BIOCEMENTATION MIXTURE FOR DUST CONTROL AND RELATED APPLICATIONS
20230227355 · 2023-07-20
Inventors
- Martin SPITZNAGEL (München, DE)
- Luitpold FRIED (München, DE)
- Saskia PAZUR (Eichenau, DE)
- Jan-Philip MERK (Hohenschäftlarn, DE)
- Florian HORNUNG (München, DE)
Cpc classification
C04B12/00
CHEMISTRY; METALLURGY
Y02W30/91
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C04B2103/0001
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention relates primarily to the use of a mixture for reducing dust formation and/or erosion. The invention relates additionally to a method for reducing dust formation and/or erosion and also to a mixture suitable for this purpose.
Claims
1. A biocementation method comprising: (A) forming a biocement mixture comprising: (i) at least one organism which is capable of forming carbonate, or of inducing and/or catalysing carbonate formation, and/or at least one enzyme which is capable of forming carbonate or of inducing and/or catalysing carbonate formation, (ii) at least one substance for forming carbonate, (iii) at least one water-soluble, water-dispersible and/or water-emulsifiable, cohesion-modifying compound, selected from the group consisting of: compounds having calcium affinity, especially compounds having at least one calcium-binding functional group selected from the group consisting of carboxylic acids, carboxylates, carbonyls, alcohols, alkoxides, thiols, thiolates, sulfates, sulfonates, amines, amides, catechols, quinones, phosphates, phosphonates; and compounds having carbonate affinity, especially compounds having at least one carbonate-binding functional group selected from the group consisting of cationic functional groups and/or neutral functional groups, especially compounds containing cations, more particularly mono- or polyvalent cations, for example quaternary ammonium compounds, mono-, di- or trivalent metal cations, carboxylic acids, sulfonic acids, peroxycarboxylic acids, thiocarboxylic acids, sulfinic acids, sulfenic acids, amides, amines, hydrazines and thiols; (iv) optionally, at least one cation source; (v) optionally, at least one adjuvant; and (B) reducing dust formation and/or erosion with the biocement mixture, pelletizing a substrate with the biocement mixture, reducing evaporation with the biocement mixture, sealing or insulating a substrate with the biocement mixture, and/or precipitating a contaminant with the biocement mixture.
2. The method of claim 1, wherein the method reduces dust formation to a greater extent than a sum of the reduction of dust formation provided individually by (i), (ii) and (iii).
3. The method of claim 1, wherein (iii) is present in an amount of at least 0.5 wt %, based on a total mass of (i), (ii) and (iii), and/or (iii) is present in an amount of at most 85 wt %, based on a total mass of (i), (ii) and (iii).
4. The method of claim 1, wherein (iii) is selected from: (iii-1)(bio)polymers selected from: cellulose and derivatives thereof, starch and derivatives thereof, lignins and derivatives thereof humic acids and derivatives thereof; chitin and its derivatives, chitosan and its derivatives, cyclodextrins and its derivatives, dextrins and its derivatives, natural adhesives, hydrogel-formers, latex, rubber, and derivatives thereof; protein sources and/or peptides which contain at least one of the following amino acids: alanine, glycine, lysine, asparagine, glutamine, glutamate, a non-proteinogenic amino acid; starch ethers and starch esters; yeasts and their derivatives and extracts; liquid and dried polymer dispersions or polymerisates comprising acids, and also their salts, cyanates, esters, ethers, oxiranes, amines, amides, sulfates, alcohols, thiols, halogens, silanes, siloxanes, phosphates, alkyls, allyls and aryls, and also derivatives thereof; (iii-2)(poly)saccharides and extracellular polymeric substances (EPS) and their respective derivatives, wherein the EPS are selected from microbial exopolysaccharides; (iii-3)carboxylic acids selected from formic acid, maleic acid, succinic acid, butanoic acid, propanoic acid, acetic acid, pyruvic acid, acetoacetic acid, levulinic acid, oxalacetic acid, citric acid, fruit acids, short-chain and medium-chain fatty acids, and lactic acid and in each case their salts and their esters; (iii-4) inorganic binders, minerals and salts, selected from cement, including its derivatives, gypsum, sodium, potassium and lithium silicates and further waterglass derivatives, calcium carbonate and its derivatives, aluminium hydroxide, calcium sulfate, calcium hydroxide, magnesium sulfate, microsilica, and kaolins; (iii-5) amino acids selected from alanine, glycine, lysine, asparagine, glutamine, glutamate, non-proteinogenic amino acids and in each case their salts and their esters and amides.
5. The method of claim 1, wherein (iii) is selected from: lignosulfonates, humic acid and salts thereof, and derivatives thereof, kraft lignins, fibres and fibre substances, selected from cellulose fibres, wood fibres and wood cellulose fibres, gum arabic, xanthan, alginates, and agar, protein sources and/or peptides selected from casein, albumin, yeast extracts, peptones, caseinate, calcium caseinate, milk powder, alanine, glycine, lysine, asparagine, glutamine, glutamate, non-proteinogenic amino acids, residual substances and industrial substances selected from of corn steep liquor, lactose mother liquors, protein lysates, molasses, protein wastes, preferably from yeast production, meat production, fruit production, vegetable production, dairy industry and papermaking, liquid and dried polymer dispersions or polymerisates selected from polyhydroxybutyrate, polylactide, polybutylenesuccinate, polyacrylic acid, polymethacrylate, poly(2-hydroxyethyl methacrylate), polyvinyl alcohol, polyvinyl acetate, polyvinylpyrrolidone, polyvinylimidazole, poly(2-ethyl-2-oxazoline), polystyrene, polyamide, styrene-butadienes, styrene-acrylates, styrenes, acrylates, acrylic acids, vinyl acetates, isocyanates, epoxides, and polyamino acids.
6. The method of claim 1, wherein (iii) is selected from: calcium lignosulfonate, sodium lignosulfonate, potassium lignosulfonate, magnesium lignosulfonate, ammonium lignosulfonate, yeast extract, albumin, starch ether, alanine, lysine, styrene-acrylate dispersion, magnesium sulfate, polyvinyl alcohol, polyvinyl acetate dispersion, styrene-butadiene dispersion, humic acid, alkali metal silicate, and combinations thereof.
7. The method of claim 1, wherein (ii) is selected from: urea and its salts; organic acids and salts thereof and esters thereof; gluconic acid and salts thereof and esters thereof; acetic acid and salts thereof and esters thereof; formic acid and salts thereof and esters thereof; propanoic acid and salts thereof and esters thereof; butanoic acid and salts thereof and esters thereof; pentanoic acid and salts thereof and esters thereof, formic acid and salts thereof and esters thereof; maleic acid and salts thereof, and esters thereof, succinic acid and salts thereof, and esters thereof, pyruvic acid and salts thereof, and esters thereof, acetoacetic acid and salts thereof, and esters thereof, levulinic acid and salts thereof, and esters thereof, oxalacetic acid and salts thereof, and esters thereof, citric acid and salts thereof, and esters thereof, fruit acids, malic acid and salts thereof and esters thereof, citric acid and salts thereof and esters thereof, fumaric acid and salts thereof and esters thereof, gluconic acid and salts thereof and esters thereof, glycolic acid and salts thereof and esters thereof, mandelic acid and salts thereof and esters thereof, oxalic acid and salts thereof and esters thereof, salicylic acid and salts thereof and esters thereof, α-hydroxycaprylic acid and salts thereof and esters thereof, and tartaric acid and salts thereof and esters thereof; peptides; amino acids, and salts thereof and esters thereof; vegetable and animal complex substrates; industrial residual substance streams; and anaerobic substrates.
8. The method of claim 1, wherein the biocement mixture is a liquid a gel, a paste, or a powder.
9. The method of claim 1, wherein (i) is selected from microorganisms, aerobic bacteria, anaerobic bacteria, facultatively anaerobic bacteria, and intermediate stages thereof.
10. The method of claim 1, wherein (i) is selected from urease, asparaginase, carbonic anhydrase, and metabolic enzymes.
11. The method of claim 1, wherein (v) is selected from: natural and chemical herbicides; fungicides, molluscicides; insecticides; hydrophobizers and wax emulsions; stabilizers, dispersants; emulsifying aids, surfactants; amines; ethanolamines; thixotropic agents; propellants; free-flow agents, crystallization seeds and crystallization modifiers; complexing agents, fatty acids; minerals and trace elements; salts; rocks sand, gravel and slate flour, rubber crumbs, rubber granules and other thermoplastic elastomers; aggregates; plant seeds, spores, plants and parts thereof; fertilizers; bacteria capable of forming polymers; and substances which modify biocementation.
12. The method of claim 1, wherein the (i) is present in and/or part of a substrate to be treated by the biocement mixture, and wherein (ii) and (iii), and optionally (iv) and/or (v), are applied separately from the (i), or wherein (i) is isolated from the substrate to be treated by the biocement mixture, cultured ex situ and then re-introduced onto and/or into the substrate to be treated either in combination with (ii) and (iii), and optionally (iv) and/or (v), or separately from (ii) and (iii), and optionally (iv) and/or (v).
13. The method of claim 1 for reducing dust formation and/or erosion comprising: (a) identifying a substrate to be treated, on/in which dust formation and/or erosion is to be reduced, (b) providing the biocement mixture, or constituents thereof, (c) applying the biocement mixture, or constituents thereof, to/into the substrate to be treated, in an amount sufficient to enable biocementation, and (d) allowing a biocement layer to form, thereby reducing dust formation and/or erosion on/in the substrate.
14. The method of claim 13, wherein the substrate is selected from organic and inorganic material and in each case derivatives and mixtures thereof, and also combinations thereof.
15. The method of claim 13, wherein (c) is carried out once or repeatedly and an amount of (iii) applied overall is at least 20 g, based on one square metre of application area, and/or an amount of the (iii) applied overall is at most 2000 g, based on one square metre of application area.
16. The method of claim 1, wherein (iii) is selected from: albumin; starch ether; alanine; lysine; styrene-acrylate; ethylene-vinyl acetate; polyvinyl alcohol; magnesium sulfate; polyvinyl acetate; styrene-butadiene; humic acid and combinations thereof, and also polymers containing monomers of the aforesaid polymers.
