PREVENTING OR REDUCING PLANT GROWTH BY BIOCEMENTATION

Abstract

The present invention primarily relates to the use of a mixture capable of biocementation as a means of preventing or reducing plant growth, preferably weed growth. The invention also relates to a method for preventing or reducing plant growth, preferably weed growth, on/in a substrate.

Claims

1. A mixture capable of biocementation of a substrate comprising: (i) one or more organisms or enzymes capable of forming carbonate, inducing carbonate formation, or catalyzing carbonate formation; (ii) one or more substances to form the carbonate; (iii) optionally, one or more cation sources; and (iv) one or more additives selected from polyhydroxybutyrate, polyacrylic acid, polymethacrylate, poly(2-hydroxyethylmethacrylate), polyvinyl alcohol, polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), copolymers, derivatives of cellulose, derivatives of lignin, lignosulfonates, derivatives of pectins, natural adhesives, in particular gum arabic, latex, derivatives of rubber, derivatives of chitin, derivatives of chitosan, derivatives of cyclodextrins, derivatives of dextrins, hydrogel formers, cold soluble and/or warm soluble (plant) glues, microbial exopolysaccharides containing or consisting of maleic acid, acetic acid, lactic acid, lactose, sucrose, glucose, fructose and/or inulin, monomers of polysaccharides, lactose, sucrose, glucose, fructose, inulin, protein sources, casein, albumin, peptones, residues and industrial materials selected from the group consisting of maize steep liquor, lactose mother liquor, protein lysates, protein waste from yeast production, protein waste from meat production, protein waste from dairy industry, silicates and derivatives thereof, water glasses and water glass-like binders, cement additives, aluminium oxide, calcium oxide, calcium hydroxide, aluminium hydroxide, microsilica, bentonites, resins and epoxides, natural and chemical herbicides, fungicides, molluscicides, insecticides, hydrophobizers, emulsifiers, thixotropic agents, crystallization nuclei and crystallization modifiers, trace elements, sulphates, bacteria capable of forming polymers, and substances that modify biocementation; wherein the mixture is free of cement, and the one or more additives are present in an amount to additionally solidify or harden the substrate or increase its stability.

2. The mixture of claim 1, wherein the mixture is a liquid, a gel, a paste, or a powder.

3. The mixture of claim 1 comprising the one or more organisms, wherein the one or more organisms are selected from microorganisms.

4. The mixture of claim 3, wherein the microorganisms are selected from microorganisms of the phylum of Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, and a mixture thereof.

5. The mixture of claim 4, wherein the microorganisms are selected from the class of Bacilli, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Actinobacteria, Cyanobacteria, and a mixture thereof.

6. The mixture of claim 1 comprising the one or more enzymes, wherein the one or more enzymes are selected from urease, asparaginase, carbonic anhydrase, and metabolic enzymes.

7. The mixture of claim 1, wherein the one or more substances to form the carbonate are selected from urea and salts thereof, organic acids and salts and esters thereof, peptides, amino acids and salts and esters thereof, vegetable and animal complex substrates, industrial waste streams, protein lysates, anaerobic substrates, and methane.

8. The mixture of claim 1, wherein the one or more cation sources are selected from organic and inorganic calcium salts, magnesium salts, manganese salts, zinc salts, cobalt salts, nickel salts, copper salts, lead salts, iron salts, cadmium salts, polymers, heavy metal cations, light metal cations, radioactive cations, and mixtures thereof.

9. The mixture of claim 1, wherein the mixture does not comprise the one or more cation sources of (iii).

10. A method for biocementation of a substrate comprising applying the mixture of claim 1 to the substrate and allowing the mixture to solidify.

11. A mixture capable of biocementation of a substrate comprising: (i) one or more organisms or enzymes capable of forming carbonate, inducing carbonate formation, or catalyzing carbonate formation; (ii) one or more substances to form the carbonate; (iii) optionally, one or more cation sources; and (iv) one or more additives selected from polyhydroxybutyrate, polyacrylic acid, polymethacrylate, poly(2-hydroxyethylmethacrylate), polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, poly(2-ethyl-2-oxazoline), derivatives of cellulose, derivatives of pectins, natural adhesives, in particular gum arabic, latex, derivatives of chitin, derivatives of chitosan, derivatives of cyclodextrins, derivatives of dextrins, hydrogel formers, cold soluble and/or warm soluble (plant) glues, microbial exopolysaccharides containing or consisting of maleic acid, acetic acid, lactic acid, lactose, sucrose, glucose, fructose and/or inulin, monomers of polysaccharides, lactose, sucrose, glucose, fructose, inulin, protein sources, casein, albumin, peptones, residues and industrial materials selected from the group consisting of maize steep liquor, lactose mother liquor, protein lysates, protein waste from yeast production, protein waste from meat production, protein waste from dairy industry; acrylates and derivatives thereof; water glasses and water glass-like binders, aluminium oxide, calcium oxide, calcium hydroxide, aluminium hydroxide, microsilica, bentonites, epoxides, natural and chemical herbicides, fungicides, molluscicides, insecticides, thixotropic agents, crystallization nuclei and crystallization modifiers, trace elements, sulphates, bacteria capable of forming polymers, and substances that modify biocementation; wherein the additives are present in an amount to additionally solidify or harden the substrate or to increase its stability.

