CA2354249A1 - Method of sustaining plant growth in toxic substrates polluted with heavy metal elements - Google Patents

Abstract

A method of sustaining plant growth in toxic substrates polluted with heavy metal elements, characterized in that it comprises amendment and remediation of the toxic substrates with an organo-zeolitic mixture. The method can also be used as a fertilizer and for beneficiation of normal, uncontaminated soils.

Description

METHOD OF SUSTAINING PLANT GROWTH IN TOXIC SUBSTRATES
POLLUTED W1TH HEAVY METAL ELEMENTS.
Activities in the industrial Age have resulted in the deposit of high levels of many metals in certain sites, to the point that human life is seriously threatened.
Metal-production activities, such as mining or smelting, as welt as the ubiquitous use of metals, have created many sites where toxic metals have become concentrated in soils.
In recent years, efforts have been made to develop phyto-remediation methods, i.e. the use of metal-accumulating plants called metallophytes to remove contaminating metals from sites.
It has been known for some time that many plant species will concentrate certain metals in their leaves, stems and roots to a varying degree.
For heavy metals, two different types of phyto-remediation methods can be distinguish:
- rhizofiltration, by concentration of heavy metals in plan roots;
- phyto-stabilisation, the roots of the plants limiting heavy metals availability and limiting mobility of said metals into the groundwater.
More than 400 phyto-remediator are known, most of them absorbing nickel. The more rarely absorbed heavy metals include manganese, cadmium and lead.
Various metallophytes have been tested, such as Brassicaceae (Thlaspi brachypetal, Thlaspi ochroleucum, Thlaspi caerulescens, Thlaspi rotundifolium, Cardaminopsis halleri), Caryophyllaceae (Minuartia verna, Polycarpea synandra), Fabaceae (Astragalus pectinatus, Astragalus bisculatus) Myriophyllium verticillatum, Pshychotrai douerrer, Vola calaminaria.
Document US-A-5.917.117 relates to a method by which hyperaccumulation of metals in plant shoot such as Brassicaceae (e.g. Brassica, Sinapsis, Thlaspi, Alyssum, Eruca) is induced by exposure to inducing phytotoxic agents such as chelating agents (e.g. Roundup ~) and high concentrations of heavy metals. The exposure to inducing agent is made after a period of plant growth, as metal accumulation into plant shoots has dramatic negative effects on plant growth.
The use of phytotoxic inducing agents as described in document US-A-5.917.117 is non-ecological and potentially dangerous for the operator.

Document US-A-5.711.784 disclose a method of extracting nickel, cobalt and other metals including the platinum palladium metal families from soil by phytomining. The conditions include 1) lowering the soil pH by addition of sulphur and use of ammonium N fertilisers, 2) maintaining low Ca in the soil by acidification of the soil with sulphur or sulphuric acid and, 3) applying chelating agents to the soil, such as NT,A.
The method described in document US-A-5.711.784 is complicated and non-ecological.
Document US-A-5.927.005 relates to a method of removing heavy metals from '10 soil using creosote plants (Lacrea tridentate). Again, to increase the rate of metal uptake in the plants, it is proposed to increase the acidity or to add chelators to the soil in which the creosote bushes are growing.
Other phyto-rernediation techniques are described in documents WO-A-00/28093, WO-A-00/31308, WO-A-98/59080, WO-A-94/01357, EP-A-0 911 387, JP-A
57.000.190, DE-A-4100758, DE-A-39.21336, US-A-5 100 455, US-A- 5.320.663, US-A-5 364 451, US-A-5 785 735, US-A-5 809 693, US-A-5 853 576, US-A-5 928 406, US-A-5.944.872, US-A-6 117 462.
Despite increasing interest and research, several problems associated with phyto-remediation remain. For example, some metals in contaminated areas may be ;?0 hardly reached via phyto-remediation because they lie beneath the rhizosphere, many of the known metal-accumulating plants being simply too small to accumulate large quantities of metals. Additionally, many of the plants thus far identified as useful in phyto-remediation are from tropical regions.
One object of the invention i:> to provide means to increase plant root growth in ;?5 toxic substrates polluted with heavy metal elements.
Another object of the invention is to provide means preventing surface erosion, especially for toxic substrates polluted with heavy metal elements.
Another object of the invention is to provide means of promoting growth of metallophytes and other plants on toxic ground polluted by the presence of heavy 30 metal elements.
Another object of the invention is to provide above mentioned means, said means being ecological and less expensive than most of known bio-remediation methods.

