CA2269301A1 - A bacterial inoculated sulfur-based fertilizer - Google Patents

Description

A BACTERIAL INOCULATED SULFUR-BASED FERTILIZER
Field of the Invention This invention relates to the treatment of sulfur-based fertilizers to enhance their breakdown in soil to provide oxidized sulfur compounds as plant nutrients.
Background of the Invention The role of sulfur oxidizing bacteria in the oxidation of elemental sulfur to various higher sulfur oxidation state sulfur compounds in the soil has long been known. The thiobacillus genus is ubiquitous in soil at concentrations of the order of 102 bacteria per gram. In the presence of elemental sulfur and under appropriate environmental conditions these bacterial populations can, over time, multiply to levels in excess of 10a bacteria per gram thus affecting the rate at which the elemental sulfur is converted into plant usable oxysulfur species.
The most common of these is the highest oxidation state sulfate (S04-). Elemental sulfur can thus be used as a controlled release form of sulfate plant nutrient.
It is important, for agronomic reasons, that the rate at which essential sulfate plant nutrient is made available to the plant be controlled. It is commonly provided as ammonium sulfate. This source is freely soluble in water and can be rapidly leached from the soil after application, especially during heavy rainfall episodes, heavy irrigation, or flooding. Such leaching can have undesirable environmental consequences in natural drainage systems. Elemental sulfur is not soluble in water. Thus losses of the sulfate produced from it are limited by the rate at which it undergoes microbiological (bacterial} oxidation to soluble sulfate. Furthermore, the rate at which many plants consume sulfate nutrient available in the soil varies with both plant type and the time in the growth cycle.
The ability to control the rate at which sulfur is made available in the soil has therefore both agronomic and environmental consequences.
A further use of elemental sulfur as a soil amendment relates to its ability to produce sulfuric acid which can be used to lower the pH of soil that is chemically basic (pH>7) to levels more suited to plant growth. It is important that this release of acid into the soil be controlled in order to avoid generation of overly acidic pockets and to obtain maximum benefit from the sulfur applied by avoiding wash out by leaching.
Another common form of plant nutrient elemental sulfur is a premicronised, regranulated particle where the elemental sulfur has been ground to a micron sized particulate and then regranulated using a water soluble binding agent. When this form of plant nutrient elemental sulfur contacts water, it degrades into its original micron sized form which can then be acted upon by bacteria at either the natural population level or an enhanced level created by inoculation.
Canadian Patents 159,849, 219,720 and 1,308,566 describe various fertilizer compositions where microorganisms have been included with the fertilizers. Canadian patent 219,720 teaches that a bacterial culture may be dried and mixed with ground sulfur, either before, during or after grinding of the sulfur. Similar techniques are described in US patents 3,186,826 and 5,366,532, as they relate to including microorganisms with a fertilizer composition.
Summary of the Invention This invention relates to how particulate elemental sulfur, in a variety of forms, may be inoculated with specific bacteria before being applied to soil as a fertilizer or in a fertilizer mixture, in order to accelerate the rate at which the elemental sulfur is oxidized to an oxysulfur chemical form which can be used by plants as a nutrient.
In an aspect of the invention, the inoculation may be carried out in a manner such as by coating with suitable polymeric compounds or the like so that the bacterial activity is sustained, in dormant form, between the time of manufacture of the inoculated product and the time of application to the soil as a active source of sulfate fertilizer.
In another aspect of the invention, the process of inoculation may be combined with treatments designed to reduce the tendency of the sulfur particulate to degrade into fines by abrasion, or other mechanical mechanism, during storage handling and transportation and, in so doing, avoid conditions for the generation of undesirable airborne sulfur dust.