12. The mixture of claim 11, wherein the mixture is a liquid, a gel, a paste, or a powder.

13. The mixture of claim 11 comprising the one or more organisms, wherein the one or more organisms are selected from microorganisms.

14. The mixture of claim 13, wherein the microorganisms are selected from microorganisms of the phylum of Firmicutes, Proteobacteria, Actinobacteria, Cyanobacteria, and a mixture thereof.

15. The mixture of claim 14, wherein the microorganisms are selected from the class of Bacilli, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Epsilonproteobacteria, Actinobacteria, Cyanobacteria, and a mixture thereof.

16. The mixture of claim 11 comprising the one or more enzymes, wherein the one or more enzymes are selected from urease, asparaginase, carbonic anhydrase, and metabolic enzymes.

17. The mixture of claim 11, wherein the one or more substances to form the carbonate are selected from urea and salts thereof, organic acids and salts and esters thereof, peptides, amino acids and salts and esters thereof, vegetable and animal complex substrates, industrial waste streams, protein lysates, anaerobic substrates, and methane.

18. The mixture of claim 11, wherein the one or more cation sources are selected from organic and inorganic calcium salts, magnesium salts, manganese salts, zinc salts, cobalt salts, nickel salts, copper salts, lead salts, iron salts, cadmium salts, polymers, heavy metal cations, light metal cations, radioactive cations, and mixtures thereof.

19. The mixture of claim 11, wherein the mixture does not comprise the one or more cation sources of (iii).

20. A method for biocementation of a substrate comprising applying the mixture of claim 11 to the substrate and allowing the mixture to solidify.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0124] FIG. 1: Suppression of weed growth by non-ureolytic biocementation using the bacterial strain B. pseudofirmus: Effect against monocotyls (annual meadow grass) and dicotyls (ribwort plantain). Average coverage rates of weed growth in the 42-day documentation period with weekly control of the control (top) versus the sample treated with biocementation mixture 1 (centre). Visual representation (bottom) of weed growth in control application (bottom left) compared to treatment with biocementation mixture 1 (bottom right) after 42 days of growth.

[0125] FIG. 2: Suppression of weed growth by non-ureolytic biocementation with the bacterial strains A. crystallopoietes, B. cohnii B. halodurans, and B. pseudofirmus: Effect against monocotyls (annual meadow grass) and dicotyls (ribwort plantain). Average coverage rates of weed growth over the 42-day documentation period for weekly control measurement versus samples treated with biocementation mixture 1.

[0126] FIG. 3: Application of ureolytic biocementation with L. sphaericus to suppress weed growth in quartz sand: Effect against monocotyls (annual meadow grass) and dicotyls (ribwort plantain) weeds. Average coverage rates of weed growth (top) in the documentation period of 42 days with weekly assessment of control versus biocementation mixture 2 and biocementation mixture 3. Visual presentation (centre) of weed growth in control application (centre left) compared to treatment with biocementation mixture 2 (centre centre) and biocementation mixture 3 (centre right) after 42 days of growth in the laboratory. Graphical representation of the solidification of the biocementation layers (bottom) by investigation of the average breaking force of the specimens.

[0127] FIG. 4: Application of ureolytic biocementation with L. sphaericus to suppress weed growth in land soil: Effect against monocotyls (annual meadow grass) and dicotyls (ribwort plantain) weeds. Average coverage rates of weed growth (top) in the documentation period of 42 days with weekly assessment of control versus biocementation mixture 2 and biocementation mixture 3. Visual presentation (centre) of weed growth in control application (centre left) compared to treatment with biocementation mixture 2 (centre centre) and biocementation mixture 3 (centre right) after 42 days of growth in the laboratory. Graphical representation of the solidification of the biocementation layers (bottom) by investigation of the average breaking force of the specimens.