According to the invention, there is provided a method of sustaining plant growth in toxic substrates polluted with heavy metal elements, characterised in that it comprises amendment of the toxic substrates with an organo-zeolitic mixture.
The heavy metal element caa~ be zinc, copper, lead, cadmium or arsenic, the orgo-zeolitic mixture being added to the said polluted substrate between 10 %
and 25 %.
The method of the present invention can be used to sustain the growth of various plants, especially plant root growth in toxic substrates polluted with heavy metal elements.
Normally, owing to the lack of available nitrogen and other essential nutrient elements, ground containing high levels of toxic metals would not sustain plant growth to a level that will prevent surface erosion. By amending the ground with the organo-zeolitic fertiliser this condition can be overcome by growing plants with very dense root systems.
The method of the present invention can be manipulated to vary the shoot to root ratio of the plant species used. In tt-~is respect, plants which concentrate heavy metals such as Zn, Cd and Cu in their shoots can be grown successfully and Groped to remove the metals from the rhizosphere.
The method of the present invention will enable the metal enriched plant tissue, on ashing, to be reduced to a small volume v~rhich can be disposed of easily by mixing with zeolite amended Portland cement and used in the production of concretes that are known to have high compressive strengths.
More precisely, after harvesting and ashing the plant, the heavy metal cations contained in the ash can be put into aqueous solution and ion-exchanged into a 2:5 zeolitic tuff. The resulting zeolitic material can be dried and used to produce blended cements which have improved compressive strength and are also known to reduce the expansion caused by alkali-aggregate reactions.
It is known that natural zeolite minerals can be used as biological fertiliser (see for instance JP-A-10210855, ,JP-A-4197110, EP-A-444392, US-A-5 082 488, US-~~0 A-5 451 242,US-A-5 900 38'l, RU-A-2 121 777, RU-A-2 132 122, RU-A-2 137 340). The preparation of an organic fertiliser incorporating zeolitic tuff is described r in document US-4.559.073, the inclusion of the zeolitic component being claimed to lower the water content of the mixture to allow effective aerobic fermentation.

Document US-A-5.106.405 disclose the property of ion-exchanging ammonium ions that, via soil microbioloc3ical reactions, would supply available nitrogen to plants growing in a substrate amended with a bio-fertiliser containing zeolitic component.
The inventor has discovered that natural zeolite materials could be used to prepare a biological fertiliser which can be applied to ground contaminated with heavy metal cations to enable the sustainable growth of plants and to control the development of shoot to root ratio in such a way that plant morphology can be adjusted to either maximise soil retention by dense root growth or increase the foliage uptake of toxic heavy metal ions.
If untreated, such ground will not support vegetation and becomes subject to surface erosion by wind and rain. Toxic material transported by these agents into local drainage patterns is thus isolated and therefore uncontrollable.
Specific implementation of the invention will now be described, by way of example, provided for illustrative proposes and not intended to limit the scope of the invention as claimed herein. Any variations in the exemplified compositions and methods which occur to the man skilled in the art are intended to fall within the scope of the present invention.
Example A clay rich toxic soil containing : 2.87 % Organic matter, 1.1 iv Calcium carbonate, 2.24 % total Iron, 28.9 mg.kg~' Copper, 915 mg.kg-' Zinc, 670 mg.kg-' Lead, 12.2 mg.kg~' Cadium and 18.9 mg.kg-' Arsenic has been amended with 16.7 % organo-zeolitic fertiliser"
Organo-zeolitic fertiliser is prepared as follows.
Animal waste, e.g. chicken manure, is composted together with crushed zeolitic tuff containing the zeolite Ca, K, Clinoptilolite in a ratio of 1.2 (by volume) i.e. tuff to manure. The materials are mixed together with enough water to make the pile damp and choppen straw is added. Air is forced through the pile from a perforated plastic pipes) laid inside the pile during construction and the reaction is carried out under cover. This could prevent saturation of the pile with rain water.

