises waste residues remaining after extraction of phycocolloids (e.g agar) from algae of the species Gracilaria spp. (e.g., Gracilaria multipartita; Gracilaria gracilis; Gracilariopsis longissima; Gracilaria edulis; and G acilaria conferva ides). In one embodiment, the algal based biomass is dry, desiccated or dehydrated.

In another embodiment, the algal based biomass is algal biochar.

In one embodiment, algae or waste residues remaining after extraction of plrycoeolloids from algae are coated with metal cations before producing biochar.

in a second aspect, the invention provides a method of producing a biosorbent for the reduction or removal of an oxyanion from water and ^' or anotlier aqueous solution including;

i. treating one or more algal based biomass with a metal cation solution for a time and under conditions sufficient to coat the algal based biomass in one or more metal cations; and

ii. separating the treated algal based biomass from the metal catio solution.

Suitably, the metal cation solution has a valence of > 2.

In one embodiment the metal cation solution is an iron solution.

Preferably, the iron solution is a ferric solution. More preferably, the ferric solution is ferric chloride solution.

hi some embodiments, the oxyanio may be a selenium-, nitrogen-, sulfur-, phosphorus-, arsenic-, chromium-, molybdenum- and/or boron-containing oxyanion.

In one embodiment the oxyanion is selected from the group consisting of selenite, seienate, sulfate, arsemte, arsenate, chromate, molybdate, borate, phosphate and nitrate.

In one embodiment, the treated algal based biomass is washed to remove any residual metal cations.

In one embodiment, the algal based biomass is treated with a metal cation solutio from up to about 48 hours.

Preferably, the algal based biomass is treated with a metal cation solution for about 24 hours.

In one embodiment, the algal based biomass is treated with a concentration of up to about 50% metal cation solution.

Preferably, the algal based biomass is treated with a concentration of about 5% metal cation solution.

In one embodiment, the treated algal based biomass is dried, desiccated or dehydrated at a temperature of up to about 0°C. Preferably, the treated algal based biomass is dried at a temperature of about

60°C.

in one embodiment, the treated algal based biomass is dried, desiccated or dehydrated for up to about 48 hours.

Preferably, the treated algal based biomass is dried for about 24 hours.

In one embodiment, the algae based biomass is algal biochar.

In one embodiment, algae or waste residues remaining after extraction of phycocolloids from algae are coated with metal cations before producing biochar.

In a particular embodiment, the algal based biomass comprises one or more algae species derived or obtained from, grown and/or cultivated in waste water. in a third aspect, the invention provides a biosorbent, produced according to the method of the aforementioned aspect.

In a fourth aspect, the invention provides a method of reducing or removing an oxyanion from water and/or another aqueous solution, including;

contacting the biosorbent of the first aspect or the third aspect with water or an aqueous solution containing an oxyanion, for a time and under sufficient conditions to absorb the oxyamon from the water or aqueous solution. In one embodiment the oxyanion is selected from the grou consisting of seletiite, selenate, sulfate, arsenite, arsenate, ehromate, molybdate, borate, phosphate and nitrate.

In one embodiment the biosorbent is contacted with water or an aqueous solution containing an oxyanion for up to about 4 hours.

Preferably, the biosorbent is contacted with water or an aqueous solution containing an oxyanion for about 4 hours.

In one embodiment, up to about 90% of an oxyanion is absorbed from the water or aqueous solution.

Preferably, more tha about 90% of an oxyanion is absorbed from the water or aqueous solution.

It may be advantageous to remove any metal cations that may have leached from the biosorbent and/or any metal cations that were originally present in the water and/or other aqueous solution before treatment with the biosorbent.

Accordingly, in a further embodiment, the water or aqueous solution is contacted with another algal based biosorbent, for a time and under sufficient conditions to absorb one or more metal cations from the water or aqueous solution. In one embodiment, the water or aqueous solution may be treated simultaneously with both the biosorbent and the another algal based biosorbent. in another embodiment, the water or aqueous solution may be treated sequentially either with the biosorbent and then the another algal based biosorbent or the another algal based biosorbent and then the biosorbent.

Preferably, the another algal based biosorbent is an algal based biomass that has not been coated with metal cations.

Even more preferably, the another algal based further biosorbent is an algal biochar that has not been coated with metal cations. In one embodiment, the one or more metal cations to be absorbed is selected from the group consisting of aluminium, sodium, cadmium, chromium, copper, lead, manganese, nickel, barium, calcium, cobalt, iron, magnesium, potassium, vanadium and zinc.

in a fifth aspect, the invention provides a method of reducing or removing one or more metal cations from water and/or another aqueous solution, .including;

contacting an algal based biomass, with water or an aqueous solution containing one or more metal cations, for a time and under sufficient conditions to absorb the one or more metal cations from the water or aqueous solution.

In a particular embodiment, the one or more metal cations to be absorbed is selected from the group consisting of aluminium, sodium, cadmium, chromium, copper, lead, manganese, nickel., barium, calcium, cobalt, iron, magnesium, potassium, vanadium and zinc.

In a certain embodiment, the algal based biomass comprises an algae species of Cladophora spp. and/or Oedagonium spp,

In one embodiment, the algal based biomass comprises one or more algae species derived or obtained from, grown and/or cultivated in waste water.

In one embodiment, the algal based biomass that has absorbed the one or more metal cations from the water or aqueous solution may be used to produce the bioabsorbent of the first aspect or the third aspect and/or according to the method of the second aspect.

In a sixth aspect, the invention provides an algal based biomass comprising one or more metal cations absorbed from water or from another aqueous solution and which is capable of absorbing one or more oxyanions from water or from another aqueous solution.

In one embodiment, the biosorbent of the firs and third aspects comprises the algal based biomass of the present aspect.

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part, of the common general knowledge in the field .

As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and '"comprised", are not intended to exclude further additives, components, integers or

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Removal (%) of Se as Se^ and Se ^{V I} by un-modified and Fe-treated biomass and biochar.

Figure 2. Removal (%) of Se as a) Se and b) Se with Fe-treated biomass.

The nominal initial Se concentrations were 500 jig L ^"1 e^ and Se ^I (see Figure 1 for measured values) with 10 g L ^" ' dried biomass. Experiments were run at 20 ^* C with an initial pH of 4 and a 4 h contact time. Species are labeled as summarized in ' Algal species and biomass preparation ⁷ .

Figure 3, Correlation between leaching of Fe into the water column during biosorption studies and % removal of Se^ (solid line, closed circles) and Se (dashed lines, open circles) with a) Fe-treated biomass and b) Fe-treated biochar. Experiments were run as described for Figure 2.

Figure 4, Removal ( ) of Se as a) se!enite and b) selenate with Fe-treated biochar derived from macroalgae. Experimental conditions and species labels are as per Figure 2.

Figure 5. Nominal and measured initial. Se concentrations in the Se ^iV and Se ⁱ treatments.

Figure 6. (a) Macroalgal species tested in the initial biosorption challenge; and (b) Nominal and measured initial Se concentrations in the Se ^l and Se ^I treatments.

Figure 7. Removal (%) of Se from freshwater by un-modified and Fe-treated dried biomass and biochar. Figure 8. Removal (%) of Se as a) Se ^, and b) Se ^{V I} with Fe-treated macroalgal biomass. Shaded bars depict brown algal species, unshaded bars show data for red algal species. The nominal initial Se concentrations were 500 ag L ^"1 Se ^l

VT ί

and Se (see Table 2 for measured values) with 10 g L ^' dried biomass. Experiments were run at 20°C with an initial. pH of 4 and a 4h contact time. Species are labeled as per Figure 6 (a).

Figure 9. Removal (%) of Se as a) setenite and b) selenate with Fe-treated biochar derived from macroalgae. Experimental conditions and species labels are as per Figure 4.

Figure 1.0. Removal (%) of Se as Se ¹ ( white bars) and Se ^[ (grey bars) with

Fe-treated biomass and biochar derived from waste residue from the Gr cil ria agar extraction process. The nominal initial Se concentrations were 500 μ§ L ^"1 Se ^a and Se ⁱ with 10 g L ^"1 dried biomass. Experiments were run at 20 C with, an initial pH of 4 and a 4h contact time.

Figure 11. Removal (%) of Se and Se " with Fe-treated biomass and biochar derived from waste residue from the Gracilaria agar extraction process at pH 4 and 8. The nominal initial Se concentrations were 500 μ L ^"1 Se ^I and Se ⁱ with 10 g L ^"1 dried biomass. Experiments were run at 20 ^s C with a 4h contact time.

Figure .12. a) Removal (%) of Se as SeVi and b) Fe -leaching by waste residue from the Gracilaria agar extraction process that is Fe-treated pre- and post- pyrolysis. The nominal initial Se concentrations were 500 L ^"! Se ^{ with 10 g L ^"1 dried biomass. Experiments wer run at 20 ^* 0 with a 4h contact time.

Figure 13. Concentrations of Se ^l (jig L ^"1 ± SE) in a (a) mock solution and (b) a real-world mine effluent; and concentrations of (mg L ^"J ± SE) of the competing oxyanions (c) SO4 ^2* and (d) NO3 ^* in. a real-world mine effluent after multiple exposures to GMB. Overall maximum percent removal for each oxyanion can be found in the top right comers of each panel .

Figure 1 _* Cumulative mass (mg) of Se ⁱ (triangles), SO ₄ ^2" (squares) and NO ₃ ^" (diamonds) removed from solution by GMB after multiple exposures.

Figure 15. Biosorptton capacity of G B for Se ^l , SO ₄ ^2" and NO3 ^" expressed as q in mM. ± SE. Se ^r (triangles, black line), SO ₄ (squares, dotted line) and NO ₃ ^* (diamonds, dashed line). Logarithmic regression lines with ^~ values of 0.831 for Se ^VI , 0,835 for S0 ₄ ^2* and 0.908 for NO3 ^* . Figure 1.6. Uptake (%) of Se ⁱ in solutions containing (a) SO; ^2" and Se ⁱ and

(b) NO3 ^" and Se at different molar ratios. T!ie grey line shows mean Se removal m the absence of SO ₄ ^" and NO ₃ ^" with 95% confidence intervals (dashed lines).

Figure 17. Experimental treatments and predicted changes in anion and cation concentrations in ADW, Arrows signify the direction and magnitude of predicted change (up = increase, down = decrease, dash = no change, ? = unknown response). Six biosorption treatments were tested: (1) Fe-biochar ("FeBC") _. ; (2) biochar ("BC"); (3) sequential Fe-biochar ("FeBC→ FeBC ^" "); (4) sequential biochar "BC → BC"; (5) sequential Fe-biochar and biochar ("FeBC → BC" and (6) simultaneous Fe-biochar and biochar ("Fe-BC + BC").

Figure 18, Modelled changes in concentrations of (a) arsenic, (b) aluminium,

(c) molybdenum, (d) nickel, (e) selenium and (f) zinc when exposed to Fe-biochar and biochar (solid and dashed lines respectively). A3 ZECC trigger levels and stocking densities which result in reduction below ANZECC concentratio indicated by horizontal grey line and dashed area, respecti ely. The initial concentration of Al, Ni and Zn tor the biochar treatment in plots (b), (d) and (i) are presented as if following application of 1.3.7 g L-l 503 of Fe-biochar. The greyed-otit area indicates biosorbent densities above the physical limit for experimentation, 60 g L-l 505 .

Figure 19. Non-metric Multi -Dimensional Scaling (nMDS) demonstrating the differential sorption of Fe-biochar, biochar, and a combination of the two, in single and sequential treatments, (A) nMDS plot (Stress <0,01) with the groups from cluster analysis superimposed- Open and closed squares and circles represent single and sequential applications of Fe-biochar and biochar, respectively. Triangles represen sequential application of Fe-biochar followed by biochar and diamonds represent simultaneous application of Fe-biochar and biochar. (B) The same nMDS with vectors superimposed, the direction and length of which indicate the strength of correlation with the treatment scenario that resulted in low concentrations for the respective element.

Figure 20. Change in solution concentration of (a) arsenic, (b) molybdenum, (c) selenium, (d) aluminium, (e) zinc, and (f) cadmium following sequential exposure to Fe-biochar and biochar. The "BC", "FeBC" and "FeBC → BC" treatments are represented as dotted, dashed and solid lines, respectively. The ' ^" FeBC + BC" treatment is represented by the circle in each panel. The "FeBC + BC" result is placed under treatment 2 to compare with, the final concentrations of the other treatments. ANZECC trigger level is represented by a horizontal grey line.

Figure 21, Scatterplots of the predicted vs. observed ADW elemental concentrations (jig L-l) following exposure of (a) Fe-biochar, (b) Fe-biochar followed by Fe-biochar, (c) biochar, and (d) biochar followed by biochar. Data has been log transformed in order to be visually presentable. Plotted line indicates the expected point placement under the ideal scenario where observed values were identical to predicted values. Points falling above the line were overestimated by the model, resulting irt higher than expected values. Points falling below the line were underestimated by the model, resulted in lower than expected values. Asterisks indicate elements with a residual of greater than ±1..

Figure 22. Scatterplots of tire predicted vs. observed ADW elemental concentrations (jig L-l) following sequential exposure to Fe-biochar followed by biochar-. Data has been log transformed in order to be visually presentable. Plotted line indicates the expected point placement under the ideal scenario where observed values were identical to predicted values. Points falling above the line were overestimated by the model, resulting in higher than expected values. Points falling below the line were underestimated by the model, resulted in lower than expected values. Asterisks indicate elements which are have a residual of greater than ±1.

Figure 23, Change i solution concentration of (a) potassium, (b) manganese, and (c) vanadium following sequential exposure to Fe-biochar and biochar. Sequential exposure of ^" the ADW to biochar, Fe-biochar, and Fe-biochar followed by biochar and represented as dotted, dashed and solid lines, respectively. Exposure of ADW to both Fe-biochar and biochar simultaneously is represented as a round dot. Simultaneous exposure only had one application yet is placed under treatment 2 to compare with the final concentrations of the other treatments. ANZECC trigger level represented by a horizontal grey line. Error bars show standard error.

Figure 24. A schematic overview of an embodiment of the method that includes cation, removal and oxyanion removal from wastewater.

DETAILED DESCRIPTION The invention provides a biosorbent, and methods of producing and using the biosorbent for reducing or removing an oxyanion from water and/or another aqueous solution.

The method of producing a biosorbent includes the treatment of algal based biomass, including for example, algal biochar and/or waste residues remaining after extraction of phycocolloids from algae, in a metal cation solution for a time and under conditions sufficient to coat the algal based biomass in one or more metal cations. The resulting biosorbent is capable of reducing or removing an oxyanion from water or an aqueous solution containing the oxyanion.

in general embodiments, the oxyanion may be selenium-, nitrogen-, carbon-, iodine-, chlorine-, bromine-, vanadium-, manganese-, sulfur-, phosphorus-, arsenic-, chromtuai-, molybdenum- and/or boron-containing oxyanion.