17. The method of claim 16, wherein wherein constituent (ii) is selected from: urea and its salts; organic acids; gluconic acid and salts thereof and esters thereof; acetic acid and salts thereof and esters thereof; formic acid and salts thereof; propanoic acid and salts thereof and esters thereof; butanoic acid and salts thereof and esters thereof; pentanoic acid and salts thereof and esters thereof; peptides; amino acids and salts thereof and esteres thereof; vegetable and animal complex substrates; industrial residual substrate streams; and anaerobic substrates.
Description
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EXAMPLE 1: ACCELERATED BIOCEMENTATION WITH IMPROVED DUST SUPPRESSION
Material and Methods:
[0197] The experiment was carried out in the laboratory in plastic vessels with a volume of 450 cm.sup.3. The application area was in each case 78.5 cm.sup.2.
[0198] The soil substrate in the experiment consisted of a silica sand having a grading of 0-2 mm. The sand had been both washed and dried by the manufacturer, and was used directly. 800 g of silica sand per plastic vessel were used as soil substrate. The plastic vessel was full to the brim.
[0199] For a control, reference mixtures were utilized, consisting of the following constituents in the following concentrations: [0200] Reference 1 (R1): Dry sand substrate without addition of aqueous component. [0201] Reference 2 (R2): Application of water. [0202] Reference 3 (R3):
TABLE-US-00002 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0203] Reference 4 (R4):
TABLE-US-00003 6.25 g/L calcium lignosulfonate [0204] Reference 5 (R5)
TABLE-US-00004 3.15 g/L calcium lignosulfonate
[0205] The biocementing reference system R3 is used for dust suppression in a form modified according to Stabnikov, V., et al. Water, Air, & Soil Pollution (2013) 224:1631. The dust-suppression tendency was studied in this publication with wind speeds of 0.39 m/s and lower. The wind speeds studied in the present example are substantially higher. The total amount of biocementing mixture delivered in the present example is greater by a factor of four. Exact replication of the literature reference produced no significant changes relative to R3.
[0206] The mixture R3 further includes trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source.
[0207] The reference mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application took place using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0208] Liquid biocementation mixtures were utilized, consisting of the following constituents in the following concentrations: [0209] Mixture 1 (M1):
TABLE-US-00005 48 g/L urea 44 g/L calcium chloride 6.25 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0210] Mixture 2 (M2):
TABLE-US-00006 48 g/L urea 44 g/L calcium chloride 3.15 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii
[0211] The mixture further included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source. Calcium lignosulfonate is the cohesion-modifying compound in the mixtures M1 and M2.
[0212] The mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application took place using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0213] All components of the present mixtures which are capable of biocementation, except for the bacteria of the strain S. pasteurii, were in solid form. The bacteria were present as a liquid culture in a culturing medium known from the prior art, as described for example in Cuthbert, M. O. et al., Ecological Engineering 2012, 41, 32-40 (see section 2.2, page 33), with 5 g/L yeast extract being used in the context of the present invention. The solid constituents and the bacteria in liquid culture were mixed directly prior to use, with the solid constituents dissolving.
[0214] Application of the reference mixtures and of the biocementation mixtures was followed by incubation over the entire observation period (in general 28 days) at an atmospheric humidity of 20% to 60% and with multiple air change per day. In this period the minimum temperature prevailing was 14.2° C. and the maximum temperature prevailing was 25.2° C.
[0215] Determined after 24 hours was the penetration depth of immersion cones of different weights (150 g, 300 g and 600 g) and also, subsequently, the dust-suppressing effect in a wind tunnel. In accordance with the test standard method DIN EN 13279-2:2014-03 (section 4.4.2.2), the penetration depth of cones with different weights (total weight of immersion cone and guide rod 150 g, 300 g, 600 g) was ascertained after 24 hours, using a described Vicat instrument with immersion cone and release device (described in DIN EN 13279-2:2014-03, pictures 2 and 3). For this purpose the sample was placed beneath the immersion rod. The immersion rod was carefully lowered until it contacted the surface of the sample. It was held for two seconds and the release device was actuated. Under the action of its own weight, the immersion cone penetrated the sample vertically. The penetration depth was read off on the scale five seconds after standstill of the cone. Sampling took place at three test sites, which were at least 3 cm distant from one another. From the three values ascertained, a mean value was formed. The measurements fluctuated by not more than 10% around the absolute value. This measurement provides information on the stiffening profile (cf. DIN EN 196-3, section 6.3.1). After the measurement, the mass of the test specimen was determined (sample mass before wind exposure) and the test specimen was placed in a wind tunnel. The mechanically stressed sample was exposed for one minute to a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. After the wind exposure, the reduced mass was determined (sample mass after wind exposure), and the emission-related weight loss was determined according to the formula indicated below. The weight of the sample vessel itself was subtracted in each case.
[0216] The dust suppression effect in a wind tunnel without prior mechanical stressing was carried out with a separate sample: the mass of the hardened sample (sample mass before wind exposure) was determined, and the sample was placed in a wind tunnel. In the wind tunnel, a flow of air was passed over the sample for one minute with a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. A measurement of the reduced sample weight was made after wind exposure (sample mass after wind exposure), and the emission-related weight loss was determined using the formula indicated below. The weight of the sample vessel itself was subtracted in each case.
[0217] The emission-related weight loss in weight percent was determined as follows:
Emission-related weight loss=[(sample mass before wind exposure.sub.day xy−(sample mass after wind exposure.sub.day xy)/sample mass before wind exposure.sub.day xy]*100
[0218] After 48 hours, the breaking strength of the layers was determined. The breaking strength (maximum value of the force measurement) can be determined by means of the following method: the method is based on the standardized test method for strength determination in cement, DIN EN 196-1:2005-05. The breaking strength is measured using a digital (breaking) strength measuring instrument in accordance with the manufacturer's instructions. A test specimen is pressed into the sample (to the point of breakage) using a crank-operation test bed, and the force applied is measured continuously. From a number of measurements (>3) the mean breaking strength is computed. The mean breaking strength is preferably between 0.5 and 1000 N, more preferably between 1 and 300 N.
[0219] Following the determination of the breaking strength, the mechanically stressed sample was placed in the wind tunnel and exposed for one minute to a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. The emission-related weight loss is determined using the formula stated above. This test serves as a reference for the long-term stability of the samples and also their dust suppression.
Results:
[0220] Where the silica sand in the dry state (R1) was subjected in the wind tunnel to a wind speed of 12 m/s for one minute, more than 50% of the weight was carried off in the form of dust. In the wet state, with the same wind speed and exposure time, the sand lost a further 1.12 weight percent of its own weight in the form of dust (R2). Under the conditions given, the sand dries out completely after 4.5 days. In this case the dust-suppressing effect dropped successively (data not shown). In the samples completely dried, the percentage emission-related weight loss after one minute of wind exposure was more than 50%.
[0221] The inventive formulations M1 and M2 showed a quicker stiffening profile than the reference systems R1, R2, R3, R4 and R5. After 24 h, a cone 150 g in weight penetrated 9 to 25 mm into the reference systems, whereas the cone with a weight of 150 g penetrated 2 and 6 mm into inventive formulations, respectively (
[0222] After 24 hours, the emission-related weight loss after one minute of wind exposure in the wind tunnel (12 m/s) without prior mechanical stressing was more than 50% for R1, 1.11% for R2, 0.41% for R3, 0.66% for R4 and 0.99% for R5. The mixture M1 gave an emission-related weight loss of only 0.03%, and the mixture M2 0.04% (
[0223] After 24 hours, the emission-related weight loss after one minute of wind exposure in the wind tunnel (12 m/s) with prior mechanical stressing (verification of the penetration depth of the 600 g cone) was more than 50% for R1, 1.12% for R2, 0.71% for R3, 1.58% for R4 and 2.78% for R5. The mixture M1 gave an emission-related weight loss of 0.16%, and the mixture M2 gave an emission-related weight loss of 0.18% (
[0224] After 48 hours reaction time, the inventive mixtures M1 and M2 showed a higher breaking strength than the associated reference systems. In this case breaking strength of the inventive mixtures was above the sum total of the individual constituents: breaking strength of R3=1.5 N, breaking strength of R5=2.2 N, breaking strength of M2=7 N. Breaking strength of R3=1.5 N, breaking strength of R4=5.1 N, breaking strength of M1=12 N (
[0225] With the increasing age of the samples, the difference in the dust-suppressing effect after prior mechanical verification became even more apparent: after 48 hours, the emission-related weight loss after one minute of wind exposure in a wind tunnel (12 m/s) with prior mechanical stressing (determination of the breaking strength) was 1.30% for R2, 0.85% for R3, 40.1% for R4 and 42.9% for R5. The mixtures M1 and M2 showed a significantly lower emission-related weight loss of 0.40% for M1 and 0.43% for M2. After even longer reaction time (10 and 28 days, respectively), the same tendencies were apparent (data not shown; in this regard see also Ex. 2).
[0226] The biocementation mixture advantageously has a similar effectiveness as many standard commercial dust suppression compositions (data not shown), with the above-described increased dust suppression after mechanical loading being comparable with that of bitumen-based systems, without the various environmental disadvantages.
[0227] Additionally, in the above-described biocementation mixtures R3, M1 and M2, the bacterial strain S. pasteurii was replaced by the same cell-count concentration of L. sphaericus in each case, with the experiments being carried out in each case as described above. The resultant liquid reference and biocementation mixtures consisted of the following constituents: [0228] Reference 6 (R6):
TABLE-US-00007 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus [0229] Mixture 3 (M3):
TABLE-US-00008 48 g/L urea 44 g/L calcium chloride 6.25 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus [0230] Mixture 4 (M4):
TABLE-US-00009 48 g/L urea 44 g/L calcium chloride 3.15 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus
[0231] The mixture additionally contained trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source. The bacteria were present as a liquid culture in a culturing medium known from the prior art, as described for example in Dick, J. et al., Biodegradation 2006, 17, 357-367 (see “Materials and Methods” section, page 359), with 5 g/L yeast extract being used in the context of the present invention. Calcium lignosulfonate is the cohesion-modifying compound in the mixtures M3 and M4.
[0232] Using the organism L. sphaericus in the inventive mixtures achieved comparable results to those using S. pasteurii (cf.