The pile reaches 50-70 °C and then the temperature drops to ambient at which stage the composted materi~ is dry, friable, odourless and ready for use as an organo-zeolitic fertiliser.
Spring Wheat (Triticum aestivum L, cv. Red Fife) was sown in two kilograms 5 substrates. Wheat grown in 'the untreated soil was used for the comparison.
The plants were grown in 255 mm diameter pots, replicated four times, under ordinary lighting conditions in a greenhouse. Watering, with de-ionised water, was by weight to field capacity (180 ml per 2 kg substrate) and the pots were placed in shallow trays to retain leachate. Watering, generally on a daily basis, prevented 1' 0 the plants from drying out and any water running from the dots was returned to the substrate surface with little loss. Plants were harvested on a regular basis each month and the shoot weights were recorded after drying to constant weight at 70 °C.
One month altar germination the substrates were leached with 400 ml of de--I 5 ionised water (pH = 8.4) and after removal of fine colloidal particles were analysed chemically. Two further leachate collections were made at monthly intervals over a three month growth period.
.?0 Leachate chemistr~at the third harvest Toxic substrate ~ N conc 0.23mg/I

Amended substrate N conc 178.00 mg/I

Toxic Substrate K conc ' 17.38 mg/I

Amended Substrate K conc 66.70 mg/I

Toxic Substrate Ca conc 20.40 mg/I

Amended Substrate Ca conc 253.00 mg/I

Toxic Substrate Mg conc 2.69 mg/I
~

Amended Substrate Mg conc 27.10 mg/1 Toxic Substrate pH 7.8 E.C. 149 ~tS/cm Amended Substrate pH 7.2 E.C. 2077 ~S/cm r These results demonstrate the degree of mobilisation of major cations in the amended soil solution.
In case of the metal trace elements a general decrease is seen in the leachates between high concentrations in the toxic substrates and low concentrations in the amended substrates. An example is shown below for Znc.
Toxic substrate: Zn conc 0.65 mg/L
Amended Substrate: Zn conc 0.10 mg/L
Following the analysis of the leachates the chemical analyses of the plant shoots express the way in which nutrient and trace metal elements are taken up from the respective substrates.
Plant shoot chemistry at the third harvest Amended substrate Toxic Substrate Ncon ~~.l9wt~ 1.16wt%

K conc 35.33 mg/g 18.20 mg/g Ca conc 5.82 mg/g 2.82 mg/g Mg conc 1.24 mg/g 0.85 mg/g Zn conc '124 ugJg 67 ~9~9 Pb conc 5 1~9~9 Cu conc 17 ~g/9 5 X9/9 The plant shoot chemistry can now be compared to the established nutrient range for Spring lNheat.