2 In another aspect of the invention, nutrients, essential to the bacterial growth, may be incorporated into the product in order to further enhance the maintenance of viability of the bacteria during storage of the product and population development after application to the soil.
In another aspect of the invention, control can be exercised on the rate at which a solid sulfur matrix breaks down and releases nutrients, by a combination of control of the physical breakdown of the matrix due to the action of the clay and the rate of microbiological oxidation and digestion of the sulfur to yield sulfate.
In another aspect of the invention, control can be exercised on the rate at which a solid sulfur particulate matrix, containing other fertilizer materials that are essential as either macro or micro plant nutrients, will break down and release these micro andlor macro nutrients in to the soil environment in which the plant is growing.
In another aspect, a selection process may be implemented to select bacterial traits for drought resistance which survive on the sulfur fertilizer.
Brief Description of the Drawings Figure 1 shows young Canola oil seed plants two weeks after germination;
Figure 2 are photographic illustrations showing comparison of the effects on seed germination in use of the treated materials of this invention;
Figure 3 is a graph showing the effect of inoculated and non-inoculated fertilizers on the amount sulfate in the soil.
Detailed Description of the Preferred Embodiments The ability to provide, together with the elemental sulfur, a preformed population of bacteria capable of oxidizing the sulfur to sulfate and to determine the size of that population is a significant aspect of this invention. It can provide a means for controlling the rate of sulfate release to the soil environment.
In addition to the bacterial population the following factors are important in determining the rate of microbiological oxidation :-Moisture Surface area of sulfur per unit weight

3 Temperature Oxygen access Recognition of the role of these factors can be relied on in optimizing the consequences of various aspects of the invention.
Elemental sulfur can be applied to the soil in a number of forms. The inoculation of all such forms is contemplated by this invention. The most common forms are blends of swelling clays and elemental sulfur in granule, pastille , pellet or other generally homogeneous geometric form. The clay component in such formulations swells on contact with water resulting in breakdown of the host sulfur matrix particle and the generation of a range of micron sized elemental sulfur particles which the bacteria will attack at a variety of rates dependent on the surface area per unit weight ratio.
It is apparent that the swelling clays function most appropriately in providing particulate fertilizer in the soil which is readily digested by the sulfur degrading bacteria. The chemistry and physical properties of the clays may be selected to optimize the particulate breakdown so as to expedite bacterial digestion.
It is thus possible, by a combination of particle size and bacterial population level determination, to affect significant control over the rate of oxidative conversion of elemental sulfur, in any form, to plant nutrient sulfate.
A variety of species of Thiobacillus bacteria exist and which have various factors affecting their ability to oxidize elemental sulfur to sulfate. This variation will, in turn, influence the rate control that can be exercised on the sulfate production process.
The work of Laishley et al. ( Can J. Microbiol. Vol. 14, 960-966, 1988) and Bryant et al. (Can.J.Microbiol. Vol. 29, 1159-1170, 1983) shows that their are at least two important species of the Thiobacillus genus which may be characterized, in general terms, by the classification acidophilic and less acidophilic. The former category function in the range of acidity indicated as pH
0.5 to 6.0 and the latter in the range pH 5.5 to 8Ø Considering the acidity levels that exist in soils at both the micro and macroscopic levels it is important

4 to select appropriate bacteria that have the capability of functioning in a wide range of acidity environments.
This invention teaches that the species T. Thiooxidans and, in particular, several of its species are preferred in order to obtain effective functioning as a sulfur oxidizing bacterium in a wide range of soil conditions. Other sulfur oxidizing species of the Thiobacilius genus may also perform the oxidizing function but with less efficacy.
In accordance with an aspect of the invention, the following conditions provide a guideline for carrying out are the inoculation process using the selected bacterial preparation.
1. The concentration of the inoculant preparation should be such that, allowing for the "kill" that may occur during and after inoculation and before application of inoculated sulfur to the soil, there still remains a sufficient population to achieve the enhanced sulfur oxidation rate desired. The following population levels have been determined .
Preferred population range in inoculating medium 106-10$ I ml.
Preferred population range on elemental sulfur inoculated 103- 10 5 / g.
2. The bacteria transferred to the surface of the sulfur are attached thereto in a manner that resists wash off by aqueous media. The presence of an aqueous medium in the immediate vicinity of the sulfur surface activates bacterial population growth and commonly the breakdown and dispersal of the elemental sulfur into a desirable range of particle size.
3. This aqueous activation step preferably occurs only when the inoculated fertilizer formulation is applied to the soil. This then requires that the surface of the elemental sulfur be kept essentially anhydrous until the formulation is applied to the soil. The bacteria, however, suffer significant "kill" if stored under prolonged anhydrous conditions. Kill rates as high as 90% have been postulated by Swaby, A.J., 1975, Biosuper-Biological Superphosphate, Australasian Agriculture pp 213-220 Edited