[0128] FIG. 5: Application of ureolytic biocementation with Sp. pasteurii to suppress weed growth in quartz sand: Effect against monocotyls (annual meadow grass) and dicotyls (ribwort plantain) weeds. Average coverage rates of weed growth (top) in the documentation period of 42 days with weekly assessment of control versus biocementation mixture 4 and biocementation mixture 5. Visual presentation (centre) of weed growth in control application (centre left) compared to treatment with biocementation mixture 4 (centre centre) and biocementation mixture 5 (centre right) after 42 days of growth in the laboratory. Graphical representation of the solidification of the biocementation layers (bottom) by investigation of the average breaking force of the specimens.

[0129] FIG. 6: Application of ureolytic biocementation with Sp. pasteurii to suppress weed growth in land soil: Effect against monocotyls (annual meadow grass) and dicotyls (ribwort plantain) weeds. Average coverage rates of weed growth (top) in the documentation period of 42 days with weekly assessment of control versus biocementation mixture 4 and biocementation mixture 5. Visual presentation (centre) of weed growth in control application (centre left) compared to treatment with biocementation mixture 4 (centre centre) and biocementation mixture 5 (centre right) after 42 days of growth in the laboratory. Graphical representation of the solidification of the biocementation layers (bottom) by investigation of the average breaking force of the specimens.

[0130] FIG. 7: Application of biocementation to suppress weed growth in open land: Effect against non-sprouted and freshly sprouted weeds on agricultural land. Average coverage rates of water control (top) compared to the area treated with biocementation mixture 6 (centre) in the documentation period of 42 days with weekly assessment. Visual representation of weed growth in control application (bottom left) compared to treatment with biocementation mixture 6 (bottom right) after 42 days of outdoor growth.

[0131] FIG. 8: Application of biocementation to suppress weed growth in open land: Effect against non-sprouted and freshly sprouted weeds in pavement joints. Average coverage rates of water control (top) compared to the area treated with biocementation mixture 6 (centre) in the documentation period of 42 days with weekly assessment. Visual representation of weed growth in control application (bottom left) compared to treatment with biocementation mixture 6 (bottom right) after 42 days of outdoor growth.

EXAMPLES

Example 1: Non-Ureolytic Biocementation with B. pseudofirmus—Suppression of Growth of Monocotyledonous and Dicotyledonous Weeds

[0132] Materials and Methods:

[0133] The experiment was carried out in the laboratory in plant pots with a volume of 450 cm.sup.3. The application area was 78.5 cm.sup.2, respectively. A total of 6 samples were treated.

[0134] The soil substrate in the experiment consisted of quartz sand with a grain size of 0-2 mm. The sand was washed and dried by the manufacturer and was used directly. 300 g quartz sand per plant pot were used as soil substrate.

[0135] Before treatment, the quartz sand was free of weed growth and contained only residues of endemic weed seeds or inflowing seeds. However, these were not sufficient for efficient weed growth. Weed sowing was carried out with 0.2 g Plantago lanceolata (ribwort plantain) and 0.1 g Poa annua (annual meadow grass) per vessel, respectively. For this purpose, the weed seeds were worked into the top soil layer at a depth of 2-4 mm.

[0136] A liquid biocementation mixture 1 was used, which consisted of the following components in the following concentrations:

TABLE-US-00001 20.0 g/l Yeast extract 0.2 M calcium acetate 0.2 M calcium lactate 6.0 g/l urea 5 × 10{circumflex over ( )}8 cells/ml B. pseudofirmus

[0137] The mixture also contains trace elements and traces of salts and sugars, for example (<1 wt. %). In this medium, urea served primarily as a source of nitrogen (and not as a carbonate source).

[0138] All components of the present mixture, which is capable of biocementation, except for the bacteria of strain B. pseudofirmus, were present in solid form. The bacteria were present as liquid culture in a culture medium known in state of the art, as described for example in Jonkers H. M. et al., Tailor Made Concrete Structures—Walraven & Stoelhorst (eds), 2008, Taylor & Francis Group, London, ISBN 978-0-415-47535-8, section 2.1, using 5 g/L yeast extract in the context of the present invention. The solid components and the bacteria in liquid culture were mixed directly before use, dissolving the solid components.

[0139] The biocementation mixture 1 and a water control were applied in three replicas to each of the test plots. The application quantity per square metre was 5 litres per replica throughout. A pipette was used for application.

