BIO-CATALYTIC CALCIUM CARBONATE CEMENTATION

Abstract

The present invention is directed to methods of preparing biocement. The methods disclosed herein employ microbial or enzymatic means first to generate acid which dissolves CaCO.sub.3, and then to degrade urea, producing CO.sub.3.sup. ions which increase the pH and lead to the reprecipitation of CaCO.sub.3. The precipitation of CaCO.sub.3 acts as a cementation process which can bind together particulate materials to yield mortars, concretes and suchlike. The invention further provides construction materials formed by the methods of the invention, and bacterial strains which can be used in the methods of the invention to generate organic acids.

Claims

1. A method of forming a construction material from CaCO.sub.3 and a particulate starting material, said method comprising: (i) enzymatically generating an acid in a CaCO.sub.3-containing preparation to decrease the pH of said preparation, thereby dissolving at least a portion of the CaCO.sub.3 to produce a dissolved calcium carbonate preparation (DCCP); (ii) combining the DCCP with a particulate starting material; and (iii) enzymatically generating an increase in the pH of said combined mixture of (ii) thereby causing at least a portion of the dissolved CaCO.sub.3 to precipitate and to bind together at least part of the particulate starting material and precipitated CaCO.sub.3, and optionally a part of any undissolved CaCO.sub.3, to form the construction material; wherein the CaCO.sub.3 and the particulate starting material may be separate or pre-mixed prior to (i), such that in step (ii) the combination of the DCCP with the particulate starting material may take place simultaneously with or after the production of the DCCP, and wherein one or more of the reagents for enzymatically increasing the pH in step (iii) may be present in the CaCO.sub.3-containing preparation, or may be added during or after any one of steps (i) or (ii), or in step (iii).

2. The method of claim 1, wherein the enzymatic generation of the pH decrease in step (i) and/or the pH increase in step (iii) is performed using a microorganism or a microbial extract which contains enzymes.

3. The method of claim 2, wherein at least the enzymatic generation of the pH decrease in step (i) is performed using a microorganism or a microbial extract which contains enzymes.

4. The method of claim 3, wherein the enzymatic generation of the pH decrease in step (i) is performed using a microorganism under aerobic conditions.

5. The method of any one of claims 1 to 4, wherein said acid is a carboxylic acid.

6. The method of claim 5, wherein said carboxylic acid is lactic acid and/or acetic acid.

7. The method of any one of claims 2 to 6, wherein said microorganism is a bacterium or said microbial extract is a bacterial extract.

8. The method of claim 7, wherein said bacterium is a lactic acid- and/or acetic acid-producing bacterium, preferably a bacterium of a strain having the NCIMB accession number 42596, 42597, 42598, 42599 or 42600, or a mutant or derivative of one of said strains, or said bacterial extract is derived from a lactic acid- and/or acetic acid-producing bacterium, preferably a bacterium with the NCIMB accession number 42596, 42597, 42598, 42599 or 42600, or a mutant or derivative of one of said strains.

9. The method of any one of claims 1 to 8, wherein all the reagents for step (iii) are present in the CaCO.sub.3 preparation.

10. The method of any one of claims 1 to 9, wherein the reagents for step (iii) are added after step (i) has been performed, being added either before, during or after step (ii), and optionally wherein any undissolved CaCO.sub.3 is removed between step (i) and step (ii).

11. The method of any one of claims 2 to 10, wherein in step (i) a microorganism is included in or added to the CaCO.sub.3 preparation, together with nutrients for growth of said microorganism and one or more substrates for generating the acid.

12. The method of claim 11, wherein said nutrients are provided in a growth or culture medium.

13. The method of claim 11 or 12, wherein said substrate for generating the acid is a sugar, preferably glucose.

14. The method of any one of claims 11 to 13, wherein said microorganism is a bacterium, preferably a lactic acid- and/or acetic acid-producing bacterium, and said acid is a carboxylic acid, preferably lactic acid and/or acetic acid.

15. The method of any of claims 2 to 14, wherein both the pH decrease of step (i) and the pH increase of step (iii) are performed using one or more microorganisms or microbial extracts, preferably one or more bacteria or bacterial extracts.

16. The method of claim 15, wherein the pH increase of step (iii) is achieved by the hydrolysis of urea by a urease enzyme.

17. The method of claim 15, wherein the pH increase of step (iii) is achieved by the metabolization of the acid produced in step (i), or by the enzymatic degradation or conversion of the acid produced in step (i), wherein the products of said metabolization, degradation or conversion are not themselves acids or are weaker acids than the acid produced in step (i).

18. The method of any one of claims 15 to 17 wherein both the pH decrease of step (i) and the pH increase of step (iii) are performed using the same microorganism or microbial extract, preferably the same bacterium or bacterial extract.

19. The method of claim 18, wherein said bacterium is of the strain having the NCIMB accession number 42600, or a mutant or derivative thereof, or said bacterial extract is derived from the strain having the NCIMB accession number 42600, or a mutant or derivative thereof.