5 by K.D. McLachlan, Sydney University Press. Thus the residual bacterial population, which determines the initial rate of sulfate production, may be affected by the elapsed time between inoculation and application to the soil.
There are, therefore, two different related embodiments in the teachings of this invention. The first is that the inoculation at the desired levels be carried out as part of the production process of the elemental sulfur fertilizer formulation, preferably avoiding any exposure of the inoculation bacterial system to temperatures in excess of 30°C. Above this temperature losses of bacterial viability may result. In this embodiment, the bacteria should be suspended in a liquid phase that contains moisture such as a gum, starch or polysaccharide phase which, on application to the surface of the sulfur will harden to form a moisture retainina coating but which, on contact with water, will soften and release the bacteria to interact with the surface of the sulfur and commence the oxidation process. Plant nutrient elemental sulfur formulations prepared in this way may have a limited shelf life depending on the conditions of storage , handling and transportation to which they are exposed.
The second embodiment relating to the manner and timing of the inoculation requires that the inoculation process will be carried out at the time of application of the plant nutrient elemental sulfur nutrient to the soil. In this embodiment the sulfur fertilizer formulation is supplied in the uninoculated form together with a separate fluid preparation containing a high bacterial population in a medium in which longer term survival of a high proportion of the population would be assure. This separate inoculant preparation would be diluted and applied to the sulfur formulation immediately prior to application to the soil. The shelf life of the formulation in this embodiment would be significantly longer.
The rate at which the bacterial population on and around the elemental sulfur fertilizer grows controls the rate of production of sulfate plant nutrient. This bacterial growth rate is influenced by the availability of nutrients essential to the growth process.
In accordance with another embodiment of this invention, the preparation of the plant nutrient elemental sulfur fertilizer formulation preferably

6 includes the incorporation of particulate sources of nutrients essential to the bacterial growth. More particularly the additional nutrients include:-Sources) of fixed nitrogen such as ammonium species as may also be incorporated at higher levels as a source of controlled release plant nutrient nitrogen.
Sources(s) of phosphate such as ammonium phosphates as may also be incorporated as a source of controlled release plant nutrient phosphate.
Calcium Chloride as a membrane stabilizer and as may also be incorporated on the surface of the sulfur fertilizer formulation for dust control purposes.
Polysaccharides as a source of carbon for other heterotropic microorganisms may be used as an agent for protecting and increasing the longevity of the bacteria.
Sources of trace amounts of iron and magnesium among other micronutrient species as may also be incorporated in the formulation as a source of such micronutrients for plant growth.
It will be seen that many of the nutrients required for bacterial growth, and hence control of the rate of microbiological oxidation, are the same as those required by plants growing in the fertilized soil. Incorporation of these nutrients, at different concentration levels in the sulfur fertilizer formulation , will therefore serve to both control the rate of bacterial growth and to provide a source of plant macro- and micro-nutrients at a controlled rate, i.e. at a rate proportional to the rate of conversion of the elemental sulfur matrix to sulfate. The incorporation of these nutrients can be achieved by addition of appropriate chemical compounds during the formulation of fertilizer.
In a further embodiment of this invention, the method of inoculation may be combined with a method of dust control. Solid elemental sulfur is subject to