dry wt . ~9I9 N P K Ca Mg Zn Cu Adequate Range : 3.0-4.50.3-0.52.9-3.80.4-1.00.15-0.320-70 5-10 Toxic Substrate : 1.16 0.5 1.80 0.3 0.10 67 5 Amended Substrate : 2.190.3 3.50 0.6 0.12 124 17 Shoot dry weight recorded at monthly harvests 15' harvest Dry weight (g/plant) Toxic Substrate: 0.26 Amended Substrate: 0.89 2"° harvest Toxic Substrate: 0.52 Amended Substrate: 5.36 3'd harvest Toxic Substrate: 1.03 Amended Substrate: 6.77 Comments on example The type of organo-zeolic fertiliser of the present invention can be adapted to grow plants with a dense root system on toxic soils that cannot normally supply ;25 sufficient plant nutrients to support such growth. This is achieved by microbiological means only as no inorganic mineral salts have been added..
It can be seen from the chemistry of the plant shoots than when the available nitrogen in the substrate is some 35 % below the adequate range a dense root system, in the case of Spring Wheat, is formed. As the percentage of organo-zeolitic material added to t:he toxic soil can be altered the concentration of available nitrogen can be adjusted ,to suit the plant species concerned. If maximum shoot growth is required then the percentage or organo-zeolitic material can be adjusted upwards to put the nitrogen concentration into the adequate range. T his would bs desired if maximum plant uptake was required in order to remove heavy metals from the rhizosphere.
The trace element concentrations of zinc and copper in the plant shoots show that the mobilisation of cations in the soil solution, due to the microbial activity of the organo-zeolitic material, m~;ke these elements available to the plant. On harvesting the soil will be partially depleted in these elements and in time the rhizosphere will become less polluted. The volume of plant material after harvest can be greatly reduced by asking and can be safely stored or possibly re-cycled.
As it is known that the addition of finely crushed zeolitic tuff to Portland Cement '10 improves its physical and chemical properties the suggestion is made that heavy metal cations remaining in the plant ash could be exchanged into zeolitic tuff which is afterwards used for such a purpose.
Possible explanation '15 Proposed explanation of the above mentioned results is given below, the detailed mechanism still being the subject of research by the inventor. During organo-zeolitic fertiliser preparation, ~~hoppen straw likely provides a source of carbon to support bacterial growth. Ammonifying bacteria as typified by Clostridium and Penicillium, acting on the organic material, decompose protins, amino sugars and ;?0 nucleic acids to ammonia. The ammonia in the cationic form NH4+ is ion-exchanged into the zeolite where it is held, loosely bound, within the pore space of the crystal lattice. The bacterial activity causes the increase of temperature up to 50-70 °C, the completion of the reaction being reached as the temperature drops to ambient.
;25 On addition of the fertiliser to a plant substrate the NH4+ ions held in the zeolite pore space diffuse at an expotential rate into the substrate. Nitrifying bacteria present in the organo-zeolitic component use the diffusing NH4+ to build very large populations and in so doing oxidise NH4+ to produce a large source of nitrate that is used in plant growth. As a consequence of t'~ bacterial reactions .30 free hydrogen ions (protons) are liberated and mobilise the soil solution causing the dissociation of metal cations present in the substrate.
The inventor has demonstrated how the organo-zeolitic fertiliser can be used with soils polluted with heavy mertals such as Zinc, Cadium, Copper and Lead. The inventor has now found that when 16-7 7 % organo-zeolitic fertiliser is added to such soils similar effects occur which greatly increase nitrate concentration and mobilise metal rations in the soil solution.
Amending a toxic soil in this way slightly lowers the pH of the soil solution but increases its electrical conductivity by an order of magnitude. At the level of amendment quoted (16-17 °/o) twice the amount of available nitrogen present in the toxic soil is provided. This increase is 35 % below the adequate range for Spring Wheat (Triticum ae;>tivum L, cv. Red Fife) and has the effect of maximising the root/shoot ratio. In this way a dense root system can be developed by increasing the amount of organo-zeolitic fertiliser added to the soil the root to shoot ratio can be decreased, in which case shoot growth is favoured.
Cation mobilisation in the soil solution provides the growing plant with nutrients such as potassium, calcium, magnesium, and zinc and the plant's requirement acts to buffer the system against concentration of these elements and diffusion from the rhizosphere. By increasing the level of plant nutrients in this way a healthy plant can be grown and sustained on toxic soils polluted with heavy metals. In the case of zinc and copper the inventor has observed that these elements are taken up by the plant at a rate that can be tolerated and the damage occurring in the same plants grown in the toxic soil is not seen. This property can be used to remove heavy metal elements from the rhizosphere by harvesting the plant. The plant material can be greatly reduced in volume by ashing without loss of the heavy metals and incorporated in a mixture of Portland Cement and finely crushed zeolitic tuff. In this vvay concrete of high compressive strength and low alkali reactivity can be made and used to store the heavy metal elements.