In one embodiment the oxyanion is selected from the group consisting of selenite, selenate, sulfate, arsenite, arsenate, chromate, molybdate, borate, phosphate and nitrate.

Preferably, the biosorbent of the invention is effective against bot of the main inorganic oxidation states of selenium (selenite and selenate) and is equally effective across a relativel wide pH range (e.g., pH 4 - 8). These characteristics are significant improvements on other existing methods of removing selenium from, for example, waste water.

Although not wishing to be bound by any particular theory, it is proposed that the deposition of the metal cations, on the surface of the biomass (e.g., algal based biomass) increases the positive valence of the biosorbent thereby increasin its affinity for the negatively charged oxyanions. This encourages the formation of inner-sphere complexes between the biosorbent surface and the dissolved oxyanion.

In another aspect, the invention provides a method of treating water or another aqueous solution with an algal based biomass to remove metal cations therefrom. This may be performed alone or together with the method fo removing oxyanions. D mliQM

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which tlie invention belongs. Although any methods and materials similar or equivalent to those described herein ca be used in the practice or testing of the present invention, preferred methods and materials are described.

It will be appreciated that the indefinite articles " " and "an" are not to be read as singular indefinite articles or as otherwise excluding more than one or more than a single subject to which the indefinite article refers. For example, "a" algae includes one algae, one or more algae or a plurality of algae.

By "about is meant a measured variation in the quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

By "algal based biomass" is meant a material comprising or derived from algae,, including but not limited, to macroalgae, such as red, brown and green algae and algal biochar, as well as waste residues, including for example, those that remain after phycocoSloid extraction from processed algal biomass.

By "coa is meant to cover, layer, embed and/ or impregnate the algal based biomass with metal cations. By "coated" is meant, the algal based biomass may be covered, layered, embedded and/or impregnated with metal cations and by coaling" is meant covering, layering, embedding and/or impregnating the algal based biomass with metal cations,

The terms '¾/? ·", "desiccated ^* and 'We¾ ¾frai ' as used herein refer to a completely dry biomass or biosorbent or one that comprises about 50%, 40%, 30%, 20% or 10%, or even about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1 %, 0.01 % fluid or water.

By "fragment is meant single algal cells or fragments thereof (e.g., 1-2 mm in diameter), or multiple algal cells or fragments thereof, which may be present in large or small formations.

An "oxyanion" or "oxoankm'' is a chemical compound of the formula A^Oy ^e' (where A represents a chemical element and O represents an oxygen atom). The oxyanion may be a selenium-, nitrogen-, carbon-, iodine-, chlorine-, bromine-, vanadium-, manganese-, sulfur-, phosphorus-, arsenic-, chromium-, mofybdenum- and/or boron-containing oxyanion. Non- limiting examples of particular oxyanions include borate, carbonate, nitrate, phosphate, sulfate, chromate, arsenate, selenate, molybdate, nitrite, phosphate, sulfite, arsenite, sele.ni.te, hypophosphite, phosphate, hyposul.fi te, perchlorate, perbromate, periodate, permanganate, chlorate, chromate, bromate, iodate, chlorite, vanadate, bromite, hypochlorite, and ypobromite.

The terra "fedueing" is used herein to describe the achievement of a lower level of an oxyanion and'or metal cation in water o an aqueous solution after treatment with the biosorbent of the invention. In some embodiments, the oxyanion and/or metal cation is reduced by about 99%, 95%, 90%, 80%, 70%, 60%, 50%. 40%, 30% _f 20% or 10%, or even about 5%, 4%, 3%, 2%, 1 %, in one embodiment, the oxyanion and/or metal cation may be completely removed from the water or aqueous solution.

The term "selenium" as used herein describes all forms of selenium, including selenite and selenate oxyamons. Selenium is a toxic metalloid that is often released in waste water effluents from mining, mineral processing and flood irrigation waters. As a result of its toxicity, there are strict regulations regarding allowable selenium concentrations in industrial effluents destined for discharge. Mining and mineral processing industries for example, require a method to quickly and cheaply remove selenium from waste water. Many existing treatment methods are effective at removing selenite but very few are effective at removing selenate. The invention provides equally effecti ve removal of both selenite and selenate from water and other aqueous solutions.

The term "wafer" as used herein includes but is not limited to fresh water, salt water, tap water, sewage water, discharged water, processing water, agricultural drainage water, animal husbandry drainage water, waste water, other grey and black water, other natural water and combinations thereof.

Biosorbent and algal based biomass

In one aspect of the invention there is provided a biosorbent comprising; algal based biomass coated with one or more metal cations, whereby the coated algal, based biomass is capable of reducing or removing an oxyanion from water and/or another aqueous solution.

The algal based biomass may be coated with any metal, cation. Preferably, the metal cation has a valence of 2. Suitably, the metal cation has a valence of 2, 3, 4, 5 or 6.

In one embodiment the metal cation has a valence of 2 or 3. In another embodiment the metal cation has a valence of 2 to 4. In one embodiment the metal cation has a valence of 2 to 5. in another embodiment the metal cation has a va!ence of 2 to 6.

Examples of .metal cations wi th a valence of > 2 include, without limitation: Ai ^m Ba ⁿ Cd ⁿ , Co" Cr ^m , Co" Fe ⁿ Fe ^m , Mn ^K ₍ Ni ⁿ , Zn ⁿ TiF, if ¹ , Zr ^w Hf^ Ti ^w _s 5n ^iV , V ^iV , V ^v and Bi ^ra

Preferably, the metal cations are iron cations. More preferably, the iron cations are ferric iron cations.

In general embodiments, the oxyanion may be selenium-, nitrogen-, carbon-, iodine-, chlorine-, bromine-, vanadium-, manganese-, sulfur-, phosphorus-, arsenic-, chromium-, molybdenum- and/or boron -containing oxyanion.

In one particular embodiment the oxyanion is selected from the group consisting of selenite, seienate, sulfate, arsenite, arsenate, chromaie, molybdate, borate, phosphate and nitrate. The biosorbent may be present as a single mass or as fragments of algal based biomass coated with one or more metal cations. The size, shape and form of the biosorbent. will vary depending on the area and location of the water or aqueous solution that requires an oxyanion to be removed. Preferably, the biosorbent forms a layer capable of contacting the water or aqueous solution for fast and efficient removal of the oxyanion.

In one embodiment the algal based biomass comprises raacroalgae. Suitably, the macroalgae is selected from the group consisting of red (Rhodophyta), brown. (Phaeophyta) and green (Chlorophyta) algae.

Preferably, the red, green and brown algal species are selected from, but not limited to, at least one or more the following: Viva spp. (e.g., Viva ohnoi; Ulva indica; Ulva conglobata; Ulva lactuca; Ulva australis; and Ulva roiiindata), Derbesia spp. (e.g., Derbesia ienuissima; Derbesia fastigiata; Derbesia marina; and Derbesia padifiae), Oedogonmm spp. (e.g., Oedoganhtm crispiim, Oedogonmm intermedium), Cladophora spp. (e.g., Cladophora vagabi da: Cladophora aegagropila; Cladophora albida; Cladophora brasffiana; Cladophora ealenal ; Cladophora eoelothrix; Cladophora cohtmhiana; Cladophora dalmatica; Cladophora fraeia; Cladophora glomerata; Cladophora graminea; Cladophora morttagneana; Cladophora ordinata; Cladophora prolifera; Cladophora rupeslris; Cladophora scopa formis; and Cladophora serieea), Padina spp. (e.g., Padina australis; Padina crassa; and Padina pavanica), Cysioseira spp, (e.g., Cytoseira trinodis; Cystoseir sauvageaueana; Cystoseira sauvageaueana; and Cystoseira sauvageaueana), Halymenia spp. (e.g., Halymenia flares ia; and Hafymema palmata), Gracilaria spp. (e.g., Gracilaria multipartita: Gracil ria verucosm; Gracilaria eduiis; Gracilaria graciiis; Gracilariopsis longissima; and GracHaria confervoides), Eucheuma spp, (e.g., Eucheuma dcnttculaium and Euch uma spimdosa), Kappap yc s spp. (e.g., Kappaphycus alvarezii), Saccharina spp. (e.g., Saccharina japonica), Sarga-ssum spp. (e.g., Sargassum aemitfum: Sargassum crispum; and Sargasmm muticum), Undaria spp. (e.g., Undaria pitmatifida; Undaria crenaia; Undaria peterseniana; and Undaria tmdarioid.es).

More preferably, the algae species is a freshwater green algae species.

Even more preferably, the algae is of the species Ciadophora vagahtmda or of a species from the genus Oedogonium spp.

in some embodiments, at least two or more algal species are selected from the following: Ulva spp. (e.g., Ulva ohnoi; Ulva indica; Ulva ctmglohata; Ulva laciuca; Ulva australis; and Ulva rolundata), Derbesia spp. (e.g., Derbesia tenuinsima; Derbesia fastigiaia; Derbesia marina; and Derbesia padinae), Oedogonium spp. (e.g., Oedogonium crispum, Oedogonium intermedium), Ciadophora spp. (e.g., Ciadophora vagabunda; Ciadophora aegagropila; Ciadophora albida; Ciadophora brasilkma Ciadophora eaienata; Ciadophora caeloihrix; Ciadophora columbiana Ciadophora dalmatica; Ciadophora fracta; Ciadophora glomerata; Ciadophora graminea; Ciadophora montagneana; Ciadophora ordhiaia; Ciadophora prolifera; Ciadophora rupestris; Ciadophora sc ^' opaeformis; and Ciadophora sericea), Padina spp. (e.g., Padina australis; Padina crassa and Padina pavonica), Cystoseira spp. (e.g., Cytoseira trinodis; Cystoseira .muvageaueana; Cystoseira saavageaaeana; and Cystoseira sauvageaueana), Halymenia spp. (e.g., Halymenia floresia and Halymenia palmata), Gracilaria spp. (e.g., Gracilaria multipartita; Gracilaria verucossa; Gracilaria eduiis; Gracilaria gracilis; Gracilariopsis longissima; and Gracilaria can fervo ides), Eucheuma spp. (e.g., Eucheuma deniiculatum and Eucheuma spimdosa). Kappaphycus spp. (e.g., Kappaphycus alvarezii), Saccharina spp, (e.g., Saccharina japonica), Sargassum spp. (e.g., Sargassum aemuium; Sargasstm crispum; and Sargassum muticum), Undari spp. (e.g., Undaria pitmatifida; Undaria crenata; Undaria peterseniana; and Undaria vmdariaides). In one embodiment, the algal based biomass comprises waste residues remaining after extraction of phycocolloids from algae.

The algal species may be selected from, bat not limited to, at least one or more the following: Padina spp. (e.g., Padina a trails; Padina crasm and Padina pavonica), Cystoseira spp. (e.g., Cytoseira trinodis; Cystaseira sauvageaueana; Cystaseira sauvageaueana; and Cystoseim sauvageaueana), Hafymenia spp. (e.g., Hafymenia floresia; d Hafymenia paimata), Gracilaria spp. (e.g., Gracilaria multipartita; GracUaria vemcosm; Gracilaria edulis; Gracilaria gracilis; Gracijatiopsis longis ima; and Gracilaria canfervaides) Eucheitma spp. (e.g., Euchmma dentieidatum and Eucheuma spmubsa), Kappaphycus spp. (e.g., Kappaphycus lvaresii), Saccharitia spp. (e.g., Saccharina Japan ica), Sargassum spp. (e.g., Sargassum aemulum; Sargassum crispum; and Sargassmn mutictim), Undaria spp, (e.g., Undaria pinnatifida; Undaria crenata; Umiaria peterseniana; and Undaria undarioides) and Phcwophyceae spp.

Preferably the algae is of the species Gracilaria spp.

Suitably, any combination and/or ratio of algae species, biomass, waste residues and/or biochar may be coated with one or more metal cations for use in reducing or removing an oxya ion from water and/or another aqueous solution.

The algae may be obtained directly from the ocean or by fanning (e.g., aquaculture ponds) or from any other known source.

In one embodiment the algal based biomass comprises one or more algae species derived from waste water. In this regard, the algae may be grown, cultivated in or derived from, for example, mining or industrial waste water treatment ponds or the like. Additionally, the algae ma have been previously used to aid in the removal of one or more metal cations, such as iron, copper, zinc and nickel, from said waste water.

In one embodiment, the algae may be dried. Algae generally contain a large quantit of water, from about 7-90% per weight. Processes for drying algae are well known in the art (Chan et al, 1997, Le Lann et al, 2008). In one embodiment, the algae may be dried at a temperature of up to about 0°C. Suitably, the algal may be dried at a temperature of about 30 ⁴ C, 4<TC, 50°C, 60'C, 7<TC, 80 ^s C or 90 ^S C. Preferably, the algae are dried at a temperature of about 60°C. The drying temperature and time will vary depending on the algae species, the drying process and. the degree of drying required .

in one embodiment, the algae may be dried for up to about 48 hours. Suitably, the algae may be dried, for about 5, 10, 15, 20, 30. 40, 50 minutes or about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, 30, 35, 40, 48 hours. Preferably, the algae are dried for a period of about 24 hours.

In one embodiment, the alga! based biomass is alga! biochar. Biochar is produced from a thermochemieal process in which algae is heated under a condition of limited or no oxygen ( . Lehmarrn et al, 2006). The pyrolysis process is often used for biochar production as. the technique is relatively simple and inexpensive and allows considerable flexibilit in both the type and quality of the biomass produced (Bird ef ai, 2011). Slow pyrolysis, which gives a maximum yield of biochar, has been widely used for decades in charcoal kilns where the combustion occurs in the absence of oxygen. A slow heating rate is maintained up to 400-700°C. At this low temperature range, a high carbon recovery from the organic biomass is obtained in the pyrolised biochar.

For a higher temperature range of 7QQ-800 C, the cation exchange capacity of biochar itself can also be improved, but a lower carbon yield (about 5% loss) is obtained.

In another embodiment, the biochar is a by product of the thermochemical processes described by Rowbotham et ah (2012), rather than by slow pyrolysis.

In one embodiment, algae is coated with metal cations before being processed to form biochar. Suitably, the biochar is produced by pyrolysis.

Method of producing the biosorkenl

In another aspect, the invention provides a method of producing a biosorbent. for the reduction or removal of an oxyanion from water and/or anothe aqueous solution including;

i. treating one or more algal based biomass with a metal cation solution for a time and under conditions sufficient to coat the algal based biomass in one or more metal cations: and

ii. separating the treated alga! based biomass from the metal cation solution. Preferably, the metal cation has a valence of > 2. Suitably, the metal cation has a valence of 2, 3, 4, 5 or 6.

in one embodiment the metal cation has a valence of 2 or 3. In another embodiment the metal cation has a valence of 2 to 4. In one embodiment the metal cation has a valence of 2 to 5. In another embodiment the metal, cation has a valence of 2 to 6.