[0233] Comparable effects on emission reduction were also achieved with slightly modified formulations of the biocementation mixtures M1, M2, M3 and M4, containing calcium acetate, calcium propionate, calcium formate, calcium lactate and/or calcium chloride at a concentration each of 0.05 to 0.3 M, not exceeding a total calcium concentration of 0.4 M (data not shown). A greater variation in the concentration of calcium lignosulfonate (e.g. 1 to 500 g/L), or of urea (e.g. 0.1 to 1.0 M) or in the amount of yeast extract (e.g. 0.1 to 30 g/L) likewise produced good emissions reduction. The dust suppression was dependent in each case on the used concentrations of the constituents of the respective biocementation mixture (data not shown). Corresponding observations were also made for correspondingly modified inventive formulations of Examples 2, 3 and 4.
[0234] Accelerating additives are used to regulate the solidification time in building materials, as for example in the case of cementitious building materials such as mortars and concretes (EP 2664596 A3). On the basis of the accelerated curing of the mixtures described in this example, a preferred use of the inventive mixtures is their use for producing building materials.
[0235] Similarly, comparable effects on emissions reduction were achieved for all of the above-stated mixtures in which the bacteria were present as powders. For this purpose, the respective bacterial cells were concentrated in the culturing medium, then expertly dried and dissolved before application in the corresponding medium.
[0236] Similarly, comparative effects on emissions reduction were achieved for all of the abovementioned mixtures in mixtures where calcium lignosulfonate was replaced by lignosulfonate acid, sodium lignosulfonate, potassium lignosulfonate or ammonium lignosulfonate, respectively, and/or where the cation source was removed (here: a calcium source such as calcium chloride).
EXAMPLE 2: ACCELERATED BIOCEMENTATION FORMULATIONS WITH IMPROVED DUST SUPPRESSION FOR NON-UREOLYTIC AND UREOLYTIC BIOCEMENTATION SYSTEMS IN COMPARISON
Material and Methods:
[0237] The experiment was carried out in the laboratory in plastic vessels with a volume of 450 cm.sup.3. The application area was in each case 78.5 cm.sup.2.
[0238] The soil substrate in the experiment consisted of a silica sand having a grading of 0-2 mm. The sand had been both washed and dried by the manufacturer, and was used directly. 800 g of silica sand per plastic vessel were used as soil substrate.
[0239] For a control, the reference mixture R3 was used, consisting of the following constituents in the following concentrations: [0240] Reference 3 (R3):
TABLE-US-00010 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0241] Reference 6 (R6):
TABLE-US-00011 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus
[0242] The mixtures R3 and R6 further included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source.
[0243] The reference mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application took place using a pipette. Following application, the surface was spread smooth with a spatula.
[0244] Liquid biocementation mixtures were utilized, consisting of the following constituents in the following concentrations: [0245] Mixture 5 (M5):
TABLE-US-00012 5 g/L yeast extract 21 g/L calcium acetate 34.9 g/L calcium chloride 46.2 g/L calcium lactate 0.40 g/L sodium hydroxide 1.07 g/L ammonium chloride 15 g/L L-alanine 25 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL B. pseudofirmus [0246] Mixture 6 (M6):
TABLE-US-00013 27 g/L urea 34 g/L calcium chloride 10 g/L yeast extract 12.5 g/L Styrene-acrylate dispersion 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus [0247] Mixture 7 (M7):
TABLE-US-00014 5 g/L yeast extract 21 g/L calcium acetate 34.9 g/L calcium chloride 46.2 g/L calcium lactate 25 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL B. halodurans [0248] Mixture 8 (M8):
TABLE-US-00015 36 g/L urea 35 g/L calcium chloride 10 g/L yeast extract 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus [0249] Mixture 9 (M9):
TABLE-US-00016 27 g/L urea 17 g/L calcium chloride 31 g/L albumin 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus [0250] Mixture 10 (M10):
TABLE-US-00017 48 g/L urea 44 g/L calcium chloride 3.9 g/L polyvinyl alcohol 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0251] Mixture 11 (M11):
TABLE-US-00018 48 g/L urea 44 g/L calcium chloride 3.9 g/L polyvinyl acetate dispersion 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0252] Mixture 12 (M12):
TABLE-US-00019 48 g/L urea 44 g/L calcium chloride 9.4 g/L starch ether 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0253] Mixture 13 (M13):
TABLE-US-00020 1 g/L yeast extract 34.9 g/L calcium chloride 25 g/L calcium lignosulfonate 21 g/L calcium acetate 46.2 g/L calcium lactate 4 × 10{circumflex over ( )}8 cells/mL B. cohnii [0254] Mixture 14 (M14):
TABLE-US-00021 1 g/L yeast extract 21 g/L calcium acetate 25 g/L calcium lignosulfonate 15 g/L L-alanine 34.9 g/L calcium chloride 46.2 g/L calcium lactate 0.40 g/L sodium hydroxide 1.07 g/L ammonium chloride 4 × 10{circumflex over ( )}8 cells/mL A. crystallopoietes [0255] Mixture 15 (M15):
TABLE-US-00022 1 g/L yeast extract 34.9 g/L calcium chloride 21 g/L calcium acetate 46.2 g/L calcium lactate 4 × 10{circumflex over ( )}8 cells/mL B. cohnii [0256] Mixture 16 (M16):
TABLE-US-00023 1.07 g/L ammonium chloride 21 g/L calcium acetate 15 g/L L-alanine 34.9 g/L calcium chloride 0.40 g/L sodium hydroxide 1 g/L yeast extract 46.2 g/L calcium lactate 4 × 10{circumflex over ( )}8 cells/mL A. crystallopoietes [0257] Mixture 17 (M17):
TABLE-US-00024 36 g/L urea 36 g/L magnesium sulfate 10 g/L yeast extract 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus [0258] Mixture 18 (M18):
TABLE-US-00025 27 g/L urea 35 g/L calcium chloride 45 g/L lysine 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0259] Mixture 19 (M19):
TABLE-US-00026 48 g/L urea 44 g/L calcium chloride 25 g/L polyvinyl alcohol 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0260] Mixture 20 (M20):
TABLE-US-00027 27 g/L urea 47 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0261] Mixture 21 (M21):
TABLE-US-00028 5 g/L yeast extract 21 g/L calcium acetate 25 g/L calcium lignosulfonate 34.9 g/L calcium chloride 46.2 g/L calcium lactate 4 × 10{circumflex over ( )}8 cells/mL B. pseudofirmus [0262] Mixture 22 (M22):
TABLE-US-00029 27 g/L urea 47 g/L calcium lignosulfonate 12 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0263] Mixture 23 (M23):
TABLE-US-00030 27 g/L urea 35 g/L calcium chloride 45 g/L lysine 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus
[0264] The mixture further included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in the mixtures M6, M8, M9, M10, M11, M12, M17, M18, M19, M20, M21, M22, M23 served primarily as a carbonate source. In the mixtures M5, M7, M13, M14, M15, M16, M21, an organic calcium salt in each case served primarily as a carbonate source. In the mixture M15, moreover, yeast extract served additionally as a carbonate source. In the mixture M16, moreover, L-alanine served as a further carbonate source.
[0265] L-alanine, calcium lignosulfonate, calcium lactate (M5, M7), calcium acetate (M1, M14), styrene-acrylate dispersion, yeast extract, albumin, polyvinyl alcohol, polyvinyl acetate dispersion, starch ether, magnesium sulfate, lysine are the cohesion-modifying compounds in the present examples, if they did not serve as a carbonate source.
[0266] All of the components of the present mixture that are capable of biocementation, except for the bacteria, were in solid form. The bacteria of the strain B. pseudofirmus were present as a liquid culture in a culturing medium known from the prior 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, with 5 g/L yeast extract being used in the context of the present invention. B. cohnii and B. halodurans were present in the same culturing medium as B. pseudofirmus, and A. crystallopoietes was present in a known culturing medium, as described for example in Hamilton, R. W. et al., Journal of Bacteriology 1977, 129(2), 874-879 (see “Materials and Methods” section, pp. 874-875). L. sphaericus and S. pasteurii were present in the culturing media described in Example 1. The solid constituents and the bacteria in liquid culture were mixed directly prior to the use, with the solid constituents dissolving.
[0267] The mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application took place using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0268] Application of the reference mixtures and of the biocementation mixtures was followed by incubation over the entire observation period (28 days) at an atmospheric humidity of 20% to 60% and with multiple air change per day. In this period the minimum temperature prevailing was 14.2° C. and the maximum temperature prevailing was 25.2° C.
[0269] After 24 hours the penetration depth of immersion cones of different weights (150 g, 300 g and 600 g) and also, subsequently, the dust-suppressing effect in a wind tunnel, were determined as described in Example 1. The dust-suppressing effect in the wind tunnel without prior mechanical stressing was carried out with a separate sample: the cured sample was exposed for one minute to a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. The emission-related weight loss in weight percent was ascertained as described in Example 1.
[0270] After 48 hours, the breaking strength of the layers was determined. The breaking strength (maximum value of the force measurement) can be determined by means of the following method: the method is based on the standardized test method for strength determination in cement, DIN EN 196-1:2005-05. The breaking strength is measured using a digital (breaking) strength measuring instrument in accordance with the manufacturers instructions. A test specimen is pressed into the sample (to the point of breakage) using a crank-operation test bed, and the force applied is measured continuously. From a number of measurements (>3) the mean breaking strength is computed. The mean breaking strength is preferably between 0.5 and 1000 N, more preferably between 1 and 300 N.
[0271] Following the determination of the breaking strength, the mechanically stressed sample was placed in the wind tunnel and exposed for one minute to a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. The emission-related weight loss is determined using the formula stated in Example 1. This test serves as a reference for the long-term stability of the samples and also their dust suppression.
[0272] On selected samples, after a reaction time of 10 days and 28 days, respectively, determinations were made of the breaking strength and also the loss of mass on wind exposure, as described above.
Results:
[0273] In the previous Example 1, the reduction in the emission-related weight loss by means of accelerated biocementation formulations was described. This example sets out how this finding can be extended to a broad group of soil-consolidating substances which accelerate the biocementation.
[0274] All of the inventive formulations described above exhibited a quicker stiffening profile than the reference systems R3 and R6. After 24 h, a cone with a weight of 150 g penetrated 14 mm into the reference systems R3 and R6, whereas the cone weighing 150 g penetrated 4 to 9.5 mm into inventive formulations (
[0275] If the weight loss in the wind tunnel is determined after the mechanical verification, then the inventive mixtures exhibit an increased cohesiveness and hence an increased dust suppression. The weight loss after 24 hours reaction time, determination of the penetration depth and one minute of wind exposure in the wind tunnel (12 m/s) is shown at the top right in
[0276] After 48 hours reaction time, the inventive mixtures showed a higher breaking strength than the associated reference systems. In this case the breaking strength of the inventive mixtures was a multiple of that of the reference system R3 (
[0277] With the increasing age of the samples, the difference in the dust-suppressing effect after previous mechanical verification was even more sharply apparent: after 48 hours, the loss of weight after one minute of wind exposure in the wind tunnel (12 m/s) with prior mechanical stressing (determination of the breaking strength) was 1.30% for R2 and 0.85% for R3.