[0140] After the application of biocementation mixture 1, incubation for 48 hours without irrigation took place. During this period, the minimum temperature was 14.2° C. and the maximum temperature was 25.2° C.

[0141] Weed growth was documented over 42 days after application. The minimum and maximum temperatures during this period were 10.7° C. and 34.0° C. The vessels were watered once to three times a week, depending on requirements. The plant pots were exposed to natural lighting with day and night rhythm.

[0142] Weed growth was documented on a weekly basis. Both the biocementation layer (layer thickness, strength) and the so-called coverage rate were determined. The weed growth coverage rates were determined by manual visual assessment of the plant pots at the specified times. The coverage rate describes in percent the area covered by weeds. From this in turn the degree of efficiency according to Abbott was calculated as follows:

Degree of efficiency=(coverage rate control.sub.day xy−coverage rate product.sub.day xy)/coverage rate control.sub.day xy

[0143] To verify the carbonate formation, 10 ml of the biocementation mixture 1 were incubated openly in a reaction vessel for 24 h at room temperature. Subsequently, the precipitated pellet was obtained by centrifugation and drying. The dried pellet was used for carbonate detection according to Scheibler.

[0144] Results:

[0145] Weed growth was almost completely reduced compared to control (FIG. 1). The average coverage rate after 42 days was 2% in the treated area (FIG. 1, centre) and 60% in the control area (FIG. 1, top). A biocementation layer was formed during treatment with the biocementation mixture 1 specified above. Weed growth occurred mainly in areas where the biocementation layer was damaged (e.g. in drying cracks). The courses of time over the 42 days can be taken from FIG. 1 (top and centre). In the course of time, an effect of biocementation in weed suppression is visible. FIG. 1 illustrates the direct comparison between a control sample (bottom left) and an application sample (bottom right) after 42 days of growth. The final degree of efficiency of the biocementation product was 96.7%.

[0146] The biocementation mixture is advantageously similarly effective as many commercially available weed suppressants (data not shown), whereby various disadvantages of such weed suppressants can be avoided.

[0147] The qualitative analysis of the carbonate formation according to Scheibler showed a positive reaction for the biocementation mixture. The control on the other hand did not show any carbonate formation (data not shown).

[0148] Comparable effects on weed growth were also achieved with slightly modified formulations of the biocementation mixture 1 containing calcium acetate, calcium lactate and/or calcium chloride in a concentration of 0.05 to 0.3 M, respectively, and not exceeding a total calcium concentration of 0.4 M in the mixture (data not shown). A variation in the urea concentration (0.0 to 0.2 M) or in the yeast extract quantity (0.1 to 30 g/l) also yielded good degrees of efficiency. Weed suppression was dependent on the used concentrations of the components of the biocementation mixture, respectively (data not shown).

[0149] The entire experiment described above was performed alternatively with weed seeds that had germinated 24 hours prior to the application of the biocementation mixture. For this purpose, the biocementation mixture was applied 1 24 hours after the start of germination. The results obtained were comparable to those described in the present example and an almost complete reduction in weed growth was achieved by applying the mixture (data not shown).

[0150] Furthermore, in the biocementation mixture 1 described above, the bacterial strain B. pseudofirmus was replaced by the same cell number concentration of B. cohnii, B. halodurans or A. crystallopoietes, respectively, the experiment being carried out as described above, respectively. B. cohnii and B. halodurans were present in the same culture medium as B. pseudofirmus (see above) and A. crystallopoietes was present in a known culture medium such as Hamilton, R. W. et al., Journal of Bacteriology 1977, 129(2), 874-879 (see section “Materials and Methods”, p. 874-875). The test results of weed suppression with these alternative biocementation mixtures are shown in FIG. 2.

Example 2: Ureolytic Biocementation with L. Sphaericus—Suppression of Growth of Monocotyledonous and Dicotyledonous Weeds

[0151] Materials and Methods:

[0152] In the present experiment, two biocementation mixtures, each with the same bacterial strain, were tested on two different soil substrates.

[0153] The experiment was carried out in the laboratory in plant pots with a volume of 450 cm.sup.3. The application area per vessel was 78.5 cm.sup.2, respectively. A total of 9 plant pots per soil substrate were treated with the two different biocementation mixtures (see below).

[0154] The first soil substrate in the experiment consisted of quartz sand with a grain size of 0-2 mm. The quartz sand was washed and dried by the manufacturer and was used directly. 300 g quartz sand per plant pot were used as soil substrate. In a further row, sifted land soil was used as the second soil substrate. Here, 250 g of land soil were used per application vessel.