20. The method of any one of claims 15 to 17, wherein the pH decrease of step (i) is performed using a first microorganism or microbial extract, preferably a first bacterium or bacterial extract, and the pH increase of step (iii) is performed using a second microorganism or microbial extract, preferably a second bacterium or bacterial extract.

21. The method of any one of claims 1 to 19, wherein the CaCO.sub.3 is in the form of limestone or chalk.

22. The method of any one of claims 1 to 20, wherein the CaCO.sub.3 is a powder.

23. The method of claim 21, wherein said powder consists of particles ranging in size from 1 to 200 m.

24. The method of any one of claims 1 to 22 wherein said particulate starting material is sand.

25. A construction material formed by the method of any one of claims 1 to 24.

26. The construction material of claim 25, wherein said construction material is a masonry unit.

27. A method of forming a cement from CaCO.sub.3, said method comprising: (i) enzymatically generating an acid in a CaCO.sub.3-containing preparation to decrease the pH of said preparation, thereby dissolving at least a part of the CaCO.sub.3 to produce a dissolved calcium carbonate preparation (DCCP); (ii) optionally combining the DCCP with a particulate material; and (iii) enzymatically generating an increase in the pH of said DCCP or said combined mixture of (ii) thereby causing at least a part of the dissolved CaCO.sub.3 to precipitate to form a cement, wherein the precipitated CaCO.sub.3 is able to function as a cement to bind together particulate or other construction materials; wherein where the method comprises step (ii) the CaCO.sub.3 and the particulate material may be separate or pre-mixed prior to (i), such that in step (ii) the combination of the DCCP with the particulate material may take place simultaneously with or after the production of the DCCP, and wherein one or more of the reagents for enzymatically increasing the pH in step (iii) may be present in the CaCO.sub.3-containing preparation, or may be added during or after any one of steps (i) or (ii), or in step (iii).

28. Use of a cement prepared according to the method of claim 27 to connect construction materials, preferably masonry units, together.

29. A method of connecting construction materials together, said method comprising applying a cement prepared according to claim 27 to at least one construction material unit and placing it together with at least one other construction material unit.

30. A bacterium which is characterised by being: (i) alkaliphilic; (ii) able to produce acid under aerobic conditions at a pH of at least 8.5, preferably in the presence of CaCO.sub.3 and more preferably also under low oxygen conditions; and (iii) able to dissolve CaCO.sub.3 to produce Ca.sup.2+ ions, preferably in the presence of glucose and CaCO.sub.3 powder.

31. The bacterium of claim 30, wherein said bacterium is suitable for use in the method of claims 1 to 24 and/or claim 27, particularly in the generation of acid in the method of claims 1 to 24 and/or claim 27.

32. The bacterium of claim 31, wherein said bacterium is of a strain having the NCIMB accession number 42596, 42597, 42598, 42599 or 42600, or a mutant or derivative of one of said strains.

33. The bacterium of any one of claims 30 to 32, wherein said bacterium is also able to produce urease.

34. The bacterium of claim 33, wherein said bacterium is of the strain having the NCIMB accession number 42600, or a mutant or derivative of said strain.

Description

[0107] The present invention may be more fully understood from the non-limiting Examples below and in reference to the drawings, in which:

[0108] FIG. 1 shows changes of pH and Ca.sup.2+ ion concentration over dissolution time.

[0109] FIG. 2 shows a consolidated sample of sand20 biocement.

[0110] FIG. 3 shows SEM images of sand20 biocement.

[0111] FIG. 4 shows an SEM image of sand40 biocement.

[0112] FIG. 5 shows SEM images of (a) 90% sand20 and (b) 50% sand20.

[0113] FIG. 6 shows elemental mapping of sand20 sample using EDS on SEM.

[0114] FIG. 7 XRD patterns of sand, limestone and the sand20 biocement sample.

[0115] FIG. 8 shows the compressive strength of consolidated samples produced using different numbers of reagent injections and having different limestone content.

[0116] FIG. 9 shows pH (A) and Ca.sup.2+ ion concentration development in limestone dissolution reactions containing different BSL-1 (B) and one BSL-2 strains (C) as a function of time. Experiments with AP-029 and UP-009 were carried out for about 16 hours, while experiments with the remaining strains and the negative control (medium only) were performed for about 21 hours.

[0117] FIG. 10 shows the results of two chalk dissolution experiments using B. safanensis strain AP-004. Changes in the pH and Ca.sup.2+ concentration of the solutions are shown in each graph. In part B two ISE and two pH electrodes were used in the solution; the results from each electrode are shown separately.

[0118] FIG. 11 shows the pH profile of chalk suspensions in defined medium following addition of acid-producing strain AP-004 (A) or AP-029 (B) and later the urease-producing species S. pasteurii.

[0119] FIG. 12 shows a picture of the setup for the consolidation experiments.

[0120] FIG. 13 shows the change over time in pH and Ca.sup.2+ concentration of the limestone dissolution solution used in the consolidation experiment. The graph shows the results of two experiments. The results of the first experiment are labelled 1 and the results of the second experiment 2A or 2B. The second experiment used two sets of probes (A and B), the results obtained from each of which are shown separately.

[0121] FIG. 14 shows two consolidated sand samples made using the method of the invention. The scales shown are in centimetres.