7 breakdown during abrasion likely to be encountered during handling, storage and transportation of a particulate sulfur fertilizer form. Treating the outer surface of the freshly produced sulfur fertilizer formulation with spray coatings of various materials that minimize the generation of fugitive fines from such abrasion breakdown, can reduce or eliminate the potential for undesirable airborne dust generation.
Simultaneously, some of these spray coatings may contain the appropriate concentrations of inoculating bacteria depending on the compatibility of the selected bacteria with the selected spray coating.
Typical of such coating materials are, calcium chloride, xanthan gum, starches, polysaccharides. It is described elsewhere in this application that many of these materials have other advantageous effects on the viability of the inoculating bacteria. In this manner, therefore, useful synergisms can be introduced into the formulations. It is also taught, however, that care must be exercised in selecting spray coating materials that will not interfere with the sulfur fertilizer particle breakdown since the ultimate sulfur particle size distribution is a key factor in controlling the rate of oxidation of sulfur to plant nutrient sulfate.
The precise manner of preparation and application of the inoculant to the sulfur will depend on the specific bacterium or bacterial mixture selected and the physical form of the elemental sulfur to be inoculated. The following is a description of a typical procedure. In addition, it is understood that the selected coating materials do not have any biocidal properties and are non-toxic and compatible with the sulfur conversion bacteria.
Growth of the selected microorganisms is carried out in a sterile nutrient media. Nutrients should include at least phosphate, ammonia, calcium, sulfate and iron with pH adjusted to that for optimum growth of the bacteria which, in this example, is pH 4.5.
A 1 % inoculum of fully grown stock culture is added to the nutrient media supplemented with 0.5% finely powdered elemental sulfur or 0.5% thiosulfate .
The optimum growth temperature will vary depending on species selected but 28°C was selected in this example. Growth is generally observed between 5°
and 30°C.
Numerical estimates of bacterial cell growth may be made by employing a standard plate count method or by using the most probable number technique to obtain an estimate of the number of viable cells per ml of culture. Other indirect methods of measurement of growth include the measurement of cellular protein or the quantitative determination of the product of metabolism namely sulfuric acid. It is important to have available a dependable method of quantifying the viable bacterial population so that an appropriate amount of inoculant is used in the fertilizer formulation.
The culture is usually fully grown in a five to seven day period under the conditions detailed above. Populations in the range of 108 to 109 viable cells per ml are obtained. The culture is then gross filtered to remove the majority of the elemental sulfur on which it has been growing. Following centrifugation, a pellet of bacterial cells is obtained which is suspended in a dilute phosphate buffer solution. This is the stock suspension from which aliquots can be taken for dilution and inoculation of commercially formed plant nutrient elemental sulfur pastilles, pellets, granules or other physical forms. If the culture has been grown on thiosulfate, centrifugation is not required inoculation of the sulfur fertilizer form can be by droplet spraying onto the formulation in a moving stream (e.g.
conveyor belt) or by passing the newly formed (cooled if necessary) sulfur particulate through a mist containing the inoculating bacteria in aerobic suspension. The bacterial cells have a strong tendency to stick to the sulfur by using a polysaccharide matrix (glycocalyx, Laishley et al. 1984) produced during growth. It is not considered essential that all sulfur particles be specifically inoculated in this process since cross inoculation of particles will occur in storage or in the soil. An inoculation efficiency of greater than 60% is preferred.
Alternatively, the inoculation can be carried out in the field by spray drop application immediately before application of the fertilizer formulation to the soil.
Also, the inoculant bacteria may be first suspended in a sprayable coating material such as a gum, starch or other polysaccharides which can then be applied to the sulfur fertilizer formulation to provide protection against extreme dehydration and thereby minimize high bacterial kill rates during storage.
It has been found that the use of xanthan gum is particularly effective in maintaining bacterial viability as applied to sulfur-based fertilizers. The bacteria used may be thiobacillus albertus. Xanthan gum acts as a stabilizer for the bacteria to maintain viability on sulfur pastilles. Viability of the bacteria is extended considerably by the use of xanthan gums which are normally sprayed onto the bacteria as applied to the fertilizers surfaces or it is admixed with the bacteria and then sprayed onto the fertilizer.
It is also understood that spontaneously occurring drought resistant mutant strains could be selected from the existing bacterial population. Such bacteria can be isolated, characterized and cultured to enhance the level of viability of the bacteria in a sulfur fertilizer mix.
It has been determined through dilution studies that a bacterial population of at least about 103 microbes per sulfur fertilizer particle of 3-4 mm diameter are required to evidence an immediate increase in microbial sulfur oxidizing activity. This translates to about 104 to 105 bacteria per gram of sulfur in this physical form.
It is important to recognize the absolute necessity of oxygen for microbial oxidation of sulfur to plant nutrient sulfate. For this reason the optimum location for the inoculant bacteria is on or near the surface of the sulfur not within the matrix where oxygen access may be limited. This oxygen requirement also points to the preference for application of the inoculated sulfur fertilizer particulate at or very near the surface of the soil. Broadcast or very shallow banding of the fertilizer is preferred.
Example I
Pot trials with and without inoculated plant nutrient elemental sulfur.
Illustrations in Fig. 1 show young Canola oilseed plants two weeks after germination that have been fertilized without (Fig. 1 a) and with (Fig. 1 b) bacterially inoculated plant elemental sulfur. The most striking feature of the two sets of plants is the difference in root growth and root complexity. The much increased degree to which the soil is adhering to the treated plant roots indicated the greater degree of root hair development that has occurred as a result of the presence of the inoculated elemental sulfur fertilizer.
The benefit of this enhanced growth in the early stages of the plant life cycle will be reflected in the earlier maturation of the plant and an earlier potential harvest. This benefit can be of significant value in regions where the growing season is limited by early and often killing frosts.
Example II
Chemotaxis Experiments on Seed Germination The photographic illustrations in Fig. 2 show the comparison of the effects on seed germination and immediately subsequent root and cotyledon growth, again for Canola. Six seeds (black dots) have been placed in a nutrient agar gel (purple) in each of three dishes where plant nutrient sulfate is supplied as follows:-Fig. 2a: as ammonium sulfate Fig. 2b: as inoculated elemental sulfur Fig. 2c: no sulfurlsulfate source supplied The importance of the availability of sulfurlsulfate in these very early stages of growth is clearly seen. With no sulfur added as in 2(c) only one of six Canola seeds has germinated and one cotyledon has sprouted and divided.
In the case of ammonium sulfate being the source of sulfate ( all immediately available as sulfate) three seeds appear to have germinated and produced cotyledons ( Fig. 2a).
In illustration Fig. 2b, however, all six of the Canola seeds around the central pellet of inoculated sulfur fertilizer have not only germinated but have developed a profuse growth of cotyledons. Note also that a yellow disc has developed around the inoculated sulfur pastille in Fig. 2b. This is a dye indication (purple to yellow) that the pH of the region around the sulfur has dropped due to acid production resulting from the microbial oxidation of the inoculated sulfur. This is direct evidence that the inoculated sulfur is already producing measurable amounts of acid sulfate within a period of six days.