[0155] Both soil substrates were free of weed growth prior to treatment. However, both soils contained minimal residues of endemic weed seeds or inflowing seeds. However, these were not sufficient for efficient weed growth. Weed sowing was carried out with 0.2 g Plantago lanceolata (ribwort plantain) and 0.1 g Poa annua (annual meadow grass) per vessel, respectively. For this purpose, the weeds were worked into the top soil layer at a depth of 2-4 mm.

[0156] Two different liquid biocementation mixtures were used in the experiment.

[0157] Mixture 2 was composed of the following components in the following concentrations:

TABLE-US-00002 20.0 g/l Yeast extract 0.25 M calcium chloride 18.0 g/l urea 4 × 10{circumflex over ( )}8 cells/ml L. sphaericus

[0158] The mixture also contained trace elements and traces of salts and sugars, for example (<1%). In this medium, urea served primarily as a source of carbonate and secondarily as a source of nitrogen.

[0159] In mixture 3, 50 ml/1 Silicade 8 (silica sol-acrylic dispersion) was additionally added as additive. The additive was used to achieve a longer lasting stability of the biocementation layer.

[0160] The components of the biocementation mixtures 2 and 3 (without bacteria) were present in solid form, respectively. The bacteria were present as liquid culture in a culture medium known in state of the art, respectively, as described for example in in Dick, J. et al., Biodegradation 2006, 17, 357-367 (see section “Materials and Methods”, p. 359). The solid components and the bacteria in liquid culture were mixed directly before use, respectively, dissolving the solid components. Silicade 8 was present in liquid form and was only added to mixture 3.

[0161] The biocementation mixtures 2 and 3 as well as a water control were applied in three replicas next to each other to the two test soils. The application quantity per square metre was 5 litres per replica throughout. A pipette was used for application.

[0162] After the application of the biocementation mixtures, incubation for 48 hours without irrigation took place. During this period, the minimum temperature was 12.4° C. and the maximum temperature was 24.2° C.

[0163] Weed growth was documented over 42 days after application. The minimum and maximum temperatures during this period were 9.7° C. and 27.9° C. The vessels were watered once to three times a week, depending on requirements. The plant pots were exposed to natural lighting with day and night rhythm.

[0164] Weed growth was documented on a weekly basis. Both the biocementation layer (layer thickness, strength) and the so-called coverage rate were determined. The weed growth coverage rates were determined by manual visual assessment of the plant pots at the specified times. The coverage rate describes in percent the area covered by weeds. From this in turn the degree of efficiency according to Abbott was calculated as follows:

Degree of efficiency=(coverage rate control.sub.day xy−coverage rate product.sub.day xy)/coverage rate control.sub.day xy

[0165] To verify the carbonate formation, 10 ml of the biocementation mixtures 2 and 3, respectively, were incubated openly in a reaction vessel for 24 h at room temperature. Subsequently, the precipitated pellet was obtained by centrifugation and drying, respectively. The dried pellets were used for carbonate detection according to Scheibler.

[0166] Results:

[0167] On the quartz sand, weed growth was completely reduced compared to the control with both biocementation mixtures 2 and 3 (FIG. 3). The average coverage rate after 42 days was 0% on the area treated with biocementation mixture 2, 0% on the area treated with biocementation mixture 3 and 31% on the control area. In both treatments (with biocementation mixture 2 and 3) a biocementation layer was formed. Weed growth occurred mainly in areas where the biocementation layer was damaged (e.g. in drying cracks). The courses of time over the 42 days can be taken from FIG. 3 (top). The effect of biocementation on weed suppression is illustrated in FIG. 3 (centre) and demonstrates the direct comparison between a control (centre left), biocementation mixture 2 (centre centre) and biocementation mixture 3 (centre right). The final degree of efficiency of both biocementation mixtures was 100%, respectively. After 42 days, the strengths of the biocementation layers were determined (as described above). The biocementation sample with mixture 2 had a layer with an average breaking force of 4.3 N, however, it is lower than with mixture 3 with 19.1 N (see FIG. 3 (below)). By incorporating the Silicade 8 additive in the biocementation layer (through biocementation mixture 3), an increased resistance to environmental parameters and thus probably longer effectiveness could be achieved. No biocement layer was present in the control sample.