[0122] FIG. 15 shows SEM images of the first consolidated sand sample. The magnification and scale are shown at the bottom of each image.

[0123] FIG. 16 shows SEM images of the second consolidated sand sample. The magnification and scale are shown at the bottom of each image.

[0124] FIG. 17 shows EDS results of the precipitated crystals in the consolidated sand samples. The results from the first sample are presented in part A, and the second sample in part B.

EXAMPLES

Example 1: Proof of Concept

Materials and Methods

Materials

[0125] Sand of 50-70 mesh size from Sigma-Aldrich (USA) was used. Jack Bean (Canavalia ensiformis) urease used in this study was supplied by Sigma-Aldrich, with a specific activity of 50,000-100,000 units/g solid. Crushed limestone used in this study was an industrial grade chalk powder, obtained from Franzefoss Miljkalk AS (Norway), with a density of 2.7 kg/dm.sup.3 and particle size ranging from 1-200 m.

Preparation of Reagent

[0126] Calcium-rich solution (CRS) was prepared by dissolving crushed limestone in 0.3 M lactic acid at room temperature (20 C.) until saturation was reached. The solution was stirred using a magnetic stirrer. The amount of limestone needed in the dissolution was estimated through PHREEQC, a geochemical modelling program. According to the program, at least 15 g of limestone is required per litre of lactic acid in order to obtain a Ca.sup.2+ saturated solution (however, as indicated above it is not necessary for the method of the invention for a saturated solution to be obtained, or used). For the purposes of this experiment, 25 g of limestone were used per litre of 300 mM lactic acid, to ensure sufficient limestone was provided. Dissolution was carried out for 24 h, with a pH and ion-selective electrode (ISE) to monitor the changes of pH and Ca.sup.2+ ion concentration in the solution. Then, the solution was filtered through filter paper to remove the remaining non-dissolved limestone, and the CRS was obtained. Next, 0.15 M of urea was added to the CRS. Ca.sup.2+ ion concentration was measured through atomic absorption spectroscopy (Perkin Elmer AAnalyst 400, USA).

Preparation of Bio-Cement

[0127] Bio-cement was prepared in a split mould of 25 mm diameter. First, 60 g of grains and 0.2 g of urease were mixed well. Then, the mixture was fed into the split mould, the two halves of which were held together tightly by screws. A layer of filter paper and porous flow channels were placed at the top and bottom of each mould. Next, the grains were compacted by tightening a screw and spring assembly on the top of the mould. The split mould was connected to a syringe pump at the inlet, and the outlet tube was placed in a beaker to drain the waste. A pressure sensor was placed at the inlet to monitor pressure changes throughout the experiment.

[0128] After that, 25 ml of reagent was injected into the mould at an injection rate of 0.5 ml/min. Reagent was pumped upwards (against gravity) through the sand in order to create more consistent results as it avoided preferential flow. The same amount of reagent was injected the prescribed number of times (Table 1). Injections were performed at 5 h intervals. After all the injections were complete, 50 ml of distilled water was injected to wash out all the by-products. Finally, the consolidated sample was taken out of the mould and dried in an oven for 2 h at 70 C. Then, the samples were sent for further characterization. Different processing conditions were used, as listed in Table 1 together with the sample coding.

TABLE-US-00001 TABLE 1 Processing conditions and material designation Grains Number of Sample code Sand (%) Limestone (%) Urease injections sand20 100 0 0.2 20 sand40 100 0 0.2 40 90% sand20 90 10 0.2 20 50% sand20 50 50 0.2 20

Scanning Electron Microscopy (SEM)

[0129] Consolidated samples were observed under a Hitachi TM3000 TableTop SEM (Hitachi High-Technologies Corporation, Japan). Elements present in the samples were identified using Quantax70 energy dispersive spectroscopy (EDS).

X-Ray Diffraction (XRD) Analysis

[0130] Crystals present in the consolidated samples were identified using Rigaku MiniFlex600 X-Ray diffractometry, with a scan range from 10 to 90 and 10/min scanning rate. The X-Ray source was Cu-K radiation with a wavelength of 0.154 nm.

Mechanical Test

[0131] The consolidated samples were cut into halves of similar height, in order to compare their properties at different parts. Mechanical properties of each layer were tested through uniaxial compression tests using a Zwicki-line testing machine (Zwick/Roell, Germany). A load cell of 1 kN and a cross-head speed of 10 mm/min were used.