[0278] The inventive mixtures M15 to M23 showed a loss of mass after mechanical verification and wind exposure of 0.04% to 0.45% (cf.
[0279] If the study of the fracture-mechanical properties and of the emission-related weight loss in the wind tunnel was carried out after a long reaction time, the difference between the reference systems and the biocementing mixtures became even more clearly apparent:
[0280] Furthermore, the reference mixture R7 and also biocementation mixture M24 were produced, and were compared with one another as described above. [0281] Reference 7 (R7):
TABLE-US-00031 50 g/L calcium lignosulfonate [0282] Mixture 24 (M24):
TABLE-US-00032 48 g/L urea 44 g/L calcium chloride 50 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii
[0283] In the mixture M24 there were additionally trace elements and traces of, for example, salts and sugars (<1 wt %). Calcium lignosulfonate is the cohesion-modifying compound in mixture M24. The bacteria of the strain S. pasteurii were present in the culturing medium described in Example 1. The mixtures were prepared and stored as described previously. These mixtures were found to consolidate within 48 hours (breaking strength not shown; in this regard, see also Example 5). With R7 a thin layer was formed, whereas for M24, a thicker, more cohesive layer was formed. These differences in the nature of the layer were reflected in a difference in emission-related weight loss. The mixtures R7 and M24 were tested after determination of the breaking strength, in a wind tunnel for one minute at 12 m/s of wind (as described above). The emission-related weight loss here was 11.3% for R7, and the emission-related weight loss of M24 was 0.21%. Selected mixtures and reference systems were allowed to react over a period of 48 hours. The results suggest to the skilled person that an increased cohesiveness produces advantages for long-lasting dust suppression.
[0284] After 10 and 28 days, the breaking strengths of the various agents were determined in comparison to the reference systems. The results achieved in this case were comparable to those described above (data not shown). The loss of mass after mechanical verification after 10 days is represented in Table 1. Here it is found that the more cohesive biocement layers had a significantly better dust suppression after mechanical testing.
TABLE-US-00033 TABLE 1 Loss of mass after 10 days reaction time, mechanical testing and one minute of wind exposure at 12 m/s wind speed for various reference mixtures and also biocementation mixtures Loss of mass after mechanical testing and one minute of wind exposure at 12 m/s Mixture or reference system wind speed, [weight percent] R3 >50 R7 50.1 M5 0.67 M6 1.56 M7 0.26 M8 0.14 M9 0.16 M10 0.96 M11 4.82 M12 0.15 M13 0.67 M14 1.82 M16 0.77 M17 6.31 M18 2.52 M19 0.04 M20 8.1 M21 0.63 M22 0.26 M23 0.11 M24 0.06
[0285] Comparable effects on emission reduction were also achieved with slightly modified formulations of the biocementation mixtures M5 to M24, containing calcium acetate, calcium propionate, calcium formate, calcium lactate and/or calcium chloride at a concentration of in each case 0.05 to 0.4 M and not exceeding a total calcium concentration of 1 M (data not shown). A greater variation in the calcium lignosulfonate concentration (e.g. 1 to 500 g/L), L-alanine concentration (e.g. 1 to 250 g/L), styrene-acrylate dispersion concentration (e.g. 1 to 350 g/L), polyvinyl alcohol concentration (e.g. 1 to 250 g/L), polyvinyl acetate dispersion concentration (e.g. 1 to 350 g/L), albumin concentration (1 to 200 g/L), starch ether concentration (e.g. 1 to 90 g/L), magnesium sulfate concentration (e.g. 1 to 300 g/L), lysine concentration (e.g. 1 to 250 g/L), urea concentration (e.g. 0.1 to 1.0 M) or in the amount of yeast extract (e.g. 0.1 to 150 g/L) likewise produced good emission reduction. The dust suppression was dependent in each case on the used concentrations of the constituents of the respective biocementation mixture (data not shown). Corresponding observations were also made for correspondingly modified inventive formulations of Examples 3, 4 and 5.
[0286] Comparable effects on emission reduction were also achieved with the biocementation mixtures M5 to M24, in which the bacteria were present as spray-dried and/or freeze-dried powder. For this purpose, the respective bacteria cells were concentrated in the culturing medium, then expertly dried and dissolved in the corresponding medium prior to application. It was found that when using dried bacteria cells, it was in fact possible to achieve a further slight reduction in the emission-related weight loss (data not shown).
[0287] Similarly, comparable effects of all the abovementioned mixtures were achieved in mixtures where calcium lignosulfonate was replaced by lignosulfonic acid, sodium lignosulfonate, potassium lignosulfonate and ammonium lignosulfonate, respectively. Furthermore, on removal of the cation source (here: calcium source) in the mixtures M5, M6, M7, M8, M9, M10, M11, M12, M13, M14, M18, M19, M21, M22 and M23, a comparable dust suppression effect was achieved. Where at the same time calcium lignosulfonate was replaced by lignosulfonic acid, sodium lignosulfonate, potassium lignosulfonate and ammonium lignosulfonate, respectively, and the cation source (here: calcium source) was removed, a comparable effect was again achieved.
EXAMPLE 3: ANALYSIS OF SELECTED MIXTURES AND ALSO REFERENCE SYSTEMS IN A WIND TUNNEL AT AN EXTERNAL TESTING LABORATORY
Material and Methods:
[0288] In an external testing laboratory, the emission-reducing effect of the reference systems R1 (dry) and R2 (water application) and also of a dust-suppressing agent R8 available commercially on the market was tested in comparison to the mixture M20.
[0289] The soil substrate used was a fine calcium carbonate with the designation ESKAL 60. This fine-particle dust is used as a test dust for various analyses in wind tunnel analysis among others. ESKAL 60 possesses a precisely defined particle distribution. The mean grain size is 60 μm. The skilled person is aware that the test dust used must be appropriate to the wind tunnel used. Plastic dishes (diameter 87 mm, height 16 mm) were filled to the brim with the soil substrate, and the precise weight of the respective vessels was ascertained.
[0290] All of the samples were then provided with the respective surface treatment agent. The treated samples were labelled in the manner of a blind test in such a way that assignment to the respective surface treatment agents was not possible.
Reference Mixture 8 (R8): 50 g/L Polymer Dispersion (Various)
[0291] The product available commercially on the market is a crust-forming agent. It was used according to manufacturer specifications and applied at 1.5 L/m.sup.2. Furthermore, the emission-reducing effect of biocementation mixture M20 was studied. Mixture 20 was applied at comparable application rates, measured in mass of solid per unit surface area, to R8.
[0292] All of the samples, apart from R2, were equilibrated under defined ambient conditions (31% relative humidity, 23° C.) in a conditioning cabinet for 24 hours and then weighed again. The samples of the reference R2 were not applied until immediately before exposure in the wind tunnel. The wetting application (R2) was treated with deionized water from a spray bottle positioned consistently, immediately before the beginning of experimentation. The mass input of water was recorded.
[0293] At the start of experimentation, the samples were positioned, in randomized order, individually and with covering, in the middle of the wind tunnel (D=0.15 m, L=5.4 m). With the beginning of experimentation, the particle counter was activated, the covering on the sample material was removed, and the wind tunnel was sealed. All of the samples were exposed each individually for 15 minutes to a flow over the sample with a mean aerosol speed of 6 m/s, measured at the height of the sample, with a determination of particle size distribution every 30 seconds. All of the experiments were repeated three times. The emission-related loss of mass was determined using the formula specified in Example 1. The measurement values reported are mean values of the three replications, and were typically in the region of 10% of the value ascertained.
Results:
[0294] The experiments show that the surface treatment agent M20 has reliably prevented dust being carried off. Emissions occurred only with the agent R8 available commercially on the market and also with the untreated samples (R1) and with the water-treated samples (R2).
[0295] In the case of the agent R8 available commercially on the market, this behaviour is manifested by the emergence of up to 180 captured particles in the first 90 seconds and by a mean loss of mass of 1.86%.
[0296] The untreated calcium carbonate samples (R1) serving for comparison had the greatest level of particles being carried off among all of the samples under review. Beginning at 2500 to 3800 particles/30 seconds, the emissions rose to 4100 to 5500 particles/30 seconds, before dropping steadily to a level of around 100 particles/30 seconds. The emission-related mean loss of mass was 74.55%.
[0297] In the case of the water-treated samples, particle release was delayed; here, particle release began only after around 200 seconds. The emission-related mean loss of mass is 66.94%. The delayed release is probably due to the evaporation of the water in the wind tunnel.
[0298] In the case of biocementation mixture M20, there was no detectable particle release, and the emission-related loss of mass was 0.003% (cf.
EXAMPLE 4: OPEN-AIR DEMONSTRATION OF THE EMISSION-REDUCING EFFECT IN A LIMESTONE MINE
Material and Methods:
[0299] In order to control the suppression of dust under open-air conditions, a biocementation mixture M20 was applied in comparison to the reference mixture R3 (as control) at three sites in a limestone quarry, illustratively. The three sites within the mine were located on a road (site 1 in
[0300] Following application, all 9 areas where application had taken place, as illustrated at the bottom of
[0301] The open-air experiment was rated for 4 weeks. The temperature during this period varied between 5.3° C. at night and 26.3° C. in the day. The relative humidity varied between 64% at night and 31% in the day. Within the experimentation period, the total amount of precipitation was 11 L/m.sup.2.
[0302] The dust suppression effect was measured at different times, after 48 h, 7 days and 28 days. After 48 hours, the dust suppression effect was verified at a number of points using a Bosch leaf blower (GBL 18V-120). The wind speeds used here were 40 m/s from a distance of one metre from the surface, and an incident angle of around 15° was used. The inspection, carried out by three mine employees, took place in the form of the classifications of “severe dusting”, “moderate dusting” and “no dusting”. All of the employees are skilled in the field of area dust suppression in mining, each having more than 10 years of relevant professional experience. “No dusting” was used when no visible particles were removed. “Severe dusting” was used when the test area formed dust in the same way as an untreated area. “Moderate dusting” was used when the dust formation was reduced in comparison to the untreated area. The expertly obtained data was additionally verified by particle analyses (data not shown).