[0168] On the land soil, weed growth was almost completely reduced compared to control (FIG. 4). The average coverage rate after 42 days was 0% on the area treated with biocementation mixture 2, 2% on the area treated with biocementation mixture 3 and 50% on the control area. In both treatments (with biocementation mixture 2 and 3) a biocementation layer was formed. Weed growth occurred mainly in areas where the biocementation layer was damaged (e.g. in drying cracks). The courses of time over the 42 days can be taken from FIG. 4 (top). The effect of the biocementation on weed suppression is illustrated in FIG. 4 (centre) and demonstrates the direct comparison between a control sample (centre left), biocementation mixture 2 (centre centre) and biocementation mixture 3 (centre right). The final degree of efficiency of the two biocementation mixtures 2 and 3 was 100% and 96%, respectively. After 42 days, the strength of the resulting biocementation layers was determined (as described above). The biocementation sample with mixture 2 had a layer with an average breaking force of 20.5 N, however, it is lower than with mixture 3 with 84.3 N. By incorporating the Silicade 8 additive in the biocementation layer (through biocementation mixture 3), an increased resistance to environmental parameters and thus probably longer effectiveness could be achieved. No biocement layer was present in the control sample.

[0169] The qualitative analysis of the carbonate formation according to Scheibler showed a positive reaction for the biocementation mixtures 2 and 3. The controls showed no carbonate formation (data not shown).

[0170] Comparable effects on weed growth were also shown in slightly modified formulations of biocementation mixtures 2 and 3 containing calcium acetate, calcium lactate and/or calcium chloride in a concentration of 0.05 to 0.3 M, respectively, and not exceeding a total calcium concentration of 0.4 M (data not shown). A stronger variation in the urea concentration (e.g. 0.1 to 1.0 M) or in the yeast extract quantity (e.g. 0.1 to 30 g/l) also produced good degrees of efficiency. Weed suppression was dependent on the concentrations of the components used in the respective biocementation mixture, respectively (data not shown).

[0171] The entire experiments described above were performed alternatively with weed seeds that had germinated 24 hours prior to the application of the respective biocementation mixture. For this purpose, the respective biocementation mixture was applied 24 hours after the start of germination. The results obtained were comparable to those described in the present example and an almost complete reduction in weed growth was achieved by applying the respective mixture (data not shown).

Example 3: Ureolytic Biocementation with Sp. Pasteurii—Growth Suppression of Monocotyledonous and Dicotyledonous Weeds

[0172] Materials and Methods:

[0173] In the present experiment, two biocementation mixtures, each with the same bacterial strain, were tested on two different soil substrates.

[0174] The experiment was carried out in the laboratory in plant pots with a volume of 450 cm.sup.3. The application area was 78.5 cm.sup.2, respectively. A total of 9 plant pots per soil substrate were treated with the two different biocementation mixtures (see below). The application area per vessel was 78.5 cm.sup.2, respectively.

[0175] The first soil substrate in the experiment consisted of quartz sand with a grain size of 0-2 mm. The quartz sand was washed and dried by the manufacturer and was used directly. 300 g quartz sand per plant pot were used as soil substrate. In a further row, sifted land soil was used as the second soil substrate. Here, 250 g of land soil were used per application vessel.

[0176] Both soil substrates were free of weeds prior to treatment. Both soils contained minimal residues of endemic weed seeds or inflowing seeds. However, these were not sufficient for efficient weed growth. Weed sowing was carried out with 0.2 g Plantago lanceolata (ribwort plantain) and 0.1 g Poa annua (annual meadow grass) per vessel, respectively. For this purpose, the weed seeds were worked into the top soil layer at a depth of 2-4 mm.

[0177] Two different liquid biocementation mixtures were used in the experiment.

[0178] Mixture 4 was composed of the following components in the following concentrations:

TABLE-US-00003 20.0 g/l Yeast extract 0.25 M calcium chloride 18.0 g/l urea 4 × 10{circumflex over ( )}8 cells/ml Sp. pasteurii

[0179] The mixture also contained trace elements and traces of salts and sugars, for example (<1%). In this medium, urea served primarily as a source of carbonate and secondarily as a source of nitrogen.

[0180] In mixture 5, 50 ml/1 Silicade 8 (silica sol-acrylic dispersion) was additionally added as additive. The additive was used to achieve a longer lasting stability of the biocementation layer.

[0181] The components of the biocementation mixtures 4 and 5 (without bacteria) were present in solid form, respectively. The bacteria were present as liquid culture in a culture medium known from the state of the art, respectively, as described for example in Cuthbert, M. O. et al., Ecological Engineering 2012, 41, 32-40 (see section 2.2, p. 33). The solid components and the bacteria in liquid culture were mixed directly before use, respectively, dissolving the solid components. Silicade 8 was present in liquid form and was only added to mixture 5.