Inventor : Dr. Peter l.Leggo Statement Work on Ryegrass (Lolium perenne L.) has shown that amendment of the plant substrate with the zeolite bio-fertilizer will enhance plant growth in a similar way to that seen with Spring Wheat (T'riticum aestivt~m L., cv.Par2gon).
Dense root systems can be sustained in soils polluted with heavy metals. The conraminated substrate used was identical to than. in the spring wheat programme as described in the above patent In the ryegrass programme un-ammoniated zeolitite (i.e. zeoliased volcanic tuff containing the zeolite mineral clinoptilolite) was added in varying proportions to the initial amended substrate containing 16 -17 (volume %) organo-zeolitic fertilizer (zeolite bio-ferdlizer).
The exchangable rations in the clinoptilolite used are Calcium and Potassium.
Zeolite minetaLs with appreciable quantities (> ca.2 Wt %) are undesirable as reactions in the plant substrate increase the sodium availability which can cause depression is growth The typical formula of the clinoptilolite used i<; : (K ,9Ca t~ Na a, Mg 0.,) al ~, Si :99 0 ;,. 23.1 H,0 It was found that the relationship between shoot dry weight and weight °,o excess zeblitite defined the (cut-aid limit of growth enhancement above which continued addition of zeolitite diminished plant growth.
This effect was found to vary according to plant density. A low density group (2~ plancslpot) having a higher cut-off limit than a high density group (ca. 600 plane /pot).
The low density plants also showed greater growth enhancement than the high density group.
Analytical data: Shoot dry weights (i) Low density group harvested at four months Plant substrate Shoot weight (g) Gp.l Toxic substrate 738 GpZ Amended substrate, (16 --17) vol % zeolite bio-fertilizer 31.46 Gp.3 Ditto + 75 wt % un-amoniatedzeolitite 32.73 Gp.4 Ditto + 1 ~0 wt % un-amoniated zeolitite 33.36 Gp.S Ditto + 225 wt % un-ammo;niated zeolitite 33.98 Gp.6 Ditto + 300 wt % un-ammoniated zeolitite 28.87 (ii) High density group harvested at five months.
Plant substrate Shoot weight (g) Gp. l Toxic substrate 6.76 Gp.2 Amended substrate (16-17) vol% zeolite bio-fercilizer 25.83 Gp.3 Ditto + ~,5 wt% ~_~o~ated zeolitite 26.49 Gp.4 Ditto + 150 wt% un-ammoniated zeolitite 25.46 Gp.S Ditto + 225 wt% un-ammoniated zeolitite 25.10 Gp.6 Ditto + 300 wt% un-ammoniated zeolitite 24.51 Statement The root systems in the low density group show a decline in root mass above an excess addition of 150 wt% un-ammoniated zeolitite.
As in the case of the shoot mass a distinct cut-off is seen in root growth. In the present case, working with heavy metals in the specified range in neutral to slightly alkaline conditions no advantage is gained in increasing tire excess zeolitite above 150 wt %.
Whereas shoot growth is maximised at 225 wt% excess zeolitite.
This again demonstrates that the tdo-fertilizer can be formulated to maacimise either shoot or root growth.
In order to formulate the bio-ferti'(izer for these effects it will be necessary to conduct initial laboratory trials as the biological factors (i.e. remedialplantspecies and density), bacterial population, soil properties, heavy metal species and concentrations will vary independently at specific sites. .
Analytical data: Root dry weights (i) Low density group harvested at five months.
Plant substrate Root weieht (g) Gp. l Toxic substirate 3.45 Gp.2 Amended substrate (16 -17) vol% zeolite bio-fertilizer 6.53 Gp.3 Ditto + 75 wt% un-ammoniated zeolitite 8.43 Gp.4 Ditto + 150 wt% un-ammoniated zeolitite 11.80 Gp.S Ditto + 225 wt% un-ammoniated zeolitite 11.67 Gp.6 Ditto + 300 wt% un-ammoniated zeolitite 7_q.4 Statement Harvested plant shoots in the high density group were analysed for trace concentrations of Zinc, Copper and Lead. By relating the trace metal chemistry to the shoot dry weight of the amended groups it was found that the cut-off limit corresponded to a concentration of 138 ug.g't Zinc. Above this value shoot dry weight dropped dramatically.
Analytical data: Trace metal chemistry in shoots Plant substrate pg. ~ t Zn Pb Cu Gp.Toxic subsuate . 74.2 6.7 16.1 l Gp.2Amended substrate (16-17) vol% zeolite106.7 6.8 22.2 bio-fertilizer Gp.3Ditto + 75 wt% un-ammoniated zeolitite138.1 6.1 26.0 Gp.4Ditto + 150wt% un-ammoniated zeolitite140.6 7.6 24.6 Gp.SDitto +'?25 wt% un-ammoniated zeolitite163.4 6.5 22.9 Gp.6Ditto + 300 wt% un-ammoniated zeolitite170.0 9.4 23.