[0303] After the first testing (48 h), the areas under study was again released for operation and the barriers were removed. At this point, care was taken to ensure that all of the areas were equally exposed. The visual inspection of the areas and also the measurement of the dust suppression effect were carried out for all of the areas after 7 days and 28 days as well as after 48 hours.
Results
[0304] After one day, the layer of the accelerated biocementation formulation M20 was perceptible, whereas that of the reference mixture 3 had not consolidated. After a reaction time of 48 hours, it was possible to reproduce the relative breaking strengths of the layers as described in Example 2 (data not shown).
[0305] The testing of the dust suppression effect using the Bosch (GBL 18V-120) led to the following rating by the experts after 48 hours: [0306] Mixture 20 (M20)—“no dusting” [0307] Reference 3 (R3)—“moderate dusting” Reference 2 (R2)—“moderate dusting”. [0308] In this case there was no difference between the dust suppression effect at each site of application.
[0309] Seven days after application, the three application sites of road, dump and pit were inspected according to the scheme described above. On the dump and in the pit, it was apparent that in the case of the mixture 20 (M20) there was still a firm layer apparent, whereas there was no layer formed in the case of the reference mixtures R2 and R3. The dust suppression tendency was rated as follows: [0310] Mixture 20 (M20)—“no dusting” [0311] Reference 3 (R3)—“moderate dusting” [0312] Reference 2 (R2)—“severe dusting”.
[0313] On the road, the effect was even more clearly apparent. This is due to the effect of the invention whereby the inventive biocementation mixture M20 has a high mechanical strength. On the road, the following rating was undertaken: [0314] Mixture 20 (M20)—“no dusting” [0315] Reference 3 (R3)—“severe dusting” [0316] Reference 2 (R2)—“severe dusting”.
[0317] Results comparable to those after 7 days were achieved after 28 days. After this time, the experiment was discontinued.
[0318] Similarly, comparable effects on emission reduction for all of the above-stated mixtures were obtained in mixtures wherein the bacteria were present as powders. For this purpose, the respective bacterial cells were concentrated in the culturing medium, then expertly dried and dissolved before use in the corresponding medium.
[0319] This example impressively shows that the inventive formulations, on account of their more rapid consolidation and higher strength, exhibit an improved dust suppression effect under mechanical loading. Moreover, crusts generated with inventive formulations can be maintained over a longer period by comparison with existing systems.
EXAMPLE 5: SYNERGISTIC EFFECT OF BIOCEMENTATION FORMULATIONS WITH COHESION-MODIFYING COMPOUNDS
Material and Methods:
[0320] The experiment was carried out in the laboratory in plastic vessels with a volume of 450 cm.sup.3. The application area was in each case 78.5 cm.sup.2.
[0321] The soil substrate in the experiment consisted of a silica sand having a grading of 0-2 mm. The sand had been both washed and dried by the manufacturer, and was used directly. 800 g of silica sand per plastic vessel were used as soil substrate. The plastic vessel was full to the brim.
[0322] For a control, reference mixtures were utilized, consisting of the following constituents in the following concentrations: [0323] Reference 3 (R3):
TABLE-US-00034 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0324] Reference 7 (R7):
TABLE-US-00035 50 g/L calcium lignosulfonate [0325] Reference 9 (R9)
TABLE-US-00036 25 g/L polyvinyl alcohol [0326] Reference 10 (R10)
TABLE-US-00037 15.6 g/L polyvinyl alcohol [0327] Reference 11 (R11):
TABLE-US-00038 9.4 g/L starch ether [0328] Reference 12 (R12):
TABLE-US-00039 50 g/L humic acid [0329] Reference 13 (R13):
TABLE-US-00040 50 g/L sodium silicate [0330] Reference 14 (R14):
TABLE-US-00041 25 g/L styrene-butadiene dispersion
[0331] The mixture R3 further included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source.
[0332] All components of the present mixtures which are capable of biocementation, except for the styrene-butadiene dispersion, humic acid, and also the bacteria of the strain S. pasteurii, were in solid form. The bacteria were present as a liquid culture in a culturing medium known from the prior art, as described for example in Cuthbert, M. O. et al., Ecological Engineering 2012, 41, 32-40 (see section 2.2, page 33), with 5 g/L yeast extract being used in the context of the present invention. The solid constituents and the bacteria in liquid culture were mixed directly prior to use, with the solid constituents dissolving.
[0333] The reference mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application of the fully dissolved samples was carried out using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0334] Liquid biocementation mixtures were utilized, consisting of the following constituents in the following concentrations: [0335] Mixture 12 (M12):
TABLE-US-00042 48 g/L urea 44 g/L calcium chloride 9.4 g/L starch ether 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0336] Mixture 19 (M19):
TABLE-US-00043 48 g/L urea 44 g/L calcium chloride 25 g/L polyvinyl alcohol 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0337] Mixture 24 (M24):
TABLE-US-00044 48 g/L urea 44 g/L calcium chloride 50 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0338] Mixture 25 (M25):
TABLE-US-00045 48 g/L urea 44 g/L calcium chloride 15.6 g/L polyvinyl alcohol 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0339] Mixture 26 (M26):
TABLE-US-00046 48 g/L urea 44 g/L calcium chloride 50 g/L humic acid 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0340] Mixture 27 (M27):
TABLE-US-00047 48 g/L urea 44 g/L calcium chloride 50 g/L sodium silicate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0341] Mixture 28 (M28):
TABLE-US-00048 48 g/L urea 44 g/L calcium chloride 25 g/L styrene-butadiene dispersion 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii
[0342] The mixture M12, M19, M24, M25, M26, M27 and M28 further included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source.
[0343] Starch ether, polyvinyl alcohol, calcium lignosulfonate, humic acid (in each case as polymer), sodium silicate and styrene-butadiene dispersion are the cohesion-modifying compound in the mixtures M12, M19, M24, M25, M26, M27 and M28. Urea served in the mixtures M12, M19, M24, M25, M26, M27 and M28 as a carbonate source.
[0344] All components of the present mixtures which are capable of biocementation, except for the styrene-butadiene dispersion, humic acid, and also the bacteria of the strain S. pasteurii, were in solid form. The bacteria were present as a liquid culture in a culturing medium known from the prior art, as described for example in Cuthbert, M. O. et al., Ecological Engineering 2012, 41, 32-40 (see section 2.2, page 33), with 5 g/L yeast extract being used in the context of the present invention. The solid constituents and the bacteria in liquid culture were mixed directly prior to use, with the solid constituents dissolving.
[0345] The mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application of the fully dissolved samples was carried out using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0346] Following the application of the reference mixtures and also of the biocementation mixtures, incubation took place over the total observation period for 28 days at an atmospheric humidity of 20% to 60% and with multiple changes of air per day. Within this period, the minimum temperature prevailing was 14.2° C. and the maximum temperature prevailing was 25.2° C.
[0347] After one, two, three, four, ten and 28 days, the breaking strength and the emission-related weight loss were conducted as described in Examples 1 and 2. Moreover, the layer thickness was measured:
[0348] After one, two, three, four, ten and 28 days, the breaking strength of the layers was determined. The breaking strength (maximum value of the force measurement) can be determined by means of the following method: the method is based on the standardized test method for strength determination in cement, DIN EN 196-1:2005-05. The breaking strength is measured using a digital (breaking) strength measuring instrument in accordance with the manufacturers instructions. A test specimen is pressed into the sample (to the point of breakage) using a crank-operation test bed, and the force applied is measured continuously. From a number of measurements (>3) the mean breaking strength is computed. The mean breaking strength is preferably between 0.5 and 1000 N, more preferably between 1 and 300 N.
[0349] Following the determination of the breaking strength, the layer thickness of the layer formed was determined. For this purpose, a manual measurement was carried out after mechanical breakage of the layer, by means of a calliper. The layer thickness was determined at six points on the broken layer; the deviation of the individual measurements was 1 mm. The layer thickness was documented as the arithmetic mean of the individual measurements.
[0350] Following the determination of the layer thickness, the mechanically stressed sample was placed in the wind tunnel and exposed for one minute to a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. The emission-related weight loss is determined using the formula stated in Example 1. This test serves as a reference for the long-term stability of the samples and also their dust suppression.
Results:
[0351] In the previous Examples 1 to 4, formulations were described which exhibited a more rapid stiffening profile and a reduced emission-related weight loss. In the course of the analysis it emerged unexpectedly that for inventive mixtures there is not necessarily a correlation between breaking strength and emission reduction. A correlation would really have been expected, and has also been observed for agents described in the prior art, with reference to the reference mixtures R9 and R10 (
[0352] In the case of inventive mixtures, after two days there was no observable direct correlation between high breaking strength and low emission-related weight loss (cf. Example 2). M24, for example, had a breaking strength of 14 N after four days, whereas the associated reference system R7 had a breaking strength of 26.5 N. R7, however, showed a significantly higher emission-related weight loss of 53%. There was almost no consolidation of the reference system 3 in this time (breaking strength R3=1.5 N), and it exhibited an emission-related weight loss, after determination of the breaking strength and wind exposure, of 51%. The combination of the two systems (mixture 24) produces an emission-reducing system in which the emission-related weight loss was only 0.87%. The breaking strength of this system was M24=14 N. In the context of the prior art, there was no expectation that this less break-strong mixture would have a significantly higher dust suppression. This is attributed to the synergistic effect between biocementation and the cohesion-modifying substances: M24, M19, M25 and M12 exhibited a significantly lower emission-related weight loss than their individual components R3 and R7, R3 and R9, R3 and R10, and R3 and R11 (
[0353] Layer thickness determination of the layer formed produced the following values: the layer thickness of R7 was 8 mm, whereas M24 had a layer thickness of 14 mm.
[0354] Similar observations were also made when using the cohesion-modifying compounds starch ether (R11, M12), humic acid (R12, M26), sodium silicate (R13, M27) and styrene-butadiene dispersion (R14, M28). In said mixtures the breaking strength of the respective biocementation mixture is less than that of the respective reference, but the emission reduction is greater (cf.