[0182] The biocementation mixtures 4 and 5 as well as a water control were applied in three replicas next to each other to the two test soils. The application quantity per square metre was 5 litres per replica throughout. A pipette was used for application.

[0183] After the application of the biocementation mixtures, incubation for 48 hours without irrigation took place. During this period, the minimum temperature was 12.4° C. and the maximum temperature was 24.2° C.

[0184] Weed growth was documented over 42 days after application. The minimum and maximum temperatures during this period were 9.7° C. and 27.9° C. The vessels were watered once to three times a week, depending on requirements. The plant pots were exposed to natural lighting with day and night rhythm.

[0185] Weed growth was documented on a weekly basis. Both the biocementation layer (layer thickness, strength) and the so-called coverage rate were determined. The weed growth coverage rates were determined by manual visual assessment of the plant pots at the specified times. The coverage rate describes in percent the area covered by weeds. From this in turn the degree of efficiency according to Abbott was calculated as follows:

Degree of efficiency=(coverage rate control.sub.day xy−coverage rate product.sub.day xy)/coverage rate control.sub.day xy

[0186] To verify the carbonate formation, 10 ml of the biocementation mixtures 4 and 5, respectively, were incubated openly in a reaction vessel for 24 h at room temperature. Subsequently, the precipitated pellet was obtained by centrifugation and drying, respectively. The dried pellets were used for carbonate detection according to Scheibler.

[0187] Results:

[0188] On the quartz sand, weed growth was completely reduced compared to the control (FIG. 5). The average coverage rate after 42 days was 0% on the area treated with biocementation mixture 4, 0% on the area treated with biocementation mixture 5 and 40% on the control area. In treatments with the mixtures a biocementation layer was formed. Weed growth occurred mainly in areas where the biocementation layer was damaged (e.g. in drying cracks). The courses of time over the 42 days can be taken from FIG. 5 (top). The effect of biocementation on weed suppression is illustrated in FIG. 5 (centre) and demonstrates the direct comparison between a control (centre left), biocementation mixture 4 (centre centre) and biocementation mixture 5 (centre right). The final degree of efficiency of both biocementation mixtures was approximately 100%, respectively. After 42 days, the strengths of the biocementation layers were determined (as described above). The biocementation sample with mixture 4 had a layer with an average breaking force of 4.1 N, the sample with mixture 5 had an average breaking force of 19.3 N (see FIG. 5 (below)). By incorporating the Silicade 8 additive in the biocementation layer (through biocementation mixture 5), an increased resistance to environmental parameters and thus probably longer effectiveness could be achieved. No biocement layer was present in the control.

[0189] On the land soil, weed growth was almost completely reduced compared to control (FIG. 6). The average coverage rate after 42 days was 0% on the area treated with biocementation mixture 4, 0% on the area treated with biocementation mixture 5 and 50% on the control area. In treatments with the mixtures a biocementation layer was formed. Weed growth occurred mainly in areas where the biocementation layer was damaged (e.g. in drying cracks). The courses of time over the 42 days can be taken from FIG. 6 (top). The effect of the biocementation on weed suppression is illustrated in FIG. 6 (centre) and demonstrates the direct comparison between a control sample (centre left), mixture 4 (centre centre) and mixture 5 (centre right). The final degree of efficiency of the two biocementation mixtures was 100%, respectively. After 42 days, the strength of the resulting biocementation layers was determined. The biocementation sample with mixture 4 had a layer with an average breaking force of 20.8 N, the sample with mixture 5 had an average breaking force of 66.8 N. By incorporating the Silicade 8 additive in the biocementation layer (through biocementation mixture 5), an increased resistance to environmental parameters and thus probably longer effectiveness could be achieved. No biocement layer was present in the control.

[0190] The qualitative analysis of the carbonate formation according to Scheibler showed a positive reaction for the biocementation mixtures 4 and 5, respectively. The controls showed no carbonate formation (data not shown).

[0191] Comparable effects on weed growth were also shown in slightly modified formulations of biocementation mixtures 4 and 5 containing calcium acetate, calcium lactate and/or calcium chloride in a concentration of 0.05 to 0.3 M, respectively, and not exceeding a total calcium concentration of 0.4 M (data not shown). A stronger variation in the urea concentration (e.g. 0.1 to 1.0 M) also produced good degrees of efficiency. Weed suppression was dependent on the concentrations of the components used in the respective biocementation mixture, respectively (data not shown).