Statement A large increase in the elecorical conductivity is seen to occur between leachate solutions from the toxic and amended substrates; which is chazacteristic of soils amended with the bio-fertilizer.
This reaction is taken to infer that the nitrifying bacteria are boosted by addition of the bio-fertilizer. .... .
A relationship is seen between shoot total nitrogen and excess zeolitite. The data shows that a fluctuation occurs between the amended substrates throughout the range of excess zeolitite.
This behaviour is not clearly understood but is apparently a function of the bacterial composition developed in the amended substrates.

Analytical data: Eleca-ical conductivity of substrate leachate solutions and shoot total nitrogen Plant substrateE.C.(u Siemens/cmShoot total nitrosen lwt,%1 (Low density) (High decLSiry) Gp.l 119 0.74 0.98 GP~2 472 1.=18 1.~~:

Gp.3 285 1.17 1.39 Gp.4 323 1.37 l.-~7 Gp.~ l89 1.09 1.38 Gp.6 488 1.13 1.32 Comments oa results The plant weight results demoruu-ate the performance of ryegiass grown in heavy metal polluted soils of neutral to slightly alkaline pH that has been amended with zeolite bio-fertil izer.
Very large increases in plant growth occur in the amended substrates. The increase obtained in root growth is particularly important for the retention of soil particles contaminated by heavy metal residues.
However, limits are reached in the addition of un-ammoniated zeolitite above which the plants suffer deleterious effects.
As the zinc concentrations found in the shoots correlates to the behaviour of the shoot dry weights this element is thought to causing a major phytotoxic effect which is limiting growth.:
Although the detailed interactions between the bio-fectilizer, soil chemistry and bacterial population are st~I under investigation it is clear that by studying the effect of varying the concentration of the un-ammoniated zeolitite the bio-fettilizer can be formulated to provide maximum growth enhancement .
r

Claims (17)

1. A method of sustaining plant growth in toxic substrates polluted with heavy metal elements, characterized in that it comprises amendment of the toxic substrates with an organo-zeolitic mixture.

2. The method of claim 1, wherein the heavy metal element is Zinc, Copper, Lead, Cadmium, Arsenic.

3. The method of claim 1 or 2, wherein the organo-zeolitic compound comprises the zeolite mineral Ca-K clinoptilolite and animal waste.

4. The method of claim 3, wherein the animal waste comprises chicken manure.

5. The method of claim 3 or 4, wherein the organo-zeolitic compound is prepared by composting animal waste with crushed zeolitic tuff.

6. The method of claim 5, wherein the ratio by volume of tuff to animal waste is roughly 1:2.

7. The method of claim 5 or 6, wherein a source of carbon is mixed with zeolitic tuff and animal waste.

8. The method of claim 7, wherein said source of carbon comprises choppen straw.

9. The method of any one of claims 1 to 7, wherein said organo-zeolitic compound is added to said polluted substrates between 10% and 25%.

10. Use of the method defined in any of claims 1 to 9 to sustain growth of Spring Wheat.

11. Use of the method defined in any of claims 1 to 9 to sustain growth of metallophyte plants, with metal-containing metallophyte plant tissues being collected and removed at appropriate intervals.

12. Use of the method defined in claim 11, wherein heavy metal cations remaining in the plant ash are exchanged into zeolitic tuff to be added to a cement or equivalent.

13. The addition of excess un-ammoniated zeolitite increased the growth enhancement factor with the zeolite bio-fertilizer in the original compositonal form, i.e., substrate containing 16-17 vol% of the organo-zeoligic mixture.

14. Cut-off limits for the amount of excess un-ammoniated zeolitite to produce maximum growth can be specified.

15 15. These limits vary according to plant density, plant species, metal contaminant species, metal element concentration, soil physical and chemical properties.

16. By analysis of the parameters given in claim 15 it is possible to formulate the amendment to achieve either maximum shoot or root growth on a specific waste site containing heavy metal residue

17. The bio-fertilizer can be used to beneficiate and fertilize non-toxic soils.