CaCO.SUB.3 .Content Measurement

[0132] A certain weight of consolidated sample was dried in an oven at 70 C. for 24h. The samples were repeatedly weighed until a constant weight was reached. Then, the samples were degraded in 0.1 M hydrochloric acid (HCl) at 30 C. and stirred with a magnetic stirrer. Changes in pH and Ca.sup.2+ ion concentration were monitored by pH meter and ISE until constant values were reached, in order to ensure a full dissolution of the CaCO.sub.3 crystals. The remaining solids were filtered out of the solutions using filter paper, washed several times with distilled water, dried and re-weighed. CaCO.sub.3 content was determined using the following equation:

[00001]$\begin{array}{cc}{\mathrm{CaCO}}_3.Math..Math.\mathrm{content}.Math..Math.\left(\mathrm{wt}.Math..Math.\%\right)=\frac{\mathrm{Weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{samples}.Math..Math.\mathrm{before}.Math..Math.\mathrm{acid}.Math..Math.\mathrm{digestion}}{\mathrm{Weight}.Math..Math.\mathrm{of}.Math..Math.\mathrm{samples}.Math..Math.\mathrm{after}.Math..Math.\mathrm{acid}.Math..Math.\mathrm{digestion}}& \left(1\right)\end{array}$

Porosity Measurement

[0133] Changes in porosity throughout the consolidation experiments were monitored by a pressure sensor attached to a tube connecting to the inlet of the split mould. Data on the pressure changes is able to provide information on porosity inside the mould.

Results and Discussion

Dissolution of Crushed Limestone

[0134] During the preparation of CRS, changes of pH and Ca.sup.2+ ion concentration were monitored throughout the dissolution process as shown in FIG. 1. It can be observed that pH increased rapidly from pH 2 to pH 5.5 within the first 10 min after the addition of limestone into lactic acid. The increase of pH is due to the consumption of hydrogen (H.sup.+) ions from the lactic acid during the dissolution process, as described in the following equation:

CaCO.sub.3+Lactic acid(HLac).fwdarw.Ca.sup.2++HCO.sub.3.sup.+Lac.sup.

[0135] This indicates a rapid dissolution of limestone at the initial stage. Then, the dissolution rate slowed down and reached a constant pH of 6.7 after around 9 h. This suggests that the solution reaches its saturation point after about 9 h. The concentration of Ca.sup.2+ ions in the solution was measured as a function of time, as illustrated in FIG. 1. The Ca.sup.2+ concentration increased rapidly with time during the first 10 min to around 0.024 mol/l, then increased at a slower rate until it reached a maximum concentration of 0.056 mol/l after 9 h. However, it was followed by a gradual decrease of Ca.sup.2+ concentration in the solution which reached a concentration of 0.0069 mol/l at 24 h. The described phenomenon can be explained by methodological reasons. In a paper by Zander and Cooper (Intensive Care Medicine. 19(6): p. 362-363), they described the interference in Ca.sup.2+ concentration as measured by ISE by the presence of metabolisable anions such as lactate, acetate or malate. Lactate ions are known to complex with and chelate Ca.sup.2+ ions, causing the chelated calcium to no longer be detected by ISE. During the dissolution of limestone, Ca.sup.2+ ion concentration increased in the solution, but at the same time chelation of calcium occurred. When the solution reached saturation point, the Ca.sup.2+ ion concentration in the solution became constant. Meanwhile, more and more Ca.sup.2+ ions were chelated by lactate and could thus no longer be detected by ISE. Hence, the measured Ca.sup.2+ concentration reduced with time after the saturation point was reached at around 9 h of dissolution. This also suggests that ISEs are not suitable for the measurement of Ca.sup.2+ concentration when lactate is present. In order to obtain an accurate measurement of the amount of dissolved calcium, the final solution was analysed by atomic absorption spectroscopy (AAS), giving a Ca.sup.2+ ion concentration of 0.065 mol/l.

Morphological Studies

[0136] After the preparation of bio-cement, consolidated cylindrical samples were obtained. A typical image of a consolidated sand sample (before cutting) is shown in FIG. 2. The cylindrical consolidated samples have diameters of approximately 25 mm and heights ranging from 80-95 mm.

[0137] To confirm reproducibility, two samples were made under each processing condition. Fairly large parts of the consolidated samples were well cemented, and retained their structure even when immersed in water. This proved the feasibility of utilising the dissolution-recrystallization mechanism to achieve cementation.

[0138] FIG. 3 shows SEM images of the consolidated sand sample, sand20. From FIG. 3A it can be seen that crystals have precipitated on the free surface and in between the sand grains. These precipitated crystals act as a cement to bind the sand grains together. It is interesting to note that most of the precipitated crystals exhibit a semi-spherical morphology, as illustrated in FIGS. 3B and 3C, with a diameter ranging from 20-100 m. From a close-up of the spherical crystal (FIG. 3D) at higher magnification, it can be observed that the spheres appeared to be spherical aggregates of smaller sub-units of around 1-5 m. Similar crystal morphology was also observed in the sand40 samples, as shown in FIG. 4. The XRD results (which will be discussed further later) confirm that these precipitated crystals are calcite, the most stable polymorph of CaCO.sub.3. Other researchers have previously reported similar CaCO.sub.3 crystal morphology, which is known as spherulitic calcite (the crystals being calcite spherulites). Calcite spherulites are formed by aggregation of the typical rhombohedral calcite crystals into a spherical orientation. It is believed that the presence of organic molecules such as lactate is the main trigger of the spherulitic growth of calcite.