[0192] The entire experiments described above were performed alternatively with weed seeds that had germinated 24 hours prior to the application of the respective biocementation mixture. For this purpose, the respective biocementation mixture was applied 24 hours after the start of germination. The results obtained were comparable to those described in the present example and an almost complete reduction in weed growth was achieved by applying the respective mixture (data not shown).

Example 4: Open Land—Suppression of Weeds on Agricultural Land and Pavement Joints

[0193] Materials and Methods:

[0194] The experiment was carried out on agricultural land and a grouted driveway. The application area was 6 m.sup.2, respectively.

[0195] The soil substrate of the agricultural land consisted of natural land soil. Before the application of the mixture according to the invention (see below), the agricultural land was cleared of established weeds by chemical treatment with glyphosate (approx. 6 months before the present experiment). After this pre-treatment, no plant residues were left on the surface.

[0196] The joint material of the driveway consisted mainly of joint gravel and joint sand. Prior to application, these areas were mechanically cleared of established weeds by a brush cutter. After this pre-treatment there were also no plant residues left on the surface.

[0197] Both soils contained the weed seeds, inflow seeds and possibly fresh seedlings or plant remains found there. No artificial weed sowing was carried out as there were enough endemic weeds present at both sites.

[0198] For the experiment a liquid biocementation mixture 6 was used consisting of the following components and concentrations:

TABLE-US-00004 18.0 g/l Urea 62.5 g/l lignosulfonate 5 × 10{circumflex over ( )}8 cells/ml Sporosarcina pasteurii

[0199] The solution also contains trace elements and traces of salts, sugars and yeast extract, for example (<1%).

[0200] The bacteria were present as liquid culture in culture medium (see description in previous example 3). The urea and the lignosulfonate were originally present in solid form. They were dissolved in water directly before use and mixed with the liquid culture of the bacteria.

[0201] The biocementation mixture 6 and a water control were applied in three replicas to each of the two test areas, respectively. The application quantity per square metre was 4 litres per replica throughout. A standard watering can (51 volume) was used for application.

[0202] After the application of the biocementation mixture 6, incubation was carried out for 48 hours without rain or artificial irrigation. During this period, the minimum temperature was 5° C. and the maximum temperature was 25° C.

[0203] Weed growth was documented over 42 days after application. The minimum and maximum temperatures were 5° C. and 33° C., respectively. The total precipitation during the documentation period was 91 mm (l/m.sup.2). Due to the weather no additional watering was necessary.

[0204] Weed growth was documented on a weekly basis. Both the biocementation layer (layer thickness, strength) and the so-called coverage rate were determined. The weed growth coverage rates were determined by manual visual assessment of the plant pots at the specified times. The coverage rate describes in percent the area covered by weeds. From this in turn the degree of efficiency according to Abbott was calculated as follows:

Degree of efficiency=(coverage rate control.sub.day xy−coverage rate product.sub.day xy)/coverage rate control.sub.day xy

[0205] Results:

[0206] On the agricultural land, weed growth was significantly reduced compared to control. The coverage rate after 42 days was 3.3% on the treated areas and 70.0% on the control area. A biocementation layer was formed. Weed growth occurred mainly in areas where the biocementation layer was damaged (e.g. in drying cracks). The courses of time over the 42 days can be taken from FIG. 7 (top, water control) and FIG. 7 (centre, treatment with biocementation mixture 6). FIG. 7 (bottom) illustrates the direct comparison between the control and the application (within the marker, respectively). The final degree of efficiency of the biocementation mixture 6 was 95.2%.

[0207] On the grouted driveway, the weed growth was also significantly reduced in comparison to the control. The coverage rate after 42 days was 3.7% on the treated areas and 40.0% on the control area. Here, too, a biocementation layer was formed. The courses of time over the 42 days can be taken from FIG. 8 (top, water control) and FIG. 8 (centre, treatment with biocementation mixture 6). FIG. 8 (bottom) illustrates the direct comparison between the control and the application (within the joints). The final degree of efficiency of the biocementation product was 90.8%.

[0208] The biocementation mixture is advantageously similarly effective as many commercially available weed suppressants (data not shown), whereby various disadvantages of such weed suppressants can be avoided.

[0209] Comparable effects on weed growth in open land were also shown with alternative mixture formulations additionally containing 0.1 M to 0.3 M CaCl.sub.2) (based on mixture 6) (data not shown). A stronger variation in urea concentration (1.0 to 0.15 M) also produced good degrees of efficiency in weed suppression (data not shown).