[0355] Comparable effects on emission reduction of the mixtures M12, M19, M24, M25, M26, M27 and M28 were also in the case of slightly modified formulations of the biocementation mixtures, that contained calcium acetate, calcium propionate, calcium formate, calcium pyruvate, calcium salicylate, calcium citrate and/or calcium chloride in a concentration of in each case 0.05 to 0.4 M and that did not exceed a total calcium concentration of 1 M (data not shown). A greater variation in the calcium lignosulfonate concentration (e.g. 1 to 500 g/L), polyvinyl alcohol concentration (e.g. 1 to 250 g/L), starch ether concentration (e.g. 1 to 90 g/L), humic acid concentration (e.g. 1 to 350 g/L), potassium and sodium silicate concentration (e.g. 1 to 450 g/L), polyvinyl alcohol, urea concentration (e.g. 0.1 to 1.0 M) or in the amount of yeast extract (e.g. 0.1 to 30 g/L) likewise produced effective emission reduction. The dust suppression was dependent in each case on the used concentrations of the constituents of the respective biocementation mixture (data not shown). Comparable effects were also achieved when the bacterial strain was replaced by L. sphaericus, B. cohnii, B. halodurans, B. pseudofirmus and A. crystallopoietes in the same cell count per millilitre (data not shown). When B. cohnii, B. halodurans, B. pseudofirmus and A. crystalllopoietes were used analogously in the same cell count per millilitre in the formulations, the basic constituents were further adapted to the requirements of the particular bacterial strain. The skilled person is aware here that with these non-ureolytically biocementing bacterial strains, the base medium must be adapted in analogy to the constituents listed in Example 2, especially in terms of a suitable metabolic starting material. The effect on emissions reduction of the mixtures was comparable to the results set out for S. pasteurii (data not shown).
[0356] Similarly, comparable effects were achieved on emission reduction for all of the above-stated mixtures in mixtures in which the bacteria were present as powders. For this purpose, the respective bacterial cells were concentrated in the culturing medium, then expertly dried and dissolved in the corresponding medium prior to use.
[0357] Similarly, comparable effects on emission reduction as for the mixture M24 were achieved in mixtures in which calcium lignosulfonate was replaced by lignosulfonic acid, sodium lignosulfonate, potassium lignosulfonate and ammonium lignosulfonate, respectively. The removal of the cation source (here: calcium source such as calcium chloride, for example) in the mixtures M12, M19, M24, M25, M26, M27 and M28 also achieved comparable results in dust suppression. Where there was both replacement of the lignin derivate (as described above, by lignosulfonic acid, for example) and removal of the cation source (here: calcium source), this also led to comparable results in dust suppression.
[0358] On the basis of the present results, it is a plausible assumption that the cation source, especially a calcium source, is optional when using cohesion-modifying compounds as disclosed here.
EXAMPLE 6: DETERMINATION OF THE MINIMUM REQUIREMENTS FOR COHESION-MODIFYING COMPOUNDS FOR SUITABILITY FOR REDUCING EMISSION-RELATED BIOCEMENT WEIGHT LOSS AND EXTENDING BIOCEMENT INTEGRITY
Material and Methods:
[0359] The experiment was carried out in the laboratory in plastic vessels with a volume of 450 cm.sup.3. The application area was in each case 78.5 cm.sup.2.
[0360] The soil substrate in the experiment consisted of a silica sand having a grading of 0-2 mm. The sand had been both washed and dried by the manufacturer, and was used directly. 800 g of silica sand per plastic vessel were used as soil substrate. The plastic vessel was full to the brim.
[0361] For a control, reference mixtures were utilized, consisting of the following constituents in the following concentrations: [0362] Reference 3 (R3):
TABLE-US-00049 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0363] Reference 15 (R15):
TABLE-US-00050 50 g/L polyvinyl acetate 20 (solid, granules) [0364] Reference 16 (R16):
TABLE-US-00051 50 g/L polycarbonate (solid, granules) [0365] Reference 17 (R17):
TABLE-US-00052 50 g/L vegetable oil (rapeseed oil) [0366] Reference 18 (R18):
TABLE-US-00053 12.5 g/L long-chain fatty acid (stearic acid) [0367] Reference 19 (R19):
TABLE-US-00054 50 g/L starch, untreated (solid, powder)
[0368] The reference mixtures contained, instead of the constituent (iii), compounds which are not water-soluble or water-dispensable or water-emulsifiable.
[0369] The mixture R3 further included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in this medium served primarily as a carbonate source.
[0370] All components of the present mixtures which are capable of biocementation, except for the bacteria of the strain S. pasteurii, were in solid form. The bacteria were present as a liquid culture in a culturing medium known from the prior art, as described for example in Cuthbert, M. O. et al., Ecological Engineering 2012, 41, 32-40 (see section 2.2, page 33), with 5 g/L yeast extract being used in the context of the present invention. The solid constituents and the bacteria in liquid culture were mixed directly prior to use, with the water-soluble solid constituents dissolving. The non-water-soluble, non-water-dispersible and non-water-emulsifiable substances, respectively, were applied uniformly to the top layer of sand in advance, in order to achieve homogeneous application and to rule out any adverse effects on the dust suppression test arising from a possible non-homogeneous application.
[0371] The reference mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application of the fully dissolved samples was carried out using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0372] Liquid biocementation mixtures were utilized, consisting of the following constituents in the following concentrations: [0373] Reference 20 (R20):
TABLE-US-00055 48 g/L urea 44 g/L calcium chloride 50 g/L polyvinyl acetate 20 (solid, granules) 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0374] Reference 21 (R21):
TABLE-US-00056 48 g/L urea 44 g/L calcium chloride 50 g/L polycarbonate (solid, granules) 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0375] Reference 22 (R22):
TABLE-US-00057 48 g/L urea 44 g/L calcium chloride 50 g/L vegetable oil 4 × 10{circumflex over ( )}8 cell/mL S. pasteurii [0376] Reference 23 (R23):
TABLE-US-00058 48 g/L urea 44 g/L calcium chloride 12.5 g/L long-chain fatty acid 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0377] Reference 24 (R24):
TABLE-US-00059 48 g/L urea 44 g/L calcium chloride 50 g/L starch, untreated (solid, powder) 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii
[0378] Polyvinyl acetate 20 (solid, granules), polycarbonate (solids, granules), rapeseed oil, long-chain fatty acid and starch prove to be non-water-soluble and non-water-dispersible and non-water-emulsifiable, and therefore could not be counted among the cohesion-modifying compounds. Urea in the mixtures R20, R21, R22, R23 and R24 served as a carbonate source.
[0379] All components of the present mixtures which are capable of biocementation, except for rapeseed oil and the bacteria of the strain S. pasteurii, were in solid form. The bacteria were present as a liquid culture in a culturing medium known from the prior art, as described for example in Cuthbert, M. O. et al., Ecological Engineering 2012, 41, 32-40 (see section 2.2, page 33), with 5 g/L yeast extract being used in the context of the present invention. The solid constituents and the bacteria in liquid culture were mixed directly prior to use, with the water-soluble solid constituents dissolving. The non-water-soluble, non-water-dispersible or non-water-emulsifiable substances were applied evenly to the top layer of sand in advance.
[0380] The mixtures were applied each in three replications to the experimental areas. The amount applied per square metre was consistently 4 litres per replication. Application of the fully dissolved samples was carried out using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, which were typically in the region of 10% of the value ascertained.
[0381] Following the application of the reference mixtures and also of the biocementation mixtures, incubation took place over the total observation period for 28 days at an atmospheric humidity of 20% to 60% and with multiple changes of air per day. Within this period, the minimum temperature prevailing was 14.2° C. and the maximum temperature prevailing was 25.2° C.
[0382] After one, two, three, four, ten and 28 days, the breaking strength of the layers was determined. The breaking strength (maximum value of the force measurement) can be determined by means of the following method: the method is based on the standardized test method for strength determination in cement, DIN EN 196-1:2005-05. The breaking strength is measured using a digital (breaking) strength measuring instrument in accordance with the manufacturers instructions. A test specimen is pressed into the sample (to the point of breakage) using a crank-operation test bed, and the force applied is measured continuously. From a number of measurements (>3) the mean breaking strength is computed. The mean breaking strength is preferably between 0.5 and 1000 N, more preferably between 1 and 300 N.
[0383] Following the determination of the breaking strength, the mechanically stressed sample was placed in the wind tunnel and exposed for one minute to a wind speed of 12 m/s. The flow direction of the air struck the surface at an angle of 12.5°. The emission-related weight loss is determined using the formula stated in Example 1. This test serves as a reference for the long-term stability of the samples and also their dust suppression.
[0384] In order to determine the water solubility, water dispersibility and water emulsifiability of the substances, the procedure adopted was as follows: to determine the water solubility of solid, pasty and gelatinous substances (for example polyvinyl acetate 20, polycarbonate, long-chain fatty acid and starch), 5 g of the substance were placed in 100 mL of distilled water and stirred at 20° C. for 24 hours. This was followed by filtration (Homyl 80-120 μm quantitative filter paper). The filter paper was expertly dried and weighed. The mass ascertained, minus the filter mass, is the mass of the residue in grams (defined herein). The difference between 5 g and the mass of the residue in grams divided by 0.1 L gives the solubility of the respective substance in g per litre.
[0385] To determine the water dispersibility of solid, pasty and gelatinous substances, 50 g of the respective substance were admixed with 1000 mL of distilled water and homogenized at 20° C. in the DISPERMAT® LC75 dissolver at 15 000 revolutions per minute for 5 minutes. The mixture was subsequently transferred to a centrifuge vessel and centrifuged at 100 g for 2 min. The supernatant was decanted off and the precipitate was expertly dried and weighed. The mass ascertained is the mass of the precipitate after centrifugation (defined herein). The difference between 50 g and the mass of the precipitate after centrifuging divided by 1 L is the water dispersibility of the substance (defined herein).
[0386] For determining the water solubility or water emulsifiability of a liquid substance (for example rapeseed oil), the following procedure was adopted. 5 g of the substance were combined with 100 g of distilled water and stirred for 24 hours. The mixture was then transferred to a separating funnel. The mixture was stored in the separating funnel for 5 minutes. If no phase separation occurred after this time, the mixture was left to stand for a further 2 hours, preferably a further 10 hours. If no phase separation occurred, the substance was deemed to be water soluble. The water solubility of the substance in this case is at least 50 grams per litre. If phase separation occurred, the phases were separated in the separating funnel and the organic phase was dried over sodium sulfate. The weight of the dried organic phase was determined (mass of the organic phase in grams, defined herein). The difference between 5 g and the mass of the organic phase in grams divided by 0.1 L gave the water emulsifiability of the liquid substance. Water solubility, water dispersibility and water emulsifiability are used synonymously in the context of the invention. The limit value for water solubility, water dispersibility and water emulsifiability, respectively, for a compound of constituent (iii) is defined as being 1 g per litre.