Examples of metal cations with a valence of > 2 include, without limitation:

A™ Ba", Cd ^Er , Co" Cr ^m , Cu ⁿ , Fe ⁿ , Fe ^m Mn". Ni ^s , ΖΒ ^π , Th ^jy , Ti ^, , Sn ^w V ^iV , V ^v an Bi ^ra .

in one embodiment, the metal cation solution is an iron solution.

Preferably, the iron solution is a ferric solution. More preferably, the ferric solution is ferric chloride solution. Also contemplated by the invention are other solutions, including but not limited to ferrous sulfate, ferric sulfate, ferric nitrate, ferrous nitrate and ferrous chloride.

In one embodiment the oxyaoion is selected from the group consisting of selenite, selenate, sulfate, arsenite, arsenate, chromate, laoiybdate, borate, phosphate and nitrate. Preferably, the oxyanion is selenate and/or selenite.

In one embodiment the algal based biomass is treated with a metal cation solution for up to about 48 hours. Suitably, the algal based biomass may be treated with a metal cation solution for about 5, 10, 15, .20, 30, 40, 50 minutes or about 1, .2, 3, 4, 5,. 6, 7, 8, 9, 10, 15, 20, 24, 30,. 35, 40, 48 hours. Preferably, the algal based biomass is treated with a metal cation solution for about 24 hours. The treatment time may vary, depending on the size and/or type of algal based biomass used for production of the biosorbent.

In one embodiment, the metal cation solution is at a concentration of up to about 50%. Suitably, the metal cation solution is at a concentration of about 0.5%, 1 %, .2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40% or 50%. Preferably, the metal cation solution is at a concentration of about 5%. The concentration of the solution ma vary , depending on the size and/or type of biomass used for production of the biosorbent and the solution used.

In one embodiment, the algal based biomass is treated with a metal carton solution at a temperature of up to about 90"C. Suitably, the algal based biomass is treated with a metal cation solution at a temperature of about 5"C, 10 ^e C, 1 5 ⁵ C, 20*C, 3<TC, 40 ^* C, 50°C _s 60 ⁴ C, 7<TC, 80'C or 90'C Preferably, me temperature is 20°C.

The solution may be applied to the algal, based biomass by any method known in the art, examples of which include cold-spray techniques (Vucko et ai, 2012), and other coating methodologies. Preferably, the algal based biomass is immersed in a metal cation solution under conditions sufficient to coat the algal based biomass in one or more metal cations.

In some embodiments, the algal based biomass is agitated whilst being contacted with a metal cation solution. Agitation may for example be in the form of swirling, shaking or stirring the algal based biomass whilst in the solution. Agitation is employed to ensure all algal based, biomass is contacted with the metal cation solution evenly.

In one embodiment, the treated algal based biomass is separated from the metal cation solution- Suitably, the separation method is filtration. However, alternative separation methods may be used and well known separation methods in the art, include such methods as flotation, centrifugation and mechanical separation ( othandaraman & Evans, 1972).

In one embodiment, the treated algal based biomass may be washed, using an aqueous solution. Preferably, the aqueous solution is distilled water. The washing step may remove residual metal cation solution which is not bound to the algal based biomass.

In some embodiments the treated algal based biomass is dried. Drying biomass is a well known technique in the ait and includes such methods as direct and indirect drying through the use of heat jets, vacuum pumps, and evaporation. In one embodiment, the treated algal based biomass may be dried at a temperature of up to about 90°C. Suitably, the treated algal based biomass may be dried at a temperature of about 30°C, 40 ^e C ₍ 50X, 6(TQ 7CTC, 80°C or 90T. Preferably, the algal based biomass is dried at a temperature of about 60°C. The drying temperature and time will vary depending on the algae species, the drying process and the degree of drying required.

In one embodiment, the treated algal based biomass may be dried for up to about 48 hours. Suitably, the treated algal based biomass may be dried for about 5, 10, 15, 20, 30, 40, 50 minutes or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 24, 30, 35, 40, 48 hours. Preferably, the treated algal based biomass is dried for a period of about 24 hours.

Suitably, the treated alga! based biomass is dried at a temperature of about 60°C for a period of about 24 hours.

in one embodiment, the algae based biomass is algal biochar,

ϊη some embodiments, algae and/or waste residues remaining after extraction of phyeocoHoids from algae are coated wit metal cations before producin biochar. Suitably, the biochar is produced by pyrolysis.

In some embodiments, biochar is treated with, a metal cation solution for a time and under conditions sufficient to coat the biochar in metal cations.

In one embodiment the algal based biomass comprises one or more algae species derived from waste water.

Methods of using the biosorbent for the reduction or removal ofox cmiom

In one aspect of the invention, there is provided a method of reducing or removing an oxyanion from water and/or another aqueous solution, including;

contacting the biosorbent of the invention with water or another aqueous solution containing an oxyanion, for a time and under sufficient conditions to absorb the oxyanion from the water or aqueous solution.

In general embodiments, the oxyanion may be selenium-, nitrogen-, carbon-, iodine-, chlorine-, bromine-, vanadium-, manganese-, sulfur-, phosphorus-, arsenic-, chromium-, molybdenum- and/or boron-containing oxyanion.

In one embodiment the oxyanion is selected from the group consisting of selenite, selenate, sulfate, arsemte, arsenate, chromate, molybdate, borate, phosphate and nitrate. Preferably, the oxyanion. is selenate and/or selenite.

In one embodiment, the biosorbent is exposed to water dfot another aqueous solution containing an oxyanion for up to about 4 hours. Suitably, the biosorbent is exposed to water and/or another aqueous solution containing an oxyanion for about 1 , 2, 3, 4, 5, 10, 20, 30, 40, 50 seconds or about 1 , 2, 3, 4, 5, 10, 15, 20, 30, 40, 50 minutes or about 1, 2, 3 or 4 hours or unti l at least part or all of the oxyanion is absorbed and removed.

In some embodiments, at least up to about 90% of the oxyanion in the water or aqueous solution is absorbed or removed. Suitably, at least about 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10%, or even about 5%, 4%, 3%, 2%, 1% of the oxyanion in the water or aqueous solution is absorbed or removed. Preferably, at least 90% of the oxyanion in the water or aqueous solutions is absorbed and removed. Preferably, all forms of selenium, including oxyanions Se ^1Xi and Se ^vi are absorbed and removed from the water or aqueous solution,

The amount and rate at which an oxyanion is absorbed and removed may vary depending on the levels and type of oxyanion in the water or aqueous solution and the size, shape and composition of the biosorbent.

Removal of metal cations

It may be advantageous to remove any metal cations that may have leached from the biosorbent and/or any metal cations that were originally present in the water and/or other aqueous solution before treatment with the biosorbent.

Accordingly, in a further embodiment, the water or aqueous solution is contacted with another algal based biosorbent, for a time and under sufficient conditions to absorb one or more metal cations from the water or aqueous solution.

As would be well appreciated by the skilled artisan, the treatment of complex effluents that contain both oxyanion and metal contaminants, such as those from mining or industrial settings, presents a challenging scenario. Furthermore, the treatment of comple effluent is difficult because the dissolved ions in the effluent may have a variety of properties, and hence, affinities for different btosorbents. As described herein, removing oxyanions that do not have a natural affinity for algal based biomass requires coating with one or more metal cations, which typically results in additional metals being released into the waste water upon contact, hi this regard, untreated algal based bioraass is generally effective in removing a range of dissolved metal cations from waste water and the affinity of dried macroaigae for metals (e.g. N ^' ) can be enhanced by converting biomass to biochar through slow pyrolysis. Accordingly, an additional step of contacting the waste water with another algal based biosorbent that is not coated with a metal cation (e.g. algae or algal biochar not coated with a metal cation), can be effective in removing one or more metal cations from the waste water. Moreover, such a step may go some way to at least ameliorating the metal cation leaching that can occur through the previous or concomitant treatment of the waste water with a metal cation coated biosorbent.

In one embodiment, the water or aqueous solution may be treated simultaneously with both the biosorbent and the another algal based biosorbent. In an alternative embodiment, the water or aqueous solution may be treated sequentially either with the biosorbent and then the another algal based biosorbent or the another algal based biosorbent and then the biosorbent.

Preferably, the another algal based biosorbent is an algal based biomass that has not been coated with metal cations.

Even more preferably, the another algal based further biosorbent is an algal biochar. that has not been coated with metal cations.

In one embodiment, the one or more metal cations to be absorbed is selected from the group consisting of aluminium, sodium, cadmium, chromium, copper, lead, manganese, nickel, barium, calcium, cobalt, iron, magnesium, potassium, vanadium and zinc.

In one embodiment, the algal based biomass comprises one or more algae species derived or obtained from, grown and/or cultivated in waste water.

An overview of an emdodiment of the method that includes cation removal and oxyanion removal is shown in Figure 24.

in a further aspect, the invention provides a method of reducing or removing one or more metal cations from water and/or another aqueous solution, including;

contacting an algal based biomass, with water or an aqueous solution containing one or more metal cations, for a time and under sufficient conditions to absorb the one or more metal cations from the water or aqueous solution.

The one or more metal cations may be as hereinbefore described.

In a particular embodiment, the one or more metal cations to be absorbed is selected fro the group consisting of aluminium, sodium, cadmium, chromium, copper, lead, manganese, nickel, barium, calcium, cobalt, iron, magnesium, potassium, vanadium and zinc.

In a certain embodiment, the algal based biomass comprises an algae species of Ctadophwa $pp„ and/or O dogonium spp.

In one embodiment, the algal based biomass comprises one or more algae species derived from waste water.

In one embodiment, the algal based biomass that has absorbed the one or more metal cations from the water or aqueous solution may subsequently be used to produce the biosorbent of the first aspect or the third aspect and/or according to the method of the second aspect. The algal based biomass that has absorbed the one or more metal cations may be used i the form of an algal based biomass or as a bioehar (Le as an untreated or natural algal based biomass or as a bioehar as hereinbefore described).

In yet another aspect, the invention provides an algal based biomass comprising one or more metal cations absorbed from water or from another aqueous solutio and which is capable of absorbing one or more oxyanions from water or from another aqueous solution.

In one embodiment, the biosorbent hereinbefore described comprises the algal based biomass of the present aspect .

So that the invention may be readily understood and put into practical effect, reference is made to the following non-limiting examples.

EXAMPLES EXAMPLE i

Materials and Methods

Algal species and biomass preparation

Eight species from the three algal divisions (red algae - Rhodophyia, brown algae - P aeophyia and green algae - Chlarophyia) were tested for potential use i the remediation of seleniferous waste waters. The selected species were l.llva ohnoi (Uo) and Derbesia iemmsima (Dt)(marine green algae), Oedogonium crispitm (Oc) and ladaphora vagabunda (Cv)( freshwater green algae), Padina australis (Pa) and Cysloselm trinodis (Ct)(marine brown algae), and Hafymenia floresi (Hf) and Gracilarki sp. {Gs)(marine red algae). The algae were identified using morphological characteristics with the aid of taxonomic keys (McCarthy and Orchard, 2007; Cribb, 1996).The species were collected from the field or large-scale marine and freshwater culture systems. All field collections were conducted at Nelly Bay on Magnetic Island in the Great Barrier Reef, Australia (19°09'45.-84 ^>s S, 146 ^* 51 ' 02.54" E). Algae taken from cultures were originally sourced from agricultural drainage ditches, aquaculture ponds or natural marine environments in the vicinity of Townsvilie,. Australia. Prior to the experiments approximately 2 kilograms ( kg) wet weight of each algal species was harvested and dried to constant mass at 60 ^S C for 24 hours (h). The algae were then coarsely milled and sieved to retain fragments of 1-2 mm in diameter.

A 50 g sub-sample of each type o biomass was converted to biochar by slow pyrolysis under conditions previously described (Bird et a!., 201 1 ). Briefly, 50 g of algae was weighed, loaded into a wire mesh basket and suspended in a sealed 2 L stainless steel container in a muffle furnace. The container was continuously purged with nitrogen gas at 4.0 L rain ^"1 and heated to a hold temperature of 45Q"C for 1 h. The resulting biochar was then cooled to room temperature and bagged in zip-lock plastic bags.

Bio rpii n experiments,

Biosorption experiments were conducted to quantify the rate and extent of Se ^n' and Se ^VI adsorption from freshwater by the dried biomass of each species as

TV V

well as their derived biochar. The nominal Se and Se concentrations for the initial biosorption experiment were 500 μ§ L ^"! . Experimental solutions were made from diluted 1000 mg L ^"1 stocks of sodium selenite (Na ₂ SeO _: ¾) and sodium selenate (NazSeG-j) respectively in deionized water. Each trial was conducted under identical conditions of pH, temperature, contact time and biomass density. The Se stock solutions were adjusted to pH 4.0 with 0.01 M HQ before use in the experiments as this has previously been determined to he an optimal pH for Selenium (Se) biosorption (Wasewar, Prasad, and Gulipalli, 2009; Gonzalez- Aeevedo et at., 2012).

Starting Se stock concentrations were determined from three replicate samples (25 ml) prior to the addition of biomass or biochar and solutions were filtered through a 0.45 μΐη syringe filter for subsequent analysis. Therefore, all data shown relate to measured values rather than nominal concentrations (see Table 2). Subsequent to sampling, each replicate consisted of a 250 ml plastic beaker with 25 ml of the relevant Se solution (selenite or selenate) and 0.25 g of dried biomass or the biochar derivative (equivalent to a stocking density of 10 g L ^"1 of biosorbent). All experiments were conducted at the same stocking density. The beakers were shaken at 1 0 rpm in the respecti ve solutions for 4 h at 20"C. A t the end of the 4 h exposure the samples were centrifuged for 15 min (4000 rcf). The supernatant was then filtered through a 0.45 um glass fiber syringe filter, in addition, samples containing no algae were processed in the same manner to sen'e as controls to quantify losses of Se to experimental glassware. All glass and lastic-ware was acid-washed in a 5% HNC bath the rinsed in deionized water prior to use in the experiments.

Experiments with Fe-loaded biomass and bioehar followed the same methods but with an additional pre-treatment of the biosorbent substrate with ferric chloride (FeO obtained from Sigma Aldrieh® as a 40% w/V Fe solution). A 5% ferric chloride solution was prepared by diluting FeCl,? in deionized (DI) water. Algal biomass was the added to the solution at a rate of 25 g Γ ¹ for 24 h on a shaker (150 rpm) at 20 ^* C. The biomass and bioehar were subsequently filtered from the solutio and rinsed three times with approximately 50 ml of DI water, then dried at 60 °C for 24 h before being used as described above in biosorption experiments.