Not only do these illustrations show the dramatic influence of bacterial inoculation on the sulfur to sulfate conversion but further demonstrate the significant growth enhancement that can be achieved by such inoculation. The failure of the ammonium sulfate test (2a) to match the performance of the inoculated sulfur (2b) is believed to be due to the fact that too much sulfate was initially available in the ammonium sulfate case creating an ionic environment less suitable for germination and early growth. In the case of the controlled slow release of sulfate from the bacterial action on the elemental sulfur, a sulfate environment that better matches the requirements of the emerging plant growth is created.
These illustrations again demonstrate the significant plant growth benefits that result from use of inoculated elemental sulfur as a fertilizer material.
Example III
Direct Evidence of Sulfate Production in Soil The most direct evidence that the inoculated elemental sulfur fertilizer is producing sulfate in the soil at a rate different from that without inoculation is presented in Fig. 3. Here are seen the results of measurement of the sulfate content of the same soil as a function of time over a period of 8 weeks of growth of Canola with elemental sulfur fertilizer added, however, with one set inoculated and one not inoculated. The only difference between the two sets of data is the presence or absence of inoculant bacteria.
In the absence of bacterial inoculation the elemental sulfur depends on the natural soil bacterial population for oxidation to sulfate. Such action, in the early stages of growth, raises the sulfate in the soil from around 13 ppm to a maximum of 35-40 ppm after which the rate of production is insufficient to meet the demands of the maturing plant and the sulfate in the soil is drawn down again.
In the case of the inoculated sulfur fertilizer, the much higher initial level of bacterial population results in a rise from 13 ppm to over 60 ppm compared with the maximum 35-40 ppm for the uninoculated sulfur, but then the sulfate in the inoculated case continues to rise to near 80 ppm even as the maturing plant demands for sulfate increase. It should be noted that 20-30 ppm sulfate is removed in one growing season by a normal Canola crop.
These illustrations of the efficacy of inoculation of plant nutrient elemental sulfur with the bacteria necessary for microbial oxidation of sulfur to plant nutrient sulfate, demonstrate the value of this technique in enhancing the manner in which the control of the supply of essential plant nutrient sulfate can be affected.
In accordance with an aspect of the invention, a combination of control of bacterial activity through control of sulfur particle size distribution, control of bacterial population by inoculation, the provision of essential bacterial nutrients, the control of oxygen accessibility through coatings and placement in the soil, the control of bacterial viability during storage, and the control of dust generation, make it possible to design a sulfur/sulfate fertilizer product that is best suited to the wide variety of soil and climatic conditions that are encountered in agricultural zones world wide.
Although preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing the spirit of the invention or the scope of the appended claims.

Claims