[0139] For 90% sand20 and 50% sand20 samples, 10 wt % and 50 wt % of sand was replaced by crushed limestone, respectively. A similar cementation effect is observed from the SEM images (FIG. 5), in which the precipitated calcite crystals act as a binder to bind the grains together. The precipitated crystals and the added limestone are both calcite, and thus are difficult to distinguish. However, on some occasions, the precipitated calcite and the limestone can be distinguished. In FIG. 5A, two distinct morphologies of calcite can be detected. Crushed limestone is known to have a rhombohedral morphology and often has facet surfaces (as labelled in the figure). Meanwhile, the precipitated crystals appear as a cluster of smaller crystals similar to those observed in sand20 and sand40 samples, but not in a spherical orientation. This can be confirmed by a visible hole that is created by a pulled-out sand grain in FIG. 5A, where the precipitated small calcite crystals formed an aggregate and covered the sand grain. For the 50% sand20 sample (5B), it is difficult to distinguish the calcite crystals because the composition of added limestone is present in a higher amount than the precipitated calcite. In both the samples with added limestone, the presence of calcite spherulites was not detected. Differences in calcite morphology between sand and sand-limestone systems are likely due to differences in the number of potential heterogeneous nucleation sites. Random orientation of precipitated calcite in the sand-limestone systems suggests that nucleation occurred too rapidly in these systems. This results in the formation of many small calcite crystals at different places, subsequently prohibiting spherulitic growth of calcite. Therefore, it is postulated that limestone provides potential nucleation sites for calcite.

Elemental Analysis and Phase Identification

[0140] Elements present in the sand20 sample were identified using EDS mapping. Distribution of different elements in the framed area is illustrated in FIG. 6. Big grains that contained silicone (Si) and oxygen (O) correspond to sand (quartz, SiO.sub.2). The spherical crystal aggregates contained calcium (Ca), carbon (C) and O and are expected to be CaCO.sub.3. This is further confirmed by using XRD analysis. FIG. 7 shows typical XRD patterns of bare sand, crushed limestone and the sand20 sample. Crushed limestone composed of the mineral calcite with the strongest XRD peak positioned at 2 of 29.3, which is known to be calcite (104). A similar calcite peak is also observed in the sand20 sample (marked as * in FIG. 7), confirming the precipitation of calcite in the consolidated sample. Calcite content in the consolidated samples is much lower as compared to sand and thus, other lower intensity calcite peaks are not noticeable.

Mechanical Properties

[0141] The consolidated samples were cut into halves with similar heights, in order to compare their properties at different parts. As described in the experimental procedures, reagents were pumped upward into the mould during the production of bio-cement. Thus, the bottom layer is the part that was closer to the injection inlet, whereas the top layer is the part that was situated further from the injection inlet.

[0142] Results from the uniaxial compression test are presented in FIG. 8. It can be seen that the bottom layer samples give an overall higher compressive strength than the top layer samples. This is attributed to greater CaCO.sub.3 precipitation at the bottom layer that is situated closer to the injection inlet, as the reagents flow in from the bottom of the mould during each injection. The region further from the injection inlet (top layer) shows lower CaCO.sub.3 precipitation. Significant improvement in compressive strength is observed when the number of injections is increased from 20 to 40, for both top and bottom layer samples. This is due to greater CaCO.sub.3 precipitation at the higher number of injections, as expected.

Example 2: Isolation of Acid-Producing Strains

Materials and Methods

Samples, Growth Media and Isolation of Strains

[0143] Soil samples were collected near an open quarry for CaCO.sub.3 in Tromsdalen, a side-valley to Verdalen, Nord-Trndelag, Norway. Sterile water was added to the samples at 1-2.5 ml/g sample (depending on the character and original water content of the sample), samples shaken vigorously to extract free-living microorganisms associated with the material and filtered through a tea strainer to remove larger particles. The resulting water extracts of the samples were heated for 10 min at 75-82 C. in order to select for spore forming microorganisms. Samples were frozen at 20 C. and freeze-dried prior to further use. For strain isolations, 1 g freeze-dried material was added to 10 mL water and stirred vigorously. An aliquot of this was used to prepare a 10-fold dilution in water for subsequent inoculation of two types of agar plates to identify acid producing microorganisms and microorganisms positive for production of the enzyme urease. For discovery of alkaliphilic, acid-producing strains, RM-9.5 medium was used (1.0 g/L yeast extract (Oxoid), 3.0 g/L peptone (Oxoid), 10.0 g/L glucose, 5.0 g/L NaCl, 2.5 ml/L thymol blue solution (1% w/v), 15 g/L agar; the pH was adjusted to 9.5 using 1.2 g/L Na.sub.2CO.sub.3). Microbial acid production resulted in the colour of the plates changing from blue to yellow. For isolation of urease positive strains, Christensen urea medium was used (Urease Test ProtocolBenita Brink: http://www.microbelibrary.org/library/laboratory+test/3223-urease-test-protocol; 1.0 g/L peptone (Oxoid), 10 g/L glucose, 5.0 g/L NaCl, 2.0 g/L KH.sub.2PO.sub.4, 20.0 g/L urea, 0.012 g/L phenol red, 15 g/L agar; the pH was adjusted to 6.5 using 1 M NaOH). Microbial production of urease resulted in the colour of the plates changing from yellow to red.