Elemental analysis

All water samples were collected as described above and analysed for final total Se and Fe content after the 4 h biosorption experiments, to quantify Se removal and Fe-Ieaching from pre-treated biosorbents respectively.

Data analyses

As the Fe-treated biomass and bioehar experiments were run as separate trials they were analysed as separate datasets. Se^ and Se ^I uptake by Fe-treated biomass and bioehar were modeled by two way of Analysis of Variance (ANOVA) with the factors 'species' (8 algae) and Oxidation state' (selenite and selenate). Data were examined for normality and homogeneity of variance through visual inspection of frequency histograms and scatterplots of residuals vs. estimates respectively (Quinn and Keough, 2002). In addition, the percent removal of Se was correlated against the leaching of Fe (as determined by analyses of total Fe in the water samples after the experimental contact period). In ail cases removal of Se from the water was expressed as a percentage based upon the initial starting concentrations. Statistical analyses and figures were produced in R using the base analysis and gplots packages (R Core Development Team 2012; Wames, 2012).

Results

elenite and_ knat _ miso ption_hy_ dried ¹ biomass _ and_ bioehar

Un-roodified dried biomass and bioehar were both ineffective as biosorbents for Se as Se ^l and Se ^l . Only $e ^n> was detectably removed from solution by dried biomass, although no species exceeded 8% removal following the 4 h exposures (Figure 1), All of the green algae removed Se ^l to some extent, (range 1.3-7,2%) however, Padina was the most effective biosorbent with 7.74% removal. Both red algae (Gracilari and Hal m nia) and the other brown algae, Cytiorseira, were ineffective against both of the Se oxidation states. Conversion of the biomass into biochar did not markedly increase the affinity of the algal species for Se ⁿ or Se^, with less than 5% removal for most biochars (Figure 1). Padina was again the most effective biosorbent, removing approximately 10% Se ^T in 4 it (Figure 1). Se ^l was not removed to any extent by the algal-derived biochars (Figure 1 ). The negative controls showed negligible change in Se concentrations over the 4 h contact period (<2%) indicating there was no loss of Se to experimental glassware.

Seletiite and seienate adsorption hy Fe-loaded biomass and biochar

Fe-loaded biomass had a much stronger affinity for both Se ^l and Se ^l under laboratory conditions than the respective un -treated biomass and biochar. samples. There was, however, considerable variation between algal species with respect to their affinity for Se ("Species x Oxidation State" F7.32 = 83.40, P < 0.001 ) with no clear effect of algal taxonomic groupings on biosorptioii performance. Biosorptioii of Se ^B was also consistently higher than for Se ^l by all species. The highest removal of Se ^F* was with Fe-treate biomass of Cladophora, Padina, Cystoceria and Ulva, each of which removed >90% Se ^R within 4 h (Figure 2a, Figure 1 ). The same three species removed between 60-90% Se across the same period and were again the best biosorbents (Figure 2b, Figure 1), Overall, Cladophora was the best performing biosorbent with >95% Se^ removal and approximately 90% Se ^vI removal (Figure 2a and b, Figure 1). The red algae were again the poorest S biosorbents, with approximately 2.5% Se ^, and 5-1.0% Se ^I removal over the 4 h contact period (Figure 2a and b, Fi gure 1).

Fe was leached from al l of the Fe-loaded algal samples during the 4h contact time. There was, however, a clear inverse relationship between leaching patterns and Se biosorption; the species with the lowest Se biosorptioii rates leached the greatest amounts of Fe (Figure 3 a). For example, the red algae leached the greatest amounts of Fe into the water, whilst the best biosorbents ( Ulva, Cladophora and Padina) leached the lowest amounts. Overall Cladophora leached the least Fe into solution and was the best biosorbent of both Se ^T and Se ^T . Fe-leaching was significantly negatively correlated with Se biosorption by Fe-treated biomass for both Se ^I (adjusted τ ² - 0.717, P - 0,005; Figure 3a) and Se ^vl (adjusted r ^{2 «} 0.746, P - 0.004; Figure 3 a).

The conversion of biomass into biochar, followed by Fe pre-treatment had variable effects on Se biosorption. There were three main patterns of response. For red algae, Se biosorption capacity increased relative to Se biosorption obtained by Fe-treated biomass. Fe-treated biochar from GracH ri removed approximately 40 and 29% of Se and Se ^* respectively, compared to 27 and 10% removal of Se and Se ^I as Fe-treated biomass (Figures 1 , 2 and 4), Fe-treated biochar from Halymenia and Qedogomum both had a much greater Se biosorption. capacity than Fe-treated biomass of these species and were effective against both Se oxidation states (Figures 3, 1 and 4). In contrast, the Se biosorption capacity of both brown algal species and l.Jlva decreased markedly as Fe-hiochar relative to the removal rates achieved as Fe- biomass (Figures 2 and 4). Regardless, Cladophora remained the most effective hiosorbent of Se ^l as Fe-treated biochar, removing 93% of Se in this oxidation state and 82% of Se ^I , with similar results for the other freshwater filamentous species Oedogonhrm ( Figures 1 and 4). Again, the controls showed negligible change in Se concentrations over the 4 h contact period (<2%) indicating there was no loss of Se to experimental glassware. Fe again leached from the Fe-treated biochar and typically at a higher rate than from the Fe-biornass. However, while there was a trend towards increased Fe-leaching from the poorest Se biosorbents, the relationship was not statistically significant for either Se ⁿ (adjusted r ² = 0.151 , P = 0.184; Figure 3b)or Se ^J (adjusted r ² - 0,299, P = 0.093; Figure 2b).

The inventors have demonstrated that sustainable and effective Se biosorbents can be created through the modification of dried algal biomass with, ferric chloride. The derived biosorbents are effective against both Se and Se and can be used in a similar manner to traditional algal biosorbents in treatment systems. Furthermore, the transformation of dried biomass to biochar. followed by the same modification with ferric chloride, can improve the performance of specific biomass types while having a negligible effect on others. Notably, the inventors found both dried biomass and biochar to be ineffective as Se biosorbents without pre-treatment with ferric chloride. While there are some small-scale evaluations that suggest marine Cladophora species may be effective Se biosorbents without pre-treatment (Tuzen and San, 2010), the inventors results add to increasing evidence that traditional metal biosorbents siteh as unmodified biomass, biochar and activated carbon (AC) are ineffective against Se regardless of oxidation state (Mane et al., 20] 1; Mahan, Majidi, and Holcombe, 1989; Niu and Volesky. 2003; Latva, Peraniemt, and Ahlgren, 2003). Consequently, the data indicate that many types of algae may provide suitable feed stock for Se biosorbents once appropriately prepared. However, there are clearly important characteristics that differentiate between the putative feed stock species, particularly in their selectivity for Se ^{1 '"} and Se ^* - as well as their relative affinities for the Fe-treatrnents required to boost Se biosorption.

Biomass derived from green and brown, algae were consistently the best biosorbents despite some variance between representative species. There was also a significant difference between rates of removal of the two oxidation states, with all Fe-treated biomass from all species showing greater removal of Se ⁱ tha Se ^T . Fe- loaded biomass of Cladophora was the best performed substrate in this context with approximately 90% removal of Se ^j and >95% removal of Se ^,f These results differ somewhat from previous comparisons between red, brown and gree algae. For example, red algae were more effective biosorbents of Cr ^m and Cr ^l than brown and green species (Murphy, Hughes, and McLaughlin, 2008), while brown algae tend to perform best as Cd biosorbents (Hashim and Chu, 2004). There is therefore likely to be some specificity for the best biosorbents depending upon the contaminant in question. Given unmodified, biomass and bioehar were relatively ineffective biosorbents for Se, the relative performance of each substrate is most likely attributable to their affinities for Fe, as supported between the strong correlation between Se biosorption capacity and the leaching of Fe from the Fe-treated biomass.

The Fe-loaded biomass substrates leached Fe into the test vessels during the biosorption trials. Furthermore, the rate of leaching was inversely proportional to the subsequent removal of Se for each species. For example, the red algae had the lowest Se removal rates and also leached the gi eatest amounts of Fe, while Cladophora had the lowest Fe leaching rates and highest Se removal capacity. This result suggests that only Fe bound to the algal substrate contributes to the bioremecliation process and that the efficacy of the various species for Se removal is likely linked to tlieir relative affinities for Fe in solution. In addition, any preparations that minimize Fe leaching should also boost Se adsorption. Further optimization of the Fe-loading technique may therefore lead to greater capacity for Se adsorption by biomass sorbents. Strategic treatment systems may also be possible whereby im-manipulated algal biomass or biochar is deployed in treatment systems after the modified Se biosorbent to reclaim any leached Fe, thereby loading biomass for subsequent deployment as Fe-treated substrates. However, somewhat surprisingly, the relationship between Fe-leaching and Se biosorption was not significant for Fe- treated biochar. The rate of Fe-leaching from Fe-treated biochar' was, in general, greater than from Fe-treated biomass which indicates that unmodified biomass of some species has a greater Fe-retention rate than the derived biochar. The results show a strong affinity for both of the inorganic aqueous forms of Se in industrial effluents following simple preparation methods.

The mechanisms associated with anionic contaminant adsorption are typically poorly understood (Gadd, 2009). Not wishing to be bound by any particular theory, it is thought that the predominant process is electrostatic attraction between the negatively charged oxyanion and a positively charged adsorbent surface. The deposition of metal cations, such as Fe, on the surface of biomass and biochar can increases the positi ve valence of the biosorbent thereby increasing its affinity for the negatively charged selenate oxyanion (Faria, 2004). This encourages the formation of inner-sphere complexes between the deposited ferric iron and dissolved selenium oxyanion, leading to strong binding to the biosorbent (Zhang et at, 2008). Most commercially available biosorbents only possess a net positive serf ace charge at very low pH and so they have limited effectiveness at higher pH. It has also been suggested that the surface modification of biochar with Fe provides a pH- independent positi ve valence o the adsorbent thus rendering metal -modi fied biochar a more effective adsorbent at a wider range of pH. However, the use of Fe as the precipitate has the additional benefit of providing a surface that can drive the reduction of Se ^l to followed by adsorption of the reduced oxyanion to the biosorbent substrate (Dobrowolski and Otto, 2012).

One of the major findings of this work is that some of the biosorbents proved to be equally or eve slightly more effective against the Se ^l oxyanion than Se ⁵ This is in direct contrast to many of the existing Se remediation technologies, including the current Best Available Technolog (BAT; ferrihydrite adsorption) that is successful at adsorbing Se ^, but much less effective against Se ^VI (Faria, 2004). This poses a major limitation on the application of these existing technologies as many waste streams are dominated by Se . Somewhat surprisingly however, the conversion of the algal biomass into biochar did not uniformly influence the rate of Se biosoiption from solution, indeed, some of the species that were relatively poor biosorbents as Fe-treated biomass became the best performed biosorbents as Fe- treated biochar. Whilst the inventors initially predicted that the conversion of biomass to biochar would boost the surface area available for Fe adsorption and therefore increase biosoiption perfonnance for all species, they found that some species were actually poorer biosorbents following conversion to biochar. Notably, the results demonstrate that filamentous green macroalgal species perform well as both Fe-treated biomass and biochar.

While there has been a rapid increase in the number of publications describing biosoiption of contaminants with substrates such as macroalgae there has been little or no uptake of the technologies within industrial settings (Gadd, 2009). Several barriers exist to implementing macroalgal -based biosoiption at scales required by industry. One major barrier is identifying a source of biomass that is both sustainable and cost-effective. While wild harvests of macroalgae were originally envisaged to be sufficient to support industrial-scale bioremediation, it is now acknowledged that this is not environmentally sustainable (Volesky, 2007). It has been proposed more recently that algae from large-scale intensive aquaculture may fill the demand for biomass, specifically that freshwater algae can be cultured directly in metal waste streams supplemented with flue gas to provide a bioremediation and carbon sequestration service (Saunders et at, 2012)( oberts et al,, 2013). While this process is effective at removing metals such as Al, Cu and Zh from waste effluents, metalloids such as Se are more slowly sequestered by live algae (Saunders et al., 2012 and Roberts et al., 2013). The results suggest that the biomass produced at algal culture facilities integrated with waste producing industries ma then form the feed stock for further bioremediation of meta lloids such as Se through passive biosorptioii at the same facility, either as Fe-treated biomass or biochar.

EXAMPLE 2

Materials and Methods Algal species and bivmass preparation

Six macroalga! species were obtained from commercial aquaculture operations and tested for application in the remediation of waste waters containing Se. The species were derived from field collections or large scale marine culture systems (Figure 6). Prior to the experiments approximately 200 g of each algal species was rinsed in deioiiized water (DI) to remove snrftcial salts and dried to constant mass at 60'C for 24 hours (h). The algae were then coarsely milled and sieved to retain fragments of 1-2 mm in diameter. A 50 g sub-sample of each biomass was then converted to biochar by slow pyrolysis under conditions previously described (Bird et etL, 201 1). Briefly, 50g of algae was weighed, loaded into a wire mesh basket and suspended in a sealed 2 L stainless steel container in a muffle furnace. The container was continuously purged with nitrogen gas at 3.5 L min ^" ' and heated to a hold temperature of 450 ^* C for 1 h. The resulting biochar was men cooled to room temperature and bagged in zip-lock plastic bags with silica gel dehydrators until use. Biawrpiion experiment

Biosorptio experiments were conducted to examine the rate and extent of biosorption of Se from freshwater by dried algal biomass and biochar form the macroalgal species. Each algal species was tested as a biosorbent for Se in 4 different forms; as unmodified dried biomass and biochar, and as Fe-treated biomass and biochar. The biosorption experiments followed the same methods as previously described (Roberts ei a!., 2013). Briefly, to produce the Fe-treated biosorbents the biomass or biochar of each specie was soaked in a 5% ferric chloride solution at a rate of 25 g Γ ¹ for 24 h on a. shaker ( 150 rpra) at 20 . The biomass and biochar were then filtered from the solution and rinsed three times with DI water, then dried at 60' for 24 h before being used in. the biosorption experiments.

Multiple oxidation states of Se were tested in the biosorption experiments, including selenite (Se ^A' ), selenate (Se ^l ). The nominal Se concentrations were 500 fig L ^*! made from diluted 1000 mg L stocks. Actual Se concentrations in the respective treatments are listed in Figure 5. Selenite and selenate solutions were made from diluted sodium selenite (NaaSOs) and sodium selenate (Na ₂ S04 _. ) respectively in deiontzed water (Sigma- Aldrich®).