[0387] A further-preferred separation technique for dispersed and undispersed fractions is centrifugation. After appropriate drying, it is possible to determine the mass of the residue in grams and also, from this, the water solubility or water dispersibility.
Results:
[0388] In the previous Examples 1 to 5, biocementation formulations were described which together with cohesion-modifying compounds exhibited a synergistic effect and showed a reduced emission-related weight loss.
[0389] With the use of the reference mixtures R20 to R24 it emerged that the use of polycarbonate, polyvinyl acetate 20, rapeseed oil, long-chain fatty acid and insoluble starch does not lead to any synergistic effect in relation to consolidation and emission reduction (
[0390] Only the application of starch to the surface resulted in a slightly increased breaking strength of the layers (11 N), but there is no synergistic effect with the biocementation (cf. R19 and R24 in
[0391] Comparable values were also obtained after 10 and 28 days (data not shown).
[0392] Comparable effects were also achieved when the bacterial strain was replaced by L. sphaericus, B. cohnii, B. halodurans, B. pseudofirmus and A. crystallopoietes in the same cell count per millilitre (data not shown). When B. cohnii, B. halodurans, B. pseudofirmus and A. crystalllopoietes were used analogously in the same cell count per millilitre in the formulations, the basic constituents were further adapted to the requirements of the particular bacterial strain. The skilled person is aware here that with these non-ureolytically biocementing bacterial strains, the base medium must be adapted in analogy to the constituents listed in Example 2, especially in terms of a suitable metabolic starting material. The effect on emissions reduction of the mixtures was comparable to the results set out for S. pasteurii (data not shown).
[0393] The skilled person therefore realizes that cohesion-modifying compounds in the sense of the invention must have a certain water solubility and/or water emulsifiability and/or water dispersibility in order to be able to produce the synergistic effect with the biocementation.
[0394] The removal of the cation source (here: calcium source) in the mixtures stated above showed comparable results in relation to dust suppression.
EXAMPLE 7: FURTHER FIELDS OF APPLICATION OF THE INVENTIVE MIXTURES
Material and Methods:
Pelletizing
[0395] The experiment was carried out in the laboratory, in a laboratory pelletizer. For this purpose, 100 g of iron ore (haematite powder) were introduced, and liquid biocementation mixtures were utilized for dust suppression/pelletization, these mixtures consisting of the following constituents in the following concentrations: [0396] Reference 3 (R3):
TABLE-US-00060 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0397] Mixture 7 (M7):
TABLE-US-00061 5 g/L yeast extract 21 g/L calcium acetate 34.9 g/L calcium chloride 46.2 g/L calcium lactate 25 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL B. halodurans [0398] Mixture 8 (M8):
TABLE-US-00062 36 g/L urea 35 g/L calcium chloride 10 g/L yeast extract 4 × 10{circumflex over ( )}8 cells L. sphaericus [0399] Mixture 9 (M9):
TABLE-US-00063 27 g/L urea 17 g/L calcium chloride 31 g/L albumin 4 × 10{circumflex over ( )}8 cells L. sphaericus [0400] Mixture 22 (M22):
TABLE-US-00064 27 g/L urea 47 g/L calcium lignosulfonate 12 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells S. pasteurii
[0401] Additionally, the mixture contains trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in the mixtures M8, M9 and M22 served primarily as a carbonate source. In the mixture M7, calcium lactate served as carbonate source.
[0402] Calcium lignosulfonate, yeast extract and albumin in the mixtures M7, M8, M9 and M22 are the (water-soluble and/or water-dispersible and/or water-emulsifiable) cohesion-modifying compounds.
[0403] All components of the present mixture that are capable of biocementation, except for the bacteria, were in solid form. The bacteria were present as described in Examples 1 to 6. The solid constituents and the bacteria in liquid culture were mixed immediately prior to use, with the solid constituents dissolving.
[0404] This experiment was carried out also with woodchips, in order to study the capacity of the biocementation mixtures to bind woodchips.
[0405] 20 mL of the respective biocement mixture were sprayed onto 100 g of iron ore (haematite powder) and left to react for 5 minutes at a rate of 30 revolutions per minute.
[0406] After five minutes, a determination was made of the breaking strength of the resultant pellets: for this purpose, first pellets of similar diameter were selected: the diameter determined with the aid of a calliper. The pellet diameter was measured at three points on the pellet; the deviation of the individual measurements was 1 mm. Pellets were selected whose diameter corresponded to 11±1 mm. The breaking strength (maximum value of the force measurement) of the pellets can be determined by means of the following method: the method is based on the standardized test method for strength determination in cement, DIN EN 196-1:2005-05. The breaking strength is measured using a digital (breaking) strength measuring instrument in accordance with the manufacturers instructions. A cylindrical test plate is mounted on the pellet with the aid of a crank-operation test bed, and then pressed into the pellet (to the point of breakage). The force applied is measured continuously. From a number of pellets (>3) the mean breaking strength is computed. The mean breaking strength of the pellets is preferably between 0.5 and 500 N, more preferably between 1 and 150 N.
Evaporation Control
[0407] The experiment was conducted in the laboratory in plastic vessels with a volume of 1000 cm.sup.3. The application area in each case was 29.2 cm.sup.2.
[0408] The soil substrate in the experiment consisted of a silica sand having a grading of 0-2 mm. The sand had been both washed and dried by the manufacturer and was used directly. 2200 g of silica sand per plastic vessel were used as soil substrate. The plastic vessel was full to the brim.
[0409] For control, reference mixtures were utilized, consisting of the following constituents in the following concentrations: [0410] Reference 2 (R2): Application of water.
[0411] Liquid biocementation mixtures were utilized, consisting of the following constituents in the following concentrations: [0412] Reference 3 (R3):
TABLE-US-00065 48 g/L urea 44 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells S. pasteurii [0413] Mixture 11 (M11):
TABLE-US-00066 48 g/L urea 44 g/L calcium chloride 3.9 g/L polyvinyl acetate dispersion 4 × 10{circumflex over ( )}8 cells S. pasteurii [0414] Mixture 16 (M16):
TABLE-US-00067 1.07 g/L ammonium chloride 21 g/L calcium acetate 15 g/L L-alanine 34.9 g/L calcium chloride 0.40 g/L sodium hydroxide 1 g/L yeast extract 46.2 g/L calcium lactate 4 × 10{circumflex over ( )}8 cells A. crystallopoietes [0415] Mixture 22 (M22):
TABLE-US-00068 27 g/L urea 47 g/L calcium lignosulfonate 12 g/L calcium chloride 4 × 10{circumflex over ( )}8 cells S. pasteurii
[0416] The mixture additionally included trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in the mixtures R3, M11 and M22 served primarily as a carbonate source. In the mixture M16, calcium lactate served as carbonate source.
[0417] Yeast extract, L-alanine, polyvinyl acetate dispersion and calcium lignosulfonate are the cohesion-modifying compounds in the mixtures M11, M16 and M22.
[0418] All of the components of the present mixtures that are capable of biocementation, except for the bacteria of the strains A. crystalllopoietes, L. sphaericus and S. pasteurii, were in solid form. The bacteria were present as a liquid culture in the culturing media described in Examples 1 to 6. The solid constituents and the bacteria in liquid culture were mixed immediately prior to use, with the solid constituents dissolving.
[0419] Before the application of the respective mixtures, the sand was wetted with water, so that the sand is fully impregnated with water when the mixtures are subsequently applied. The respective mixtures were then applied each in three replications to the experimental areas. The application rate per square metre was consistently 10 litres per replication. The fully dissolved samples were applied using a pipette. Following application, the surface was spread smooth with a spatula. The measurement values reported are mean values of the three replications, lying typically in the region of 10% of the value ascertained.
[0420] The applied mass of the water was determined gravimetrically. For this purpose, the mass of the sand-filled sample vessel was determined before and after application of the water and of the respective biocementation mixture (mass before application, mass after application, both defined herein). The difference in the mass after application and the mass before application, minus the solid contained in the respective biocementation mixtures (cf. M11, M16, M22), is the applied amount of water (defined herein). The solid present in the respective biocementation mixture is given from the respective solid concentration multiplied by the respective application volume. The sum total of the mass before application and the solid contained in the respective biocementation mixture is the total solids mass of the beaker (defined herein).
[0421] The application of the reference mixtures and also of the biocementation mixtures was followed by incubation over the entire observation period for 168 days at an atmospheric humidity of 20% to 60% and with multiple changes of air per day. Within this period the minimum temperature prevailing was 14.2° C. and the maximum temperature prevailing was 25.2° C.; all of the mixtures were exposed to exactly the same external conditions. The mass of the sample vessel at various points in time was measured and documented (sample mass.sub.day xy).
[0422] The relative soil humidity on the respective day of measurement, in percent (day xy), was determined with the following formula:
Relative soil humidity.sub.day xy=[(sample mass.sub.day xy−total solids mass of the beaker)/applied amount of water]*100
[0423] This experiment was carried out likewise with woodchips, mine tailings and rural earth. For this purpose, the uppermost five centimetres of the sand layer were replaced by woodchips, mine tailings or rural earth, respectively, and this as the soil substrate was treated as described above with the mixtures R3, M11, M16 and M22. The total solids mass was adapted as a result of the weight of the woodchips, mine tailings and rural earth, respectively.