[0144] Agar plates were incubated at 30 C. for up to 7 days, and in order to obtain pure isolates, colonies corresponding to the desired acid or urease production phenotype, respectively, were picked as they appeared and plated on new agar plates and incubated at 30 C. until colonies appeared. From plates with pure isolates, one single colony was picked and transferred to a 12-well plate (Costar 3513) containing 1.2 ml of the medium used in the respective type of enrichment/isolation, and incubated in a shaking incubator at 30 C., 85% humidity, and 250 rpm. Cultures growing in the 12-well plates were harvested after 5 days of incubation and used as an inoculum for shake flask cultures, as well as for analyses on agar plates. The shake flask cultures were incubated shaking at 30 C. and 150 rpm, the agar plates as described above. Samples from the shake flasks were supplemented with 15% glycerol (v/v, final concentration), and agar plate cultures were harvested using 5 mL of the respective growth medium containing 15% glycerol. The glycerol cultures were stored at 80 C.

Analytical Methods

[0145] Acid production and urease activity in liquid culture and on agar plates were qualitatively detected by colour change of the pH indicators included in the medium (see above). For quantification of acids produced and glucose utilised, HPLC analysis was performed. HPLC analyses were carried out using a Shimadzu HPLC equipped with an HPX-87H Aminex column using 5 mM H.sub.2SO.sub.4, flow rate 0.6 ml/min at 45 C. Bacterial culture growth was monitored using optical density measurements at 660 nm wavelength with a Shimadzu UV-1800 spectrophotometer. Taxonomic analysis was carried out by PCR amplification and sequencing of the 16S rRNA gene followed by bioinformatic classification of the gene sequence. PCR amplification was carried out using Expand High Fidelity PCR system from Roche. The isolates were grouped according to BLAST homology analyses' closest hit and ranged within groups based on acid production and growth phenotype.

Limestone Dissolution Experiments

[0146] Pre-cultures were prepared in 9 cm petri dishes containing an agar layer made of solid RM-9.5 medium with 30 g/L glucose, topped with 10 mL liquid RM-9.5 medium with 30 g/L glucose. 15 l cell suspension from glycerol stocks was used as inoculum and the pre-culture incubated stationary at 30 C. When the medium turned yellow (indicating pH decrease due to acid production) and turbidity was observed (indicating culture growth), 1 mL of the liquid phase was transferred to 25 mL fresh RM-9.5 medium containing 30 g/L glucose and 10 g/L CaCO.sub.3 powder in 50 ml tubes, and placed for 1-3 days at 30 C. in tilted racks in a shaking incubator at 200 rpm.

[0147] For limestone dissolution experiments, 15 g of CaCO.sub.3 powder was mixed with 6 mL RM-9.5 medium containing 30 g/L glucose and 10 g/L CaCO.sub.3 in 50 mL tubes, to which 120 l bacterial culture was added. A respective setup without bacterial culture added was used as a negative control. Calibrated pH and Ca.sup.2+ electrodes (ELIT electrodes, NICO2000) were placed inside the tube, the opening was sealed with Parafilm to prevent evaporation, and continuous measurements were performed every 5-10 minutes for 16-21 hours using the ELIT 4-channel Ion-Analyzer with its corresponding software.

Results and Discussion

Screening and Selection of Strains

[0148] From processing of five soil samples, a strain collection of in total 65 isolates was established, consisting of 41 isolates from acid-production screening and 24 from urease-activity screening. The individual pure strains were arrayed in the wells of a 96-well microtiter plate, representing a master plate for subsequent analyses and selection of the best performing strains.

[0149] The microbial isolates were analysed and selected for properties useful for the production of bio-cement according to the method of the invention, in particular the ability to grow and produce high levels of acids at reduced oxygen level and high pH associated with the presence of high concentrations of limestone. Growth, acid production and urease activity on solid and/or liquid medium were tested, as well as performance in the presence of CaCO.sub.3 powder in the medium and at low oxygenation. In addition, isolates were taxonomically classified using 16S rRNA gene sequence analysis.

[0150] Of the 65 strains of the strain collection, 13 strains (Table 2) were selected as potential candidates for use in Bio-cement application testing based on their ability to grow at high pH and the production of significant amounts of acid. Two of these strains (UP-009, URF-016), derived from urease screening, were in addition confirmed for their ability to produce and secrete urease. All 13 strains were able to grow and produce acid at reduced oxygen levels and in the presence of limestone powder in the medium. Surprisingly, the addition of limestone to the growth medium enhanced glucose utilization and total acid production. The total acid production of the strains was in the range of 32-75 mM acid, with lactic acid being the dominant acid, constituting 70-80% of the total acid produced. In addition, acetic acid and minor amounts of succinic acid were detected.

[0151] Taxonomic analysis of the strains revealed that the strains were all related to different species of the genera Bacillus/Jeotgalibacillus, which are known to contain spore-forming environmental strains. Three strains (ARF-002, UP-009, URF-016), were found to be closest related to strains that were previously described as human pathogenic species related to food poisoning and therefore require handling at Biosafety level 2, unlike the residual strains that were closest related to harmless species. Six of the 13 strains were selected for tests for their capability to dissolve limestone in high solids reactions.