The biosorption trials were conducted under identical conditions of pB, temperature, contact time and biomass stocking density. The Se stocks were adjusted to pH 4.0 with 0.01 M HCl before use in the experiments. Each replicate consisted of a 250 ml plastic beaker with 25 ml of the relevant Se stock and 0.25 g of dried biomass or the biochar derivative (equivalent to a stocking density of 10 g L ^"1 of hiosorbent). The beakers were shaken at 150 rpm in the respective solutions for 4 h at 20 C. At the end of the 4 h exposure the samples were filtered to remove large particulates (75 μηι) and eentrifuged for 15 niin (4000 rcf). The supernatant was then filtered through a 0,45 pm glass fiber syringe filter. Actual starting Se stock concentrations were determined from 3 water samples that were first filtered through a 0.45 um swinge filter and all data shown relate to actual measured vahies rather than nominal concentrations. In addition, samples containing no algae were processed in the same manner to serve as controls to quantify losses of Se to experimental glassware. All glass and plastic-ware was acid-washed in a 5% HMG ₃ bath then rinsed in deionized water prior to use in the experiments.

J ockar ch mcieri iion

Each biochar was characterized for a range of properties following procedures previously described (Castine ef aL, 2013). Loss accompanying pyrolysis of each algal sample was measured to provide an estim te of organic and carbonate contents of the biomass. This was achieved by weighing the biomass before and after biochar production as described above. The sulphur, phosphorus, iron, manganese, magnesium, potassium, calcium and sodium contents of each biochar sample were then determined by inductively coupled plasma mass spectrophotometry (ICP-MS). Total nitrogen (TN) and total organic carbon (TOC) were determined following PN and PC methods as described above. Electrical conductivity (EC) and pH were determined in 0; watensample mixtures according to Australia standard methods for soil analysis (Rayment and Higginson 1992). Cation exchange capacity (CEC) was determined using silver thiourea extracts (Rayment and Higginson, 1992). and Brunauer, Emmet and Teller (BET) surface area was determined by nitrogen adsorption (Particle and Surface Sciences Pty Ltd., in Gosfoixi New South Wales, Australia).

Elemental analysts

All water samples were collected as described above and analysed for final total Se and Fe content after the 4 h bio sorption experiments, to quantify Se removal and Fe-leaehmg from pre-treated biosorbents respectively. Statistical analyses

Se ^lv and Se ^yt uptake by algal biomass and biochar were modeled using a nested Analysis of Variance (ANOVA) with 'species' nested within, 'division'. In addition, Se speciation was included as a factor in the model ('speciation'., two levels). In all cases removal of Se from the water was expressed as a percentage based upon initial starting concentrations. Data were examined for normality and homogeneit of variance through visual inspection of frequency histograms and seatterpiots of residuals vs. estimates respectively and transformed when necessary (Quinn and eough, 2002). Statistical analyses and figures were produced in R using the base analysis and gplots packages (R Core Development Team 2012; Warnes, 2012).

Results

Selemte and se!emite adsorption

Unmodified biomass and biochar from brown and green macroalgal species were ineffective biosorbents for selenium regardless of oxidation state (Figure 7). A small percentage of Se ^I could be removed by two of the three brown algal species as biomass, but Se ¹ concentrations remained unchanged after contact with the biomass samples. Conversion of the biomass to biochar slightly increased removai of Se ⁵ , with Saccharina japonica achieving approximately 10% removal (Figure 7). However, none of the biochars removed Se ^vl .

Fe-loaded algal biomass of brown macroalgal species was an effective biosorbents for Se, particularly as Se ⁱ (Figure 8). The highest removal was achieved with Fe-treated biomass of Saccharina japonica and Undaria pinnaiifida, both of which removed 90% Se ^lv and >80% Se ⁱ within 4 h. Sargassum sp. removed approximately 90% Se ^t and 30% Se ^I in the 4 h exposure period (Figure 8). As previously demonstrated, red macroalgal species were the poorest Se biosorbents,

TV VI

with <30% Se removal. Only Gracilaria sp. was able to remove Se , achieving 10% removal over the 4 h contact period (Figure 8).

The conversion of biomass to biochar had opposing effects on the Se sorption capacity of red and brown algae. Red algae became more effective biosorbents while brown algae became less effective. Following conversion to biochar, Euche ma was the best biosorption substrate with 80 and 65% removal of Se and Se respectively (Figure 9). Of the brown algae, Sacchanna japonica was the most effective with approximately 70% removal of Se ^t and Se ^vl respectively (Figure 9).

EXAM PLE 3

Materials and Methods

4 tg ^a l species and Mam s* preparation

De-alginated Gracilaria waste (DGW) (Sampah rumput laut) was obtained from Agarrado ^'8* and converted to biochar under optimized conditions as previously described (Bird et «/., 201 1), In brief, approximately 200g of waste Gracilaria biomass was weighed, loaded into a wire mesh basket and suspended in a sealed 2 L stainless steel container in a muffle furnace. The container was continuously purged with nitrogen gas at 3.5 L mm ^"1 and heated to a bold temperature of 450"C tor 1 h. The resulting; biochar was then cooled to room temperature and bagged in zip-lock plastic bags. Fe-loaded biomass and biochar were prepared through a pre-treatment of the biosorbent substrate with a 5% ferric chloride solution. Biomass and biochar were added to the ferric chloride solution at a rate of 25 g Γ for 24 h on a shaker ( 150 rpm) at 20"C. The biomass and biochar were then filtered from the solution and rinsed three times with DI water, then dried at 60 'C for 24 h before being used as described below in biosorption experiments.

Initial screening experiments

An initial biosorption experiment was conducted to examine the extent of

Se ⁿ and Se ^I adsorption from freshwater by Fe-treated DGW biomass and biochar. The nominal Se ¹ and Se^ ¹ concentrations were 500 μ L ^"1 made from diluted 1000 mg L ^"1 stocks of sodium se!emte Na^SC ) and sodium selenate a^SC^) respectively in deionized (DI) water. The Se stocks were adjusted to pH 4,0 with G.G1M HC1 before use in the experiments. Each replicate consisted of a 250 ml plastic beaker with 25 ml of the relevant Se stock and 0.25 g of dried biomass or the biochar derivative (equivalent to a stocking density of 10 g L ^"1 of biosorbent). The beakers were shaken at 150 rpm in the respective solutions for 4 h at 20 ^e C. At the end of the 4 h exposure the samples were filtered to remove large particulates (75 um) and centrifuged for 15 min (4000 ret). The supernatant. was then filtered through a 0.45 μιη glass fiber syringe filter. Actual starting Se stock concentrations were determined from 3 water samples that were first filtered through a 0.45 μηι syringe filter and all data shown relate to actual measured values rather than nominal concentrations. In addition, samples containing no algae were processed in the same manner to serve as negative controls to quantify losses of Se to experimental glassware. All glass and plastic-ware was acid-washed in a 5% HM¾ bath then rinsed in de ^' ionized water prior to use in the experiments.

in a second experiment the effect of pH o selenate biosorption was assessed following the same general methods as described above. Two pH values were assessed including 4 and 8. The pH was adjusted to 4 with 0.1M HC1 and 8.0 with 0. !M NaOH respectively.

An experiment was also conducted to determin if leaching of iron fro the biosorbent could be reduced by first loading the iron onto the biomass, then converting it to biochar (as opposed to iron-treating biochar directly). Experiments took the same form as described above, except that iro treatments were applied to the biomass before it was converted to biochar.

DGW biochar was characterized for a range of properties following procedures previously described (Castine ef aL, 2013). Loss accompanying pyrolysis was measured to provide an estimate of organic and carbonate contents of die biomass. This was achieved by weighing the biomass before and after biochar production as described above. The sulphur, phosphorus, iron, manganese, magnesium, potassium, calcium and sodium contents of each biochar sample were then determined by inductively coupled plasma mass spectrophotometry (ICP-MS). Total nitrogen (TN) and total organic carbon (TOC) were determined following PN and PC methods as described above. Electrical conductivity (EC) and pH were determined in 10; 1 watensample mixtures according to Australia standard methods for soil analysis (Rayment and Higginson 1992). Cation exchange capacity (CEC) was determined using silver thiourea extracts (Rayment and Higginson 1992), and Bnraauer, Emmet and Teller (BET) surface area was determined by nitrogen adsorption (Particle and Surface Sciences Pty Ltd., in Gosford, New South Wales, Australia).

Elemental analysts

All water samples were collected as described above and analysed for final total Se and Fe content after the 4 h btosorption experiments, to quantify Se removal and Fe-leaehmg from pre-treated biosorbents respectively. Data analyses

The initial Se biosorption experiment was analysed by 2 -way ANOVA, including the factors "treatment" (Fe-treated biomass and biochar) and "speciation"

TV VT

(Se and Se ). Data were expressed as percent removal and analysed with the R base package (R Core Development Team 2012).

Results

SeienUe and selenate adsorption by Fe~loaded DGW biomass and biochar

Fe-loaded DGW biomass was marginally effective as a biosorbent for Se as both Se ^I arid Se , achieving 32 and 38% removal of Se as Se ^r " and Se ^I respectively (Figure 10). When converted to biochar and treated with Fe ₅ the DGW

TV

displayed much higher Se removal with 90 and at least 96% removal of Se and Se ^w respectively (Figure 10). Se ^n* removal may in fact have exceeded 96% as total Se in the residual sample was less than the limit of detection (10 [ig Γ ¹ ). These results were confirmed through repeat assays which yielded near identical results. Fe-biochar was slightly more effective at removing Se ^I than Se ^I , whilst the opposite was true for Fe-biomass (Figure 10, 'treatment x speciatton': Fi,g = 20.125, P = 0,002). On the basis of these results Fe- biochar was selected for further experimental work in the following sections.

The effect of p on (he removal ofS

Biosorption of selenium as selenate was equally effective regardless of pH, with 97.14% selenate removed at pH 4 and 98.30% selenate removed at pH 8 (Figure .1 1).

Comparison, qfSe-removat b biosorbents treate with iron pre- d posi-pyrolysis Biosorption of selenium was slightly higher when the Fe-treatment was applied to the substrate before rather than after pyrolysis, with approximately 97% removal by pre-treated biosorbents and 93% with post-treated biosorbents (Figure 12a). The leaching of iron was, however, significantly greater if the iron treatment was applied after pyrolysis than before (Figure 12b). This indicates that further efficiencies in. the biosorption process may be attained by optimizing the sequence of treatments applied to boost Se uptake. Furthermore, the leaching of Fe can be mi nimized through strategic preparations of the biosorpti on substrates.

Throughout the specification the aim has been to describe the preferred embodiments of the mvention without limiting the invention to any one embodiment or specific collectio of features. It will therefore be appreciated by tliose of skill in the art that, in light of the instant disclositre, various modifications and changes can be made in the particular embodiments exemplified without departing from the scope of the present invention.

All computer programs, algorithms, patent and scientific literature referred to herein is incorporated herein by reference.

EXAMPLE 4

This Example studied the comparative ability of Gracilaria Modified Biochar (GMB) (i.e. produced by treating a waste product generated after the commercial extraction of agar from cultivated seaweeds with ferric chloride (FeCl.s) and

V Ϊ

converting it to biochar through pyrolysis) to remove Se from a single-element mock solution and a mine effluent that is contaminated with the potentially inhibiting, oxyanions SC ^" and NOV. The specific aims were to 1) compare the biosorpt on capacity of GMB for Se in both a single-element mock solution and in a real-world mine effluent, 2} compare the btosorption capacity of GMB for target (Se ^VT ) and non-target (SO4 ^2" and NO ₃ ^" ) constituents of mock solutions, and 3) determine the btosorption capacity of the GMB for Se as a function of differing relative concentrations of non-target (SO ₄ " ^" and NO3 ^" ) compounds to quantify the interactions between elements.

Methods

Real-world mine effluent

A sample of a mine effluent was obtained from a Canadia coal mine operated by Anglo American Coal at Peace River, British Columbia. The sample was collected in June 2013 from an effluent sedimentation pond and cold-shipped to James Cook University in Townsville, Australia. The effluent was stored in a fridge at 4 ^C' C until use. The concentration of total Se was 108 ig L ^"1 and speciation analyses showed this to be 99% Se with a small amount of Se ^n' . The elemental composition of the effluent is shown in Table 1.

Biomms.prepgmtiw ⁾

De-alginated Gracilaria waste was obtained from Agarlndo Bogatama in

Indonesia (for details see Roberts et al., in press). Prior to use as a biosorbeni, the de- alginated Gracilaria waste was rinsed with MilHQ water and dried in an oven at 60°C for 24 hrs. The dry biomass was then loaded with a 5% FeCis solution (Sigma Aldrich) at a rate of 25 g biomass L ^"1 for 24 h at 15 ^S C be ore being rinsed twice with ilHQ water to remove excess FeCh. After a second dr ing in the oven at 60°C for 24 h, the FeC¾~loaded biomass was converted into GMB by a pyrofysis process where the biomass was pyrolysed at a temperature of 450°C for 1 h while being continuousl pinged with N ₂ gas at a rate of 4 L mm ⁵ . The biochar was then cooled to room temperature under continuous N? flo and stored for use.

Performance o GMB in Se ^l mock solution and mine effluent

A biosorption experiment was performed to compare the performance of GMB in a Se mock solution and a real-world mine effluent containing Se and the oxyanions NOV and SO4 ^2' . The concentration of Se ^l in the mock solution was prepared to reflect that in the effluent (1.08 pg L ^"1 ) at 100 pg L ^"! . Total Se was measured to allow a direct comparison of performance in the two solutions. Since both solutions contained 99% Se ^{1' 1} , data for total Se are hereafter referred to as Se .

A 1 g L ^'1 Se ^v! solution was made by mixing NaiSeil* (Sigma Aldrich) with MilliQ water after which the solution was diluted to 100 μg L ^"1 . The mock solution and the mine effluent were treated multiple times with GMB to measure sequential reductions in dissolved Se ^I after multiple deployments of biosorbents. Each treatment involved exposing the Se source (mock or effluent) to a number of deployments of new GMB (J , 2, 3, 4, 6, 8. 10, 1.2 or 14 sequential deployments). Each replicate consisted of a 250 ml container containing 1 g GMB and 100 ml of mock Se^ solution or mine effluent. The GMB was exposed to the mock Se ^l solution or effluent for 1 h at 15°C in a shaker incubation cabinet (100 rpm). After 1 h the solution was filtered using a 75 μηι. filter mesh and transferred into a ne container with 1 g of new GMB . The final solution for each treatment was collected for analysis of total Se after the final deployment. The solution was filtered out from the GMB as described above, after which it was filtered with a 0.45 μηι syringe filter and collected in falcon rubes (what size). Stock solutions were sampled at the start of the experiment to determine initial starting concentrations.