Decontamination
[0424] Liquid biocementation mixtures were utilized, consisting of the following constituents in the following concentrations: [0425] Reference 9 (R9)
TABLE-US-00069 25 g/L polyvinyl alcohol [0426] Reference 25 (R25):
TABLE-US-00070 48 g/L urea 4 × 10{circumflex over ( )}8 cell/mL S. pasteurii [0427] Mature 20 (M20):
TABLE-US-00071 27 g/L urea 47 g/L calcium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0428] Mature 29 (M29):
TABLE-US-00072 1 g/L yeast extract 25 g/L calcium lignosulfonate 21 g/L sodium acetate 46.2 g/L sodium lactate 4 × 10{circumflex over ( )}8 cells/mL B. cohnii [0429] Mature 30 (M30):
TABLE-US-00073 48 g/L urea 50 g/L humic acid 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0430] Mature 31 (M31):
TABLE-US-00074 27 g/L urea 47 g/L sodium lignosulfonate 4 × 10{circumflex over ( )}8 cells/mL S. pasteurii [0431] Mature 32 (M32):
TABLE-US-00075 48 g/L urea 25 g/L polyvinyl alcohol 4 × 10{circumflex over ( )}8 cells/mL L. sphaericus
[0432] In addition, the mixtures R25, M20, M29, M30, M31 and M32 include trace elements and traces of, for example, salts and sugars (<1 wt %). Urea in the mixtures M20, M30, M31 and M32 served primarily as a carbonate source; sodium acetate and sodium lactate in the mixture M29 served primarily as a carbonate source. These mixtures optionally contained one of the following metal salts (0.1 M): nickel(II) chloride, iron(III) chloride, copper(II) chloride. If iron(III) chloride was used, hydrochloric acid (0.1 M) was likewise present. Each metal salt was combined with each mixture. The designation used was as follows: metal salt+respective mixture. For the metal salts, the following designation is used: iron(III) chloride=FeCl3, nickel(II)chloride=NiCl2, copper(II) chloride=CuCl2. The mixture in which, for example, as well as agent 20 there was also copper(II) chloride present is listed as CuCl2+M20 (cf.
[0433] All of the components, including the respective bacteria, were in solid form. In the cases of the pulverulent bacteria, the powder was a powder expertly dried. All of the components apart from the respective bacterial powder were mixed directly before use, with the solid constituents dissolving. As soon as the components were fully dissolved, the respective bacterial powder was added and dissolved.
[0434] After the mixtures had been combined with the bacterial powder, the mixture was stirred for 5 minutes and then left to react for 24 hours. The resultant precipitate was subsequently separated by centrifugation (3000 g, 10 min) and decanted off. The mass of the moist, heavy metal-containing precipitate was ascertained—moist mass of the heavy metal-containing precipitate (defined herein). The moist, heavy metal-containing precipitate was subsequently dried in a stream of nitrogen and the mass of the heavy metal-containing precipitate (defined herein) was determined. The presence of the respective heavy metal ion was confirmed quantitatively by atomic spectroscopy. As a control, the respective mixtures were produced without the presence of the respective metal salt (M20, M29, M30, M31 and M32) and treated according to exactly the same procedure (5 min stirring, 24 reaction, centrifugation, decanting, drying). The mass of this precipitate after decanting is the moist mass of the control precipitate (defined herein). After drying, the mass of the control precipitate was determined (defined herein). The respective supernatant was also studied by absorption spectroscopy and/or atomic spectroscopy for the presence of heavy metal ions. In this case a suitable wavelength with appropriate sensitivity was used. The concentration of the respective heavy metal ion as obtained from this analysis is the residual heavy metal ion concentration (defined herein). The residual heavy metal ion concentration divided by 0.1 mol per L multiplied by 100 is the residual heavy metal ion content of the supernatant in percent (defined herein). The respective metal salt solution was likewise stirred for 5 minutes, incubated for 24 h and centrifuged for 3000 g for 10 min, followed by determination of the residual heavy metal ion concentration.
Results:
[0435] In the testing of the inventive biocementation mixtures on various substrates, further unexpected observations were made, which are developed further below. This gives rise to further, potential fields of application, which are set out further in the present example:
Pelletizing
[0436] In the application of the inventive formulations to a moving ion ore sample, with the intention of preventing dust formation, it was found that small agglomerates or pellets were formed after application. This observation was taken up in order to form pellets with the biocementation mixtures M7, M8, M9 and M22 in a laboratory pelletizing plate.
[0437] As well as reduced emissions during production, these pellets also exhibited a greater strength than was possible with the reference formulations (R3). A pellet in each case produced using different agents M7, M8 and M9 (from left to right) is shown at the top in
[0438] The breaking strength of the pellets was as follows for the various agents: M7=28 N, M8=29 N, M9=30 N, M22=27 N—and was therefore above that of R3. Pellets produced using R3 had a breaking strength of 3 N 5 minutes after production. Processing the pellets produced with reference 3 proved to be difficult, since the pellets produced with R3 shattered very easily. This is presumably because of the absence of the cohesion-modifying compound. Cohesion-modifying substances therefore also permit the production of a biocement which can be used for pelletizing.
[0439] The mixtures according to the invention are also capable of binding and aggregating woodchips.
[0440] It was also found that when using the mixtures in which all of the components, including the bacteria, were present as powders, similar results were achieved in respect of emissions reduction. For this purpose, all of the pulverulent components were mixed and the above-described amount of water was added in the laboratory pelletizer (data not shown). Comparable effects were also achieved when the bacterial strain was replaced by L. sphaericus, B. cohnii, B. halodurans, B. pseudofirmus and A. crystallopoietes in the same cell count per millilitre (data not shown). Where B. cohnii, B. halodurans, B. pseudofirmus and A. crystallopoietes were used analogously in the same cell count per millilitre in the formulations, the basic constituents were further adapted to the requirements of the particular bacterial strain. The skilled person is aware here that in the case of these non-eurolytically biocementing bacterial strains, the base medium has to be adapted in analogy to the constituents listed in Example 2, particularly in relation to a suitable metabolic starting material. In this case it was found that effective pelletization was achieved with all of the bacterial strains (data not shown).
[0441] Unexpectedly, it emerged that after formation of the biocement, some substrates dried more slowly in the laboratory pelletizer, and consequently the effect of the biocementation mixtures on evaporation was studied at a deeper level.
Evaporation Control
[0442] The drying rate of the sand was reduced by effective layer formation. This is apparent from the higher relative soil humidity of the samples in which the mixtures M11, M16 and M22 in comparison to water application (R2) (
[0443] The altered porosity might also be relevant for other applications where porosity plays a part. This is especially so for use in insulating materials, catalyst beds and/or battery materials. As a result of the reduced porosity, the material is also suitable as sealing material.
[0444] It was also found that when using the mixtures in which all of the components, including the bacteria, were present as powders, similar results were achieved in respect of emissions reduction. For this purpose, all of the pulverulent components were mixed and incorporated into the uppermost layer of and. The corresponding liquid volume was applied subsequently (data not shown). Comparable effects were also achieved when the bacterial strain was replaced by L. sphaericus, B. cohnii, B. halodurans, B. pseudofirmus and A. crystallopoietes in the same cell count per millilitre (data not shown). Where B. cohnii, B. halodurans, B. pseudofirmus and A. crystallopoietes were used analogously in the same cell count per millilitre in the formulations, the basic constituents were further adapted to the requirements of the particular bacterial strain. The skilled person is aware here that in the case of these non-eurolytically biocementing bacterial strains, the base medium has to be adapted in analogy to the constituents listed in Example 2, particularly in relation to a suitable metabolic starting material. In this case it was found that effective evaporation reduction was achieved with all of the bacterial strains (data not shown).
[0445] When various mine tailings were used, it was found that soils with a high loading of copper(II), iron(II), iron(III) and nickel(II) ions showed substantially quicker layer formation. In the case of the use of tailing/sand layering, similar results were achieved as for the pure sand sample (data not shown). Here as well, the mixtures with cohesion-modifying substances exhibited slower drying.
Decontamination
[0446] The skilled person is aware that carbonate ions which are produced by bacteria from urea, for example, can be utilized for precipitating metal ions (Phillips et al, Engineered applications of ureolytic biomineralization: a review, Biofouling, 2013, Vol. 29, No. 6, 715-733). This is probably the reason for the observation in the previous example (Example 7, Evaporation control) that layer formation began more rapidly when heavy metal-loaded soils were used. Testing was therefore carried out as to whether the cohesion-modifying substances are also suitable for improving heavy metal ion precipitation.
[0447] In each of the mixtures used, the mass of the control precipitate is less than the mass of the heavy metal-containing precipitate. This shows that the mixtures according to the invention are capable of binding and precipitating heavy metal ions. The presence of the respective metal salt was confirmed by atomic spectroscopy.
[0448] The moist mass of the heavy metal-containing precipitate when using R9, R25 and R32 for the precipitation of iron(III) chloride was FeCl3+R9=0.00 g, for FeCl3+R20=0.75 g and for FeCl3+M32=12.4 g (before drying). In the case of FeCl3+M32, a bulky gel is formed as a result of the bacterial activity. This gel is very difficult to dry in the stream of nitrogen. The mass of the heavy metal ion-containing precipitate for FeCl3+R9=0.00 g, for FeCl3+R25=0.05 g and for FeCl3+M32=6.53 g (here the assumption is that drying was incomplete owing to the gelatinous character). The residual heavy metal ion content was 50% lower for FeCl3+M32 than for FeCl3+R25.
[0449] Where M20 was used to precipitate FeCl3, NiCl2 and CuCl2, it was found that the addition of cohesion-modifying compounds resulted in an increased precipitation of the metal salts: the moist mass of the precipitate of M20 is 0.02 g. The moist mass of the heavy metal-containing precipitate is for FeCl3+M20=0.40 g, for NiCl2+M20=1.44 g and for CuCl2+M20=0.24 g. The residual heavy metal ion content in the supernatant is shown at the bottom in
[0450] Unexpectedly it emerged that the presence of cohesion-modifying substances also increases the precipitation from solution. The use of the mixtures not explicitly stated in the results section showed comparable results in relation to precipitation efficiency (data not shown). The use of the bacteria which were present in the liquid media of Examples 1 to 6 likewise showed very good precipitation efficiency (data not shown).
[0451] A feature of cohesion-modifying compounds is that synergistically with biocementation they produce a particularly cohesive, low-emission biocement. Unexpectedly it was found that this also occurs in solution and therefore produces a particularly efficient precipitation of heavy metal ions. This was unexpected especially because polymers in particular have a tendency to bind polyvalent ions, including especially divalent metal cations such as Ca(II), Cu(II), Mg(II) and also Ni(II), to disperse them in solution and so to increase their solubility. On the basis of this affinity for divalent metal cations, the expectation was that, especially if the water-soluble and/or water-dispersible cohesion-modifying compound was a polymer, it would stabilize polyvalent metal cations, and also aggregates and agglomerates thereof, in solution and so there would be less efficient metal ion precipitation (cf. Tadros T F 2016, Nanodispersions, ISBN-978-3-11-029033-2, especially Section p. 25ff steric stabilization).
[0452] The removal of the cation source (here: calcium source) in the mixtures stated above showed comparable results in relation to the effects studied.