TABLE-US-00002 TABLE 2 Strains selected as potential candidates for use in bio-cement application testing. Total acid produced was determined by end-point measurements. Strains in bold were selected for limestone dissolution tests. Closest relative based on Total acid Biosafety ID 16S rRNA gene sequence produced (mM) level (BSL) AP-001 32 1 ARF-002 Bacillus mycoides/ 75 1/2 Bacillus weihenstephanensis AP-004 37 1 AP-005 Bacillus pumilus 65 1 AP-006 Bacillus simplex 69 1 AP-007 Bacillus pumilus 75 1 AP-011 Bacillus pumilus 35 1 AP-024 Bacillus pumilus 34 1 AP-029 35 1 AP-044 60 1 ARF-017 38 1 UP-009 62 2 URF-016 Bacillus cereus 65 2

Limestone Dissolution Experiments

[0152] Six of the 13 strains were selected for tests for their capability to dissolve limestone in high solids reactions. The tests showed that all of these strains were capable of reducing the pH in the reactions and releasing Ca.sup.2+ from the limestone powder. Both pH and Ca.sup.2+ concentration evolution profiles were significantly above background level, determined by a negative control reaction, containing medium only (FIG. 9). However, the strains displayed different behaviour in both acid production (observed as decrease in pH) and dissolution of limestone (observed as increase in free Ca.sup.2+ ions). Within a period of 16-21 hours of continuous measurements, the BSL-2 strain UP-009 was clearly the best total acid producer, resulting in the highest free Ca.sup.2+ concentration and the lowest pH. Among the BSL-1 strains, ARF-017 and AP-004 performed best in limestone powder dissolution in that period. However, the amount of Ca.sup.2+ ions released by these strains was approx. an order of magnitude smaller than by the UP-009 strain in that time period. The remaining three strains (AP-001, AP-024 and AP-044) started to dissolve limestone later than the first three and continued to produce acid when the experiment was stopped.

Example 3: Preparation of Dissolved Chalk Solutions Using B. Safenensis AP004

Materials and Methods

[0153] Inoculum of B. safanensis strain AP-004 was prepared in rich medium from a culture grown on an agar plate. 40 g of chalk was added into 1 L rich medium already containing 10 g/L CaCO.sub.3 (rich medium (RM): glucose.H.sub.2O 33 g/L, NaCl 5 g/L, Na.sub.2CO.sub.3 4 g/L, peptone 3 g/L, yeast extract 1 g/L, thymol blue (1% w/v) 2.5 ml/L (i.e. resultant concentration of 0.0025% w/v), pH 9.5 (10 g/L CaCO.sub.3 added after pH adjustment) in a beaker, and mixed well using a magnetic stirrer.

[0154] 30 ml of AP-004 inoculum was added to the mixture. The mixture was continuously stirred using a magnetic stirrer. Changes in pH and Ca.sup.2+ ion concentration were monitored using pH/ISE electrodes.

Results and Discussion

[0155] The results of two experiments are presented. The pHs of the solutions decreased and the Ca.sup.2+ ion concentrations of the solutions increased with time, indicating dissolution of chalk by the acid produced by the AP-004 bacteria. The pHs dropped from their initial levels of 9.5 to 5.5-6.5 due to acid production. Results in terms of the Ca.sup.2+ ion concentrations were slightly inconsistent between individual experiments, but both showed the same trend. The results of the two exemplary experiments are shown in FIG. 10.

Example 4: Production of a Defined Medium for Acid-Producing Strains

Background

[0156] Use of a complex medium for acid production, containing relatively large amounts of glucose as well as yeast extract and peptone, was found to have the disadvantage that after acidification and subsequent re-alkalization of the solution with S. pasteurii, there were still significant amounts of organic substrate, possibly including glucose, left in the solution, and a secondary re-acidification phase was thus occasionally observed as the leftover glucose was metabolised.

[0157] A chemically defined medium based on pure chemical compounds such as glucose, mineral salts (NH.sub.4Cl, K.sub.2HPO.sub.4, MgSO.sub.4, etc.) and if necessary limited amounts of organic growth factors (vitamins, specific amino acids, etc.), has the advantage that, if supplied in the right quantities, the only available carbon source after the acidification step will be organic acids. Any secondary growth on organic acids will lead to a pH increase, thus fortifying the pH increase due to hydrolysis of urea by S. pasteurii.

Materials and Methods

Bacterial Strains

[0158] Four acid-producing (AP) isolates were selected for the study. They have been assigned to species as follows: AP-004Bacillus sp. closely related to B. pumilus and B. safanensis; AP-006Bacillus sp. closely related to B. simplex; AP-029B. pumilus; and AP-044B. licheniformis. All are believed to be Biosafety level 1. All were cultured at 30 C. For the alkalization step Sporosarcina pasteurii DSM 33 was used.