Comparative, sessment _ of ^' the biosorption . capacity _ < GMBjor $( ¹ _. , _. . SO/ ^" and NO 3 A biosorption experiment was conducted to evaluate the relative biosorption capaci ty of the GMB for the target Se ^{v l} and non-target compounds SG ^" and ^' NO3 ^* in equimolar solutions. Biosorption capacity, in this study, is defined as q, with a higher q signifying a higher biosorption capacity of the GMB to an oxyanion and the highest biosorption capacity referred to as q _mK . q is expressed as the amount of an element removed per gram biosorbent (e.g. 1.2 pg Se ^l g ^"1 G B).

To identify the biosorption capacity of (1MB for each of the compounds, a q _msK kinetic experiment was undertaken. Seven concentrations of Se^, SO ₄ ^2"" and NO ₃ ^" were prepared (0,01 , 0,05, 0. 1 , 0.5, 1 , 1.5 and 5 m ) in Mil!iQ water from three stock solutions (NazSeC^, NazSC^ and aNC , respectively) (Sigma Aldrich). The concentrations were selected to encompass tire equilibrium concentration that delivers a Se ^I q _imK value for the GMB (Roberts et al, 2012, J App Physiol), To run the experiment, 0,3 g of GMB was first weighed out into 250 ml acid washed plastic containers and then exposed to 30 ml of one of the solutions for 1 h at ! 5°C and 100 rpm. The biomass was filtered out directly following the exposure using a 75 pm filter mesh and collected in falcon tubes and dried in a drying oven at 60°C. The solution was filtered usin a 0.45 pm syringe filter and collected in falcon tubes before being analyzed. Samples of the seven stoc k solutions were taken at the start of the experiment to determine initial starting concentrations. The q value at each point was calculated according to the equation:

r [(W^ /) x V]/M;

s the initial concentration (mg .L ^" 1 Vi ■ ^

where, W, _; i ) of Se , SO ₄ " and NO3 ^" , W _/ is the final concentration (nig L ^" ) of Se* , SO ₄ ^*" an NO? ^" , V is the volume (in L) of water used in the experiment, and M is the mass of biochar (g) used in the experiment.

The effect gfSO ^' and NDj o Se ^1'7 biosorption

An experiment was conducted to examine the effects of the non-target compounds SO ₄ ^2" and NQf on the uptake of Se ^l by GMB. To do this, the GMB was exposed to 100 pg L ^'x Se ^l in die presence of non-target compounds (SO ₄ " ^" and NO ) at different molar ratios. The concentration of Se ^l was prepared to match the concentration of Se ^! in the mine effluent so that the capacity of the biosorbent could be evaluated under realistic conditions. The molar ratios of Se :SO.| ^2" and Se ^l :NOs ^" were determined to include the existing ratios of these compounds in the mine effluent as described below.

The mine effluent contained 643 mg L ^"1 SO4 ^2" (6.7 mM L ^"1 ) and 158 mg L ^"1

NO3 ^" (2.5 mM L ^"1 ). With a concentration of 108 pg I, ^"1 Se ^VI (0.0007 mM L ^"1 ), the effluent had a Se^rSQ* ^2" ratio of approximately 1 :9500 and a Se ^Vi :NO.-¾ ^* ratio of approximately 1:3600. The biosorption of Se ^Vi was therefore investigated at molar ratios of 1 :0 (Se ^VI only as a control) and 1 :0.5, 1 : 1 , 1 :10, 1 : 100, 1 : 1 ,000 and 1 :10,000. With an initial Se ^I concentration of 0.0007 mM L ^"5 , the molar concentrations of Se ^J S04 ^2' and Se :NO in the treatments were 0.0007:0, 0.0007:0.00035, 0.0007:0.0007, 0,0007:0,007, 0.0007:0.07, 0.0007:0.7 and 0,0007:7 mM.

A 100 mM stock solution was prepared for SO/ ^" and NO.? ^" by dissolving Na^S0 ₄ and NaN0 ₃ (Sigma Aldrich) in Mi!liQ water. The S0 ₄ ^2" and N0 ₃ ^" stock solutions were then diluted with MilliQ water to give 1 L of each of the 7 concentrations. The Se ^{% f} was then added to each solution by pipetting i 100 μ of a 1 g L ^"1 Se ^! tock solution (made from Na ₂ Se04 Sigma Aldrich), Three replicates of each molar ratio were run by adding GSVlB to an aliquot of the solution (10 g I ¹ ) and placing it on a shaker plate (1 h at 15°C and 100 rpiii). After one hour the solution was filtered (0.45 μηι syringe filter) and analysed according to the previously described methods. Stock solutions were taken at the start of the experiment to determine initial starting concentrations. The removal of Se ^t was calculated as the difference between initial and final concentrations for each treatment.

Elemental analysis

Analysis of Se ^VI was carried out by the Advanced Analytical Centre at James Cook University using a Varian 820-MS inductively Coupled Plasma Mass Spectrometer (Melbourne, Australia). The isotope "Se was used for the quantification of Se ' as other isotopes of Se ^t were interfered with by ClA or ArAr External calibration used a series of Se standard solutions where indium was used as the internal standard to correct for instrument drift and potential matrix effects. A 1% HC1 solution was spiked with 1 ppb Se and measured three times for quality control. A recovery of between 98.5 and 1 10% showed no significant, interferences. Procedural controls without G B showed no change over the experimental period indicating no measurable loss of Se ^T to experimental plastic- ware. SO/ ^" and NO were analysed by Tro Water at James Cook University, using an OI Analytical Flow IV Segmented Flow Analyzer and a Thermo Scientific Gallery Discrete Analyzer. SO/ ^" was analysed with the standard method 4500- SO/ ^" - E and NO¾ ^" was analysed with the APHA standard method 4500-ΝΟ F method. Statistical analysis To examine the adsorption capacity of the GMB for Se ^VI , SO ^2" and NO 5 ^' over multiple deployments, one-way ANOVAs were run for each oxyanion separately using square-root transformed data to meet the assumptions for normality and homogeneity of variance. Tukey's Post Hoc test was used to detect differences between exposure times.

The affinity of GMB for Se ^' , S<V and NO was analysed separatel for each oxyanion, The percent uptake from initial to final concentration in each replicate, and the means of each exposure time, were analysed separately by one-way ANOVA. Data were square-root transformed to meet the assumptions for normality and homogeneity of variances after which Tukey's post hoc test was used to detect differences between exposure times, q was expressed a the amount of an element removed per gram biosorbent tor each element at 7 different concentrations, where ¾ nii¾K was the amount of each element per gram biosorbent at the highest concentration tested. A line of best fit (logarithmic) was fitted to the data to characterise the trajectory of GMB saturation.

The hiosorption of Se ^" * ¹ as a .function of the oxyantons SO4* ^" and NO/ by the GMB was analysed as percent uptake from initial to final concentration for Se ^M in each replicate and the means of each exposure time was analysed by one-way ANOVA. Tukey's post hoc test was used to detect differences between elemental ratio treatments. All data were checked for homogeneity of variances and no transformation was needed. Dat were plotted as percent Se uptake at 7 ratios of Se to SO/ ^" and. NO/ and a line of best fi t was fitted to the data (exponential).

Results

Performance o GMB in e ¹⁷ mock solution and mine effluent

The vast majority of Se in the mock solution was adsorbed by the GMB within minute of exposure to the biosorbent. After the first deployment of G B into the mock solution, 98% of the Se ^vl had been removed, significantly reducing the concentration of Se ^vt from 100 to 1.6 μ L ^'] (ANOVA: F _£i, 20 = 2094.11 , P - <0.001) (Fig 1.3a).

In contrast to the rapid removal of Se from the mock solution, a single deployment of GMB in the real-world mine effluent removed only 3% of the $e ^T f om die effluent (Fig 13b). At the same time, the first deployment of GMB removed 4% of Oi ^' and 24% of NO/ (Fig l c and d). Given the difference in the initial concentrations of the three elements, this equated to q values of 0.31 pg Se ^I removed g ^'1 GMB, 3.27 rag SO4 ^2' g ^'1 GMB and 5.01 mg NO/ g ^"1 GMB. After the second deployment concentrations in the mine effluent had been reduced by 1.2% for Se , 7% for SO/ ^" and 33% for NO/. By the last deployment (14 sequential exposures to unused GMB) the concentrations of Se ^l , SO ₄ ^2" and NO/ in the real- world mine effluent had reduced by 63, 27 and 85%, respectively (Fig lb, c and d). This equated to a cumulative removal of each of the oxyanions from the effluent of 7 pg Se ^vi , 21.7 nig SO/ ^' and 17.6 mg NO/ (Fig 14). A minimum of 4 deployments were required for Se ⁱ to show a significant difference to the initial Se concentration of 108 pg L ^"1 (ANOVA: F = 50.15, P <0.001 and Tukey's HSD; P - 0.002). A significant reduction in S0 ₄ ^2" was noted between all deployments and the initial concentration of 643 mg L SC^ ^2" , with the exception of the first deployment (A OVA: F<>, 1 = 1 10.06, P - <0.001). Furthermore, the concentration of NO/ in all deployments were significantly less than the initial concentration of 158 mg L ^"1 O and showed a slight decrease i the rate of uptake after 8 deployments (ANOVA: F _{9< i5} > = 591.08, P <0.001).

Comparative assessment of the biosorption capacity of GMB for Se ^1'1 , SO/ ^' and NOf

There was a considerable difference in the biosorption capacity of the GMB for Se , SO,/ ^" and NO in equimolar mock solutions. The GMB had a q _m&K of 0.03 mM g ^{" 1} for Se , 0.01 for SO and 0.04 for NO/ (3.80, 1.23 and 2.55 mg g ¹ , respectively) (Fig 15). Overall, GMB had the highest biosorption capacity for NO/ followed by Se ^f and SO ₄ ^"" . Somewhat surprisingly, at low initial concentrations (0,05 and 0.1 mM) GMB had a higher q value for SO ₄ ^2' demonstrating that the relationship is non-linear and a function of initial concentrations (Table 2). The response was non-linear for all three elements.

The lower concentrations of Se _s SO ₄ ' ^" and NO./ showed the highest percent change between initial and final concentrations. However, the biosorption capacity of the GMB was greatest at the highest concentrations (Fig 15). The greatest percent removal of Se ^I was 99% at both the 0.01. and 0.05 m concentrations. The highest removal of SO ₄ ^2" was at 93% at 0.05 mM and at 43% at 0.5 mM for NO/. The removal of Se at both 0.01 and 0.05 mM was significantly different to the other concentrations tested (ANOVA: F<», 14 - 418.31, P <0,001). There was significant differences in the uptake of SO/ ^' between the concentrations at which the highest (0.05 mM) and lowest (5 mM) uptake was detected (ANOVA: F _{6i i 4} = 302.60, P <0.001). The removal, of MC¾ ^" was relatively consistent across concentrations and only the 0.01 and 0.5 mM concentrations were significantly different to the percent uptake at 5 mM. (ANOVA: F _{6> i} = 4.51 , P =·· 0.01.0). The line of best fit was a logarithmic relationship between the concentration of elements in solution and on die GMB with ² values of 0.83 for Se ^vl , 0.83 for SO ₄ ^2" and 0.91 for N0 ₃ \

The effect of SO/ ^' and MOj on Se* biosorption

The biosorption capacity of GMB for Se ^I was affected by increasing levels of SG ", but not by the presence of NOV, across the molar ratios tested in this experiment (Fig 16a and b). The GMB removed 93% of Se ^l from solution in the absence of non-target compounds. The biosorption of Se^ was reduced by 2% to 91% when the concentratio of SO ₄ ^2" was less than the concentration of Se ^I (i.e. at molar ratios of 1 :0.5) (Fig 16a). There was a further decrease in the biosorption of Se to 33-89% when Se :S(V ^" concentrations were 1 : 1 or 1 : 10. At. molar ratios 1:100, 65% of the Se ^I was removed compared to a <10% removal of Se ^vl at molar ratios of 1 :1 ,000. No removal of Se ^l from the solution was detected at the molar ratio representing that of the mine effluent, 1 : 10,000. The relationship between the biosorption of Se ^Xi and the molar ratio of Se ^l ;S04 ^2" was well described by the equation:

y = 85.762.3*e ^~ ° ^'0027x

where- y is the percent removal of Se * from a 1.00 i L ^"1 solution and x is the molar ratio of 5e ^Vi :S0 ₄ ² \

There was no effect of NO3 ^* on the removal of Se ^{v l} from solution at molar ratios up to and exceeding those revealed in the effluent. The GMB removed >90% Se ⁱ from the Se ^ O;f solution i all treatments tested (Fig 16b) which was surprising considering the relatively high affmity of GMB for NO3 ^" (Figs 15 and 16b).

Discussion

Algal-based biosorption has long been proposed as a sustainable and cost- effective means of treating industrial effluents, however, there has been little application of the approach at scale (Gadd, 2009, J Cheni Technol Biotechnoi; Vijayaraghavan and Joshi, 2013, Journal of Environmental Science and Health; Volesky, 2001 , Hydrometallargy). There are two possible explanations for this limited application. Firstly, biosorption is rarely empirically tested in real-world effluents and so there are uncertainties regarding the true potential to treat complex effluents with biosorbents. Secondly, biosorbents typically display limited selectivity for target elements in multi-element solutions. Examining the efficacy of algae for the biosorption of contaminants in real-world industrial effluents is therefore essential to understand how biosorption wil l occur in complex multi -element sources as a pre-cursor to developing commercial strategies to make this process economically 'viable.

in this Example, GMB had an almost 100% immediate effectiveness in mock

Se ^l solutions that contained concentrations of Se ⁱ typical of coal-mining operations (108 μ L ^~l ). However, in the real-world mine effluent with the same initial concentration of Se ⁱ , the biosorption of Se was significantly impeded by the presence of high concentrations of SO ₄ ^*' . To improve the biosorption of Se from the mine effluent it is critical to reduce the concentration of SO ₄ ^*" . Our data show that the critical Se ^l :S0 ₄ ^2" molar ratio, that can deliver considerable biosorption, is 1 : 10. Therefore, we predict that the GMB will only be effective at removing 89% of the available Se when the SO ₄ ' ^" concentration is <0.67 mg L ^" in the mine effluent. Currently, the concentratio of S(V ^~ in the effluent is approximately 640 mg L meaning that a reduction of 99.9% of the current SO ^~ concentration is required for GMB to reduce the concentration of Se in the effluent by any considerable amount. GMB can be used as an efficient biosorbent to reduce concentrations of Se ^I with 89% in mine effluents if the molar concentration of SO ^" is 10-fold that of Se ^l . Interferences by SO ₄ ^2" on the removal of Se ⁱ are not uncommon, Yamani et af, (2014) recorded interferences from S0 ₄ ^2" on the biosorption of Se ^VI using impregnated chitosan beads as a biosorbent in agricultural drain water. They found that the biosorbent removed 80% of the available Se in the absenc of SO^ ^' compared to onl 10% when the concentratio of SO ₄ ^*" was 1 -fold that of Se . Interestingly, they also noticed that at low levels (1:0.1 ) S0 ₄ ^~" had a minor impact on the uptake of Se ^VI by the biosorbent.