Culture Media

[0159] Defined Medium 1 (DM1): Taurine 12.5 g/L; glucose.H.sub.2O 11.0 g/L; NH.sub.4Cl 2.3 g/L; trisodium citrate.2H.sub.2O 1.0 g/L; K.sub.2HPO.sub.4, 0.70 g/L; MgSO.sub.4.7H.sub.2O 0.40 g/L; trace mineral solution 1 (TMS1, see below), 5 ml/L; Wolfe's vitamin solution (WVS, see below) 10 ml/L, pH 9.5.
Defined Chalk Suspension Medium (DCSM): Glucose.H.sub.2O 11.0 g/L; NH.sub.4Cl 2.3 g/L; K.sub.2HPO.sub.4 0.70 g/L; MgSO.sub.4.7H.sub.2O 0.40 g/L; TMS1 5 ml/L; WVS 10 ml/L; trisodium citrate.2 H.sub.2O 0 g/L, 0.2 g/L or 1.0 g/L. One part medium was mixed with two parts chalk (CaCO.sub.3).
Semi-Defined pasteurii Medium (SDM): Urea 20 g/L; taurine 6.25 g/L; glucose.H.sub.2O 5.5 g/L; NH.sub.4Cl 2.3 g/L; trisodium citrate.2H.sub.2O 1.0 g/L; vitamin-reduced casamino acids 1.0 g/L; K.sub.2HPO.sub.4 0.70 g/L; MgSO.sub.4.7H.sub.2O 0.40 g/L; trace mineral solution 1 for S. pasteurii (TMS1-pasteurii, see below) 5 ml/L; WVS 10 ml/L, pH 9.5.
Complex Medium for S. pasteurii (CM): Bacto Tryptone 15.0 g/L; Bacto Soytone 5.0 g/L; NaCl 5.0 g/L; urea 20 g/L; pH 7.3.
Rich Medium for AP Strains (RM): as defined above in Example 3.
Defined pasteurii Concentrate (DC): Glucose.H.sub.2O 55 g/L; urea 20 g/L; vitamin-reduced casamino acids 10 g/L; TMS1-pasteurii 5 ml/L.
WVS: Folic acid 20 mg/L; pyridoxine.HCl 10 mg/L; thiamine.HCl 5 mg/L; riboflavin 5 mg/L; nicotinic acid 5 mg/L; calcium D-(+)-pantothenate 5 mg/L; p-aminobenzoic acid 5 mg/L; thioctic acid 5 mg/L; biotin 2 mg/L; vitamin B12 0.10 mg/L.
TMS1: FeSO.sub.4.7H.sub.2O 5.0 g/L; ZnSO.sub.4.7H.sub.2O 0.44 g/L; CuSO.sub.4.5H.sub.2O 0.39 g/L; MnCl.sub.2.2H.sub.2O 0.15 g/L; CoSO.sub.4.7H.sub.2O 20 mg/L; Na.sub.2MoO.sub.4.2H.sub.2O 10 mg/L; conc. HCl 50 ml/L.
TMS1-pasteurii: As above for TMS1 with additional 0.5 g/L NiCl.sub.2.6H.sub.2O.

Experimental Procedures

[0160] The ability of the AP isolates to grow in DM1 was screened in 8-well tissue culture plates. Cells from RM agar were inoculated into wells with 5 ml DM1 with and without 1.0 g/L casamino acids and with a varying concentration of taurine (3-100 mM). Growth was assessed visually and pH recorded as a function of time.

[0161] The ability of strains able to grow in DM1 without addition of casamino acids to acidify chalk suspensions in DM1 (without taurine) was characterised in plastic containers (120 ml) closed with a screw cap. The strains were cultured in shake flasks at 30 C. in DM1 with 100 mM taurine as buffer, harvested by centrifugation, washed once with DM1 without taurine, and inoculated into the chalk suspensions. pH was measured as a function of time at room temperature (22-23 C.) and 30 C.

[0162] The acidic chalk suspensions were re-alkalized by addition of S. pasteurii and DC, and the pH in the re-alkalized chalk suspension followed for 25 days. S. pasteurii was cultured in shake flasks at 30 C. in SDM, harvested by centrifugation, washed once with water, and inoculated into the acidic chalk suspensions. The studies were performed at room temperature and 30 C.

Results

Ability of Acid-Producers to Grow in Defined Mineral Medium

[0163] All fours strains grew well and lowered pH in DM1 when it contained 1.0 g/L casamino acids, but only two of the strains, AP-004 and AP-029, grew well and lowered pH without casamino acids in the medium. Interestingly, a small amount of CaCl.sub.2 (1 mmol/L) in the medium appeared to stimulate the growth of the cells. It was also observed that cell growth rate increased when the buffer capacity was reduced, indicating that the cells grow better at a lower pH than 9-9.5. In wells with the highest buffer capacity (100 mM Taurine), pH did not decrease below pH 8.2-8.3.

[0164] When culturing bacteria at pH 9-9.5, significant amounts of ammonium in the medium may be lost to the atmosphere as ammonia. The ability of AP-004 and AP-029 to use nitrate (NO.sub.3.sup.) instead of ammonium (NH.sub.4.sup.+) as a nitrogen source was therefore tested. Both strains could utilize nitrate as N-source, although they grew more rapidly with ammonium as N-source.

Acidification of Chalk Suspensions

[0165] The acidification of a chalk suspension in DM1