To combat the effects of the interacting oxyanion SO4 ^2" on the biosorption of Se , one bioremediation approach would be to reduce the concentrations of SO ₄ ^"" (Yamani et al., 2014, Water Research; Lalvani 2004). A high concentration of SO4 ^"" is a common problem in mine effluents (Rodriguez et al, 2012, International Biodeterioration and Biodegradation) and therefore there are numerous methods for the reduction of SO ₄ ^" (Gadd, 2009, J Chem Technol Biotechnol; Rodriguez et al., 2032, International Biodeterioration and Biodegradation; Kaksonen and Puhakka, 2007, Engineering in Life Sciences), One of the primary methods for reducing the concentration of SO4 ^2* is the biological reduction of SO4 ^2' using S04 ^2' -reducmg bacteria (SRB) in a passive anaerobic respiration process (Kaksonen and Puhakka, 2007, Engineering in Life Sciences). Other methods include passive reactive barriers and anaerobic ponds and the more effective, but costly, active treatment system of bioreactors (Gadd, 2009, J C em Technol Biotechnol; Rodriguez et al., 2012, international Biodeterioration and Biodegradation; Kaksonen and Puhakka, 2007, Engineering in Life Sciences). Due to the similar chemical properties of Se ^I and SC^ ^" , Lalvani (2004) suggested a two-step immobilization approach for drain water to reduce both SO4 ^2" and Se ⁱ , by removing SO ₄ ^2" with barium chloride (BaCfe) and using nanoparticles of Fe to reduce the concentrations of Se . For industries with excess Se ^1' and high levels of SO ₄ ^2" , the primary action should be to reduce SO 4* ^' levels before attempting the biosorption of Se .

While N(¾ ^~ concentrations of 5 mg L ^"1 and above have been reported to inhibit the biosorption of S.e ^l in ground water by soil (Bailey et al, 2012, Journal of Environmental Quality), our data demonstrate that NOV had no effect on the biosorption of Se ^I by GMB. Despite NO3 ^" concentrations rangin between 0.02 and 434.03 mg L ^"1 and being 1,500 times higher than the concentration of Se* ¹ , NO ₃ ^" had no impact on the uptake of Se ^Vi . This means that not all oxyanions are problematic and that for new water sources it will be necessary to identify oxyanions which can interfere with biosorption of target element.

In light of the foregoing, GMB is an efficient biosorbent for Se ^! in mock solutions and can successfully remove 98% of the Se ^vl present and achieve a maximum biosorption capacity (q _xmx ) of 3.80 mg Se " removed gf GMB. In contrast, the biosorption capacity of GMB to Se in a mine effluent is significantly reduced due to the presence of the oxyanion S( ^' V ^" . The antagonistic relationship between Se*' and SO ^'"" shows that any bioremediation of Se ^, ½ an effluent with high levels of SO.«. ^"' will be unsuccessful unless the levels of SOV ^" are reduced. Under the current mine conditions, with 108 μ L ^" Se and 640 mg L ^" SO ^*' , a reduction of 99% of the available SO.f ^" by the GMB is needed to achieve a considerable reduction of Se ^I . Conversely, high levels of the oxyanion N<¾ ^" do not impair the biosorption of Se ⁱ by GMB despite the biosorption capacity of M , ^" to GM B being higher than that of Se and $(¾ ^*" . This study clearly demonstrate that, although valuable in understanding kinetics, results and conclusions drawn from a single- element mock experiment are of little use in predicting the performance of biosorbents in real-world effluents when competing compounds are present. As demonstrated here, GMB is an efficient biosorbent for Se^, however, it requires that high concentrations of the competing oxyanion SO4 ^"' are reduced prior to bioremediation.

Table 1. The concentratio of all elements in the real-world mine effluent. Bold values exceed CCME (Canadian) or ANZECC (Australian) water quality criteria.

Etetrs«ir S£ CCME Aumct

A! 0.28 ± 0.00 55

As 0,50 t 0.00 S 24 mi 3 (V)

B 58.63 ± 8,83 1,500 370

Cd 0,11 ± 0.00 0.2

Co 2,08 * 0.02

Cr 0.51 i 0 02 1 <V1), 8.9 {111} 1

C 0,58 ± 0.13 1 .4

Fe SO.00 i aoo 300

Mg 111,300.08 4 ¾34&21 1,900

Mr 2.85 ± 0 24

M© 26 05 * 2.87 73 34 m .m ± LOS 11

Ph 0.03 i- 0.00 3.4

Se 108,08 ± S.41 1 11

$f 333 SO * 0.05

V 1.48 * 0,49

m 2.50 ± 0.00 30 8

188*388.87 ± UM

so 642.500.00 ± 6.034.82 Table 2. q in mM g ^l for Se , S<¾ ^2" and Ο at 7 mM concentrations (0.01,1, 0,5, 1, 1.5 and 5). f/ _misx for each element is in bold.

0.81 0.001S x o.oooca 0,0005 ± 0.00002 0.0005 ± -0.00005

o 0.0080 ± o.oooce aoos8 ± -0.00004 0.0014 0.00036

0.1 0.0123 -o.oeois 0,0062 :i -o.oooii 0.003S * -0.00129

as f! Ot l? ± O.OOOC54 0,0125 ± -0,00104 0.0229 ssmm

1 0.0116 x o.o:i)i2 0,0080 ± -&OO208 0.0210 ± -0.00083 i,S 0.0169 t 0.00013 0.0094 .t -0.OCOS? 0.0249 is -ilCSM

s MOM t 0 _* 00X2? ± 0ίββ85? 0<0δ825

EXAMPLE 5

Materials and Methods

Industrial Effluent

This study focused on Ash Dam Water (ADW) from Tarong co l- fired power station in south-east Queensland, Australia (26,76°S _} 151.92°E). Tarong is one of Queensland's largest coal-fired power stations with a generation capacity of 1400 MW and a 46,000 ML AD to store waste -water from ash disposal processes on site. The ADW was sourced directly from the AD and transported to James Cook University (JCU), Townsville in clean plastic 1000 L intermediate Bulk Containers (IBCs) and stored at ambient temperature in 12,000 L storage tanks until use. The ADW was collected and shipped with the permission, and assistance of Stairwell Energy Corporation.

Algal biosorbenfs production ami preparation

Oedoganmm sp. (Genbank: KF606974) (hereafter Oedagom ), was used as the feedstock for the biosorbents. Qedogonium is a filamentous, freshwater macroaiga that is native to Tarong AD. The biomass for this study was cultivated in f/2 media in 2500 L tanks during the austral summer months (January - March) in the aquaculture facility on. the Townsville campus of JCU ( 19.33°S, 146.76°E). Prior to experiments, 2 kg of algae was harvested from the tanks and o ven dried at 60°C for 48 hours (h). The biomass was then converted into biochar by slow pyro lysis under conditions previously described (Bird et al., 2 1, Bioresour Technol). Briefly, Oedogonatm was suspended in a muffle furnace and purged with ; gas at 4.0 L mi ¹ while being heated to 450°C for 1 h. A sample of the biochar was converted to Fe-biochar by soaking it in a 5% Fe ^J ~ solution (diluted Sigma Aklrich® 45% w/v FeCl ₃ stock solution) at a density of 25 g L ^*1 for 24 h on a shaker plate (100 rpm) at 20°C. The Fe-biochar was filtered from the FeC solution and rinsed three times with deionized DI) water at a rate of 20 ml g ^"1 and then dried at 60°C for 48 h. Derivation of predictive sorption model

A model was developed using data from a previous study with Tarong ADW (Table 5) to predict the change in concentration of 21 elements after the deployment of each biosorbent. Most metal and metalloid sorption occurs within the first hour of exposure and the most effective sorption occurs when the pH of the ADW is im- manipulated (pH -7, 1). The biosorption data collected under these conditions was used to constract the predictive model. Following I h of exposure of Fe-biochar or biochar to ADW the q-value (the mass of an ion [ trg] adsorbed from, or released into, solution per unit of biosorbent [g]), was calculated according to Volesk (2007). The q-value was determined for each combination of biosorbent (Fe-biochar and biochar) and element (Al, As, Ba. B, etc) to derive a model that predicted the additive effect of multiple biosorbents, applied sequentially or simultaneously, on the r gle effl.ue.nt:

Where, Mi is the mass of an element in solution (pg) following treatment number i; C is the initial concentration of the element, in solution (ug U ¹ ); V is the volume of effluent solution (L): q _x is the q-value of the element for biosorbent, A ^* (tig g ^~! ); S _x ,i is the mass (g) of biosorbent, x ₇ added for treatment, i.

Biosorption experiments

The aim of the biosorption experiment was to maximise tire removal of oxyanionic elements (As, Mo and Se) with Fe-biochar, followed by the removal of cationic contaminants with secondary, or simultaneous, deployment of biochar. As described above, this sequential treatment strategy is logical because the targeted removal of metalloids by Fe-biochar also contributes some metals into solution. This requires a second phase of biosorption with biochar to remove existing and leached metals in ADW.

In total there were 6 biosorption treatments: (I.) Fe-biochar ("FeBC"); (2) biochar ("BC"); (3) sequential Fe-biochar ("FeBC→ FeBC ¹ '); (4) sequential biochar ("BC ~→ BC"); (5) sequential Fe-biochar and biochar ("FeBC→ BC"); and (6) simultaneous Fe-biochar and biochar ("FeBC + BC") (Figure 1 7). As the aim of the biosorption experiment was to achieve remediation of metalloids and metals, the primary treatments of interest are the sequential and simultaneous application of Fe- biochar and biocha (treatments 5 and 6 respectively. Figure 17). Treatments 1-4 were included as controls to assess the effects of Fe-biochar and biochar in isolation for comparison with the sequential and simultaneous treatments. Each treatment strategy was repeated 3 times to evaluate the model predictions.

The predictive model was used to select the loading densities of each biosorbent for the two main treatments and controls. Metalloids were targeted for removal by using a density of Fe-biochar that was predicted by the model to result in removal of Mo. the most abundant metalloid, to below ANZECC water quality criteria. The result was that Fe-biochar would be used at a density of 13.7 g L ^"1 (see further justification in results section "Development of treatment scenarios using the predictive model "), A limitation of the experiment was that the biochar density predicted by the model to remove all metals (1 10 g U ^l ), is not feasible as the maximum possible loading density of biochar or Fe-biochar was 60 g L ^"1 . Therefore, all biochar treatments were loaded at a density of 60 g L ^"1 and all Fe-biochar treatments were loaded at a density of 13.7 g L ^{" 1} . The simultaneous "Fe-BC + BC" treatment included 13,7 g L ^"! Fe-biochar and 46.3 g L ^"!" biochar to give the maximum 60 g L ^_i of biosorbent (Figure 1 7).

Each replicate consisted of a 250 ml plastic beaker with 50 ml of ADW and the appropriate mass of biosorbent. The pH of the A0W was unaltered (7.06 ± 0.01) for all treatments. Fe-biochar or biochar (depending on treatment) was added to the ADW and placed in a shaker cabinet (Eppendoif Innova® 44R) at 100 rpm in 20°C fo 1 h. After 1. h, the biosorbent was separated from ADW by centrifugation (7000 rcf, 5 min), followed by two stages of filtration (75 μνα nylon filter paper, then 0.45 um syringe filter). The filtered solution was transferred to another 250 ml plastic beaker for the next treatment (biochar or Fe-biochar depending on the scenario). The process of the second sequential treatment was identical to the first except that the mass of biosorbent was adjusted to account for any loss of solution during the separation process to ensure the same density was applied. After the second treatment the solution was processed as described and the water samples retained for analysis,

It is important to note that testing multiple treatments in the biosorption experiments provides an opportunity to validate the performance of the model when initial elemental profiles differ from those under which the model was produced. For example, after the first stage of the "BC— > BC" treatment, the effluent is predicted to have low metal, but unchanged metalloid, concentration. If the model accurately predicts the performance of the second BC treatment this indicates the model is robust to variations in initial effluent characteristics. Such a finding would support the use of the model across diverse effluents with a wide range of elemental profiles. Elemental analysis The concentrations of metals and light metal ions (Al, Ba, Ca„ Cd, Co, Cr, Cu, Fe, K, Mn, Mg, Na, Hi, Pb, Sr, V, and Zn) and metalloids (As, B, Mo, Se) were measured using a Bruker 82G-MS Inductively Coupled Plasma Mass Spectrometer (ICP-MS; Al, Ba, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb, Sr, V, Zn) or a Varian Libeny series II Inductively Coupled Plasma Optical Emissions Spectrometer (JCP-OE5; Ca, , Mg, Na). An external calibration strategy was used for both instruments _. , where a standard solution of 0.45 μχη filtered A ^' DW was used as the vector to calculate the concentration of elements. Collisional Reaction interface (CRl) was used for As (CRl gas: Hi) and V (CRl gas: He), while * ^* Se isotope was used for Se quantification, to eliminate polyatomic interferences for these elements. A 1 % HC1 solution was spiked with 1 ppb As, Se and V and measured three times for quality control; recovery between 98.5 and 11.0% indicated no significant interferences. All analyses were conducted at the Advanced Analytical Centre at J ^' CU, Townsville. Data an lysis,

Multivariate patterns in biosorption were visualized using non-metric Multi-

Dimensional Scaling (nMDS) from a Bray-Curtis similarity matrix following forth - root transformation. A Permutational Multivariate Analysis of Variance (PERMANOVA) was conducted in Primer 6.1.14 between the elements and the factor of treatment (Figure 17). Univariate analysis took the form of one-way Analysis of Variance (A OVA), with the final concentration for each sequential treatment analysed. Data were examined for normality and homogeneity of variance using normal-probability plots of raw residuals and predicted-residual scatter plots. When necessary the data were log-transformed. The PCA and ANOVA tests were conducted in Statistica (Ver. 10, Statsoft Inc.), The log-predicted final concentrations of each element from the biosorption model were plotted against the log-observed values on a scatterplot for validation of the model. A trend line of y - x was plotted to indicate where the points would be expected to lie if the observed and perfect values were in perfect agreement Any point with a residual value > ±1 was highlighted as a deviation from the predicted concentration.

Results

Characteristics of AD W

Twelve. (Al, As, B, Cd, Cr, Cu, Pb, Mn, Mo, Ni, Se, and Zn) of the 21 elements measured in the ADW have trigger levels established by the Australian and New Zealand Environmental Conservation Couneil (ANZECC, Table 3), Nine of these elements (Al, As, B, Cd, Cu, Mo, i, Se and Zn) were present in untreated ADW at concentrations in excess of the ANZECC trigger values for the protectian of aquatic life at the 95% level (Table 3). These elements are therefore the focus of the following results and discussion.

Development of treatment, scenarios using the predictive model

The model was used to predict the amount of Fe-biochar and biochar required to achieve comprehensive remediation of metalloids and metals to below ANZECC trigger values. Allowing for a 15% deviation in removal capacity, the model predicted 13.7 g L ^"1 of Fe-bioehar would be sufficient to reduce the concentrations of As, Mo and Se in ADW to below the trigger values (Figure 18a, c and e). As the Fe- biochar leaches some metals into solution, it was applied first in the sequential treatments so that the subsequent treatment of biochar could adsorb both the existing and leached metals from ADW (Figure 18b, d, f). The density of biochar required during the second treatment for removal of the metals was therefore calculated assuming 13.7 g L ^" ' Fe-biochar had been used in the fust stage of treatment. A density of 10 g L ^"5 biochar was predicted for the removal of the leached and existing metals. Given physical limitations the maximum stocking density that could be achieved was 60 g L ^'1 . Consequently, all biochar treatments were applied at this density (Figure 17). The simultaneous treatments were tailored to result, in the maximum removal of both metalloids and metals within the physical constraints of the biochar system. Therefore, Fe-biochar was applied at a density of 13.7 g L ^_i and biochar was added at 46.3 g L ^"1 , giving the maximum stocking density of 60 g ^' L ^"1 in treatment 6 ("FeBC +BC", Figure 17). The simultaneous treatment ("FeBC +BC", Figure 17) was included despite the need for different stocking densities to determine if a mixed biosorbent consisting of Fe-biochar and biochar has potential as a single- step biosorption treatment for complex effluents. As biosorption and metal leaching occurs relatively rapidly (15 min), it is possible there are rapid interactions between leached and bound elements in the simultaneous exposure that are not evident in the sequential treatments.

Evaluation of treatment scenarios with biosorpiion experiments

Treatment of ADW with Fe-biochar and biochar resulted in a significantly different final composition of dissolved elements than treatment with only Fe- biochar or only biochar (PER ANQVA: "treatment" pseudo-Fasf^.l, P ⁼ 0.001). ADW treated with Fe-biochar (treatment 1 "FeBC" and treatment 3 "FeBC → FeBC", Figure 17) had lower concentrations of metalloids (As, Mo and Se) than untreated ADW (Figure 19). ADW treated with biochar (treatment 2 "BC" and treatment 4 "BC→ BC", Figure 17) had lower alkali, alkaline and transition metal concentrations (Na, Mg, Si; Ca, Cr, Pb, Fe, Ai, Ni, Cu, Zn. and Co) than untreated ADW (Figure 1 ). When ADW was treated with both Fe-biochar and biochar (treatment 5 "FeBC -* BC" and treatment 6 "FeBC + BC", Figure 17), it had a unique elemental composition compared to the other treatments, achieving significantly lower concentrations of both metalloids and metals thai untreated ADW (Figure 19), with the best outcome being from the sequential treatment (treatment 5 "FeBC→ BC") which attained the lowest concentrations of metalloids and metals of all the treatment scenarios (Table 4).

a) Sorption by Fe-biochar alone ("FeBC" and "FeBC→ FeBC")

The metalloids (As, Mo and Se) were removed from ADW by Fe-biochar

(Figure 20a-c; Table 4). Two applications of Fe-biochar in sequence (treatment 2 "FeBC→ FeBC", Figure 17) resulted in greater removal of metalloids than any other treatment, but also resulted in the highest concentrations of dissolved metals being released (Figures 20 and 23), For example, Mo decreased from 3 13 to 43 ,ug L ^'! in ADW (Figure 20b, Table 4), while Zn increased from 36 to 2.167 μ§ L ^" ' (Figure 20e, Table 4). The first deployment of Fe-biochar was more efficient at removing the metalloids than the second sequential deployment, which yielded lower removal rates of As, Mo and Se per unit Fe-biochar used (Figure 20a-c). Therefore, ADW exposed to "FeBC" and "FeBC— *· FeBC" treatments was characterised by lower metalloid and higher metal concentrations than untreated ADW (Table 4). h) Sorption fry biochar alone ("BC " and "BC—> BC ")

Metals (Al, Cd, Cu, Mn, Ni, Pb and Zn) were removed from ADW by biochar (Figure 20d-f, Table 4). The final concentrations of Al, Cu, Mn, Ni, Pb, and Zn were lowest following the sequential biochar treatment ("BC — + BC") (Figure 20d-e, Table 4). Al, Ni and Zn were all decreased to below their trigger levels in ADW during the sequential treatment "BC -→ BC" (Figure 20d-e, Table 4). As was also reduced in concentration in the "BC → BC" treatment, however, the final concentration was higher than the final concentration in the "FeBC — *· FeBC" treatment (Figure 20a, Table 4). The change in the concentration of was the opposite to the other metals, with substantial leaching occurring, resulting in a total increase in the concentration in ADW of over 1,800,00.0 μg L ^"1 in the "BC -→ BC" treatment (Figure 23, Table 4).

c) Sequential and simultaneous treatments ("Fe.BC→ BC" and "FeBC ·*· BC ^' ")

The sequential treatment of ADW using Fe-biochar to remove metalloids followed by biochar to target the existing and leached metals (treatment 5 "FeBC— *· BC") resulted in the most comprehensive treatment of the ADW (Table 4). As described above, metalloids were removed by the initial application of Fe-biochar while the metals, Al, Cr, Cu, Ni, Pb and Zn all leached off the biosorbent into solution (Figure 20). However, during the subseqitent application of biochar. most of these metals were adsorbed to below initial concentrations (e.g. Figure 20d-e), with ^■ the exception of Cu and Ni (Table 4). Of the eight ANZECC metals (Al, CcL, Cr, Cu, n, Ni, Pb, Zn), four (Al, Cd, Pb, Zn) were reduced in ADW to the lowest concentrations measured across all treatments, and an additional three (Cu. Ni, Mn) were reduced to the second lowest concentrations in ADW in the "FeBC→ BC" treatment (Tables 4 and 6).

When. Fe-biochar and biochar were used simultaneously (treatment 6 "FeBC + BC") there were higher final concentrations of As. Mo and Se in ADW compared to ' ^' 'FeBC→ FeBC" and "FeBC→ BC" treatments (Figure 20a-c). B was reduced to its lowest concentration in the "FeBC ^" + BC" treatment, however, this only constituted a 4% drop from the initial concentration (Tables 4 and 6), Similarly, Cr was reduced to the lowest concentration following a treatment with "FeBC + BC" of 1 ,2 ± 0.04 μg L ^"1 , however, this concentration is still an order o magnitude higher than the initial concentration of Cr in ADW, 0.1 ± 0.02 μ L ^"1 (Tables 4 and 6). Both Cu and Ni had equivalent concentrations between the treatments of "FeBC→ BC" and "FeBC ^" + BC" (Tables 4 and 6). An. interesting result was the response of V, which reduced in concentration following sequential and simultaneous treatments, but increased slightly in concentration with individual treatments of biochar and Fe- biochar (Figure 23).

Evaluation of the model

The final concentrations of elements following tire "FeBC" and FeBC→ FeBC" treatments were close to those predicted by the model (r =· 0,97 and 0.96 respectively, Figure 21a-b). The treatment of ADW with Fe-biochar delivered greater than expected reductions in Mo, but was less effective than predicted for As (in both single and sequential treatments) and Se (in sequential treatments) (Figure 21a and b). The model was slightly less accurate for predicting the concentrations following "BC" and "BC → BC" treatments, as demonstrated by the weaker correlations between predicted and observed concentrations (r =0.88 and 0.69 respectively. Figure 21c, d). Despite the weaker correlation, the concentration of 14 of the 21 elements was accurately predicted for a single application of biochar (Figure 21c). The treatment of ADW in the "BC" treatment resulted in higher than predicted concentrations of B and V and lower than expected concentrations of Mn (Figure 21c). The deviations from model prediction were compounded in the "BC→ BC" treatment as 11 elements (As, B, Ba, Cr, Fe, Mn, Na, i, Se, V & Zn) out of the 21 had residual values > ±1. Na had the greatest deviation from expected concentration (Figure 2 Id; Table 4). B, V and Mn had similar deviations from predicted concentrations in the "BC→ BC" treatment (Figure 21 d).

In the "FeBC→ BC" treatment 17 of the 21 elements were remediated close to, or below, predicted concentrations (Figure 22). Twelve elements out of 21 (Al, As, B, Co, Cr, Cu, Fe, Mo, Pb, Se, V & Zn) had residuals > ±1 (Figure 22). However, of these most were removed from ADW more effectively than was predicted by the model As, B, Se and V had higher concentrations than predicted after the "FeBC→ BC" treatment (Figure 22). Despite the high rate of leaching of Fe from the Fe-biochar treatment, the subsequent biochar treatment removed all of this Fe, resulting in a concentration (5 pg I., ^"1 ) much lower than was predicted (39,451 pg L ^*1 ) (Figure 22; Table 4).

Discussion

The inventors have demonstrated that the sequential application of multiple biosorbents provides a comprehensive treatment of a complex effluent. The combination of targeted remediation of metalloids by Fe-biochar and metals by an imeoated biochar resulted in a greater number of elements being treated compared to the application of either biosorbent independently. Furthermore, the reduction of metalloids followed by metals, by the sequential application of Fe-biochar then biochar, demonstrates that the effects of these biosorbents are additi ve. The inventors model predicted the concentration of most elements following treatments with Fe- biochar, although the model was slightly less effective at predicting the concentrations of elements whe biochar was involved in the sequence. Regardless, the selectivity of the biosorbetrts was consistent and accurately modelled across the six water types (i.e. ADW treated by Fe-biochar, biochar, and combinations thereof) in which the initial element composition varied greatly.

Treatments that combined Fe-biochar and biochar ("FeBC → BC" and "FeBC + BC") achieved the most comprehensive remediation of the greatest number of elements. However, sequential treatments with Fe-biochar ("FeBC -* ^■ FeBC") and biochar ("BC→· BC") were more effective at removing their target constituents (metalloids and metals, respectively). In the same way that the removal of a. given element will be greater in isolation than when that element is present in a comple mixture, removing multiple elements from an effluent occurs at the expense of tire sorption capacity of the biosorbent for each individual element (Figueira et al., 1997, Bioteehnol Bioeng).

Predicting the concentrations of elements following treatments of Fe-biochar and biochar with the model was very successful, with all of the sequential and single treatment scenarios resulting in at least a moderately strong correlation between predicted and observed values. Modelling has been used extensively i biosorption studies, however often only to predict and quantify the mechanism by which the element is being absorbed onto the biosorbent using Langmuir and Freundiich isotherms (Moghaddam et al. 2013, Chem Eng J; Mehta and Gaur, 2005, Crit Rev Biotechnol; Ali and Gupta, 2006, Nat Protoc; Yang and Volesky, 1 99, Removal and concentration of uranium by seaweed biosorbent). The empirical model described herein performed particularly well when considering that the starting concentrations of most elements were significantly different to those in the effluent that was used to derive the model, and that the water quality after each treatment was unique, spanning 6 types of water treated with different combinations of biosorbents, resulting in a broad range of initial concentrations (Table 4). In this context, the model is as a robust tool for predicting the treatment of a broad range of waste water profiles by biosorbents derived from Oedogonh , and as a tool to develop working treatment strategies for a variety of waste water sources with known elemental compositions. The majority of existing data has assessed elements in isolation, while an industrial effluent typically contains a multitude of co-existing, interacting elements. Given the large quantities of biomass that will inevitably be required to treat industrial effluents, it will also be necessary to select and cultivate biomass locally for the express purposes of bioremediarion. The inventors have demonstrated that different types of biosorbents (Fe-biochar and biochar) can be produced from a single feedstock that is native to an industrial facility (the freshwater .macroalga O d goniwn) and used to sequentially treat metalloids and metals from a complex effluent. Through the use of a predictive model we demonstrate that sequential biosorption is largely predictable and consistent across a wide range of initial conditions. Targeting metalloids that do not have a natural affinity for biochar requires Fe-treatment, which inherently results in a suite of additional metals being released into solution. This metal leaching can in aim be addressed through the use of biochar as a final treatment. Therefore, while biosorption has been widely cited as a sustainable and cost-effective means of treating waste waters, our data clearly show that the reality is far more complex than is typically acknowledged, but remains achievable.

Throughout the specification, the aim has been to describe the preferred embodiments of the invention without li miting the invention to any one embodiment or specific collection of features. Various changes and modifications may be made to the embodiments described and illustrated without departing from the present invention.

The disclosure of each patent and scientific document, computer program and algorithm referred to in this specification is incorporated by reference in its entirety.

Table 3. Concentration of elements in Ash Dam Water and associated ANZECC trigger values. Bold values exceed the ANZECC trigger value for protection of aquatic life at the 95% level.

ANZECC Initial

Element Trigger Concentration [ ¾

lixg l ¹ ) L ^"1 (±SE)j

Aluminium 55 89 (2.1)

Arsenic 13 34 (0.2)

Boron 370 3823 (18)

Cadmium 0.2 2.1 (0.01)

Chromium 1 0.1 {0.02}

Copper 1.4 2.1 (0.03)

Lead 3.4 0.03 (<0.01)

Manganese 1900 0.9 (0.2}

Molybdenum 34 3913 (7.2)

Nickel 11 38 (0.3)

Selenium 11 97 (0.3)

Zinc 8 36 (2.0)

Barium - 100 (0.3)

Calcium - 335 ^ 3 .3 (12,012)

Cobalt - 0.3 (<0.01)

Iron - 5.0 (<0.01)

Magnesium - 99,800 (334S)

Potassium - 44,867 (2178)

Sodium - 446,333 (11,169)

Strontium - 4080 (21)

Vanadium - 982 (8.1)

Change in element concentrations in ADW after exposure to Fe-biochar and hiochar biosorbents (see Figure 17 for explanation of treatments). Table 5. Change in dissolved elemental concentration (jig L- l) in ADW following treatment with bioehar and Fe-biochar for 1 h at a solution pH of 7.1 , and the derived q-vaiues (μ$ g- 1 }.

Table 6. Summary table for ANOVA tests run on each of the 21 elements investigated. Oneway analysis of variance tests were run on final elemental concentration with the factor of Treatment. Type 11.1 sum of squares was used. All. tests met the assumptio of homogeneity of variance, normality of residuals and independence. Transformation of the data were required for some elements, the transformation applied is listed next to the title. Factors in bold indicate significance under alpha of 0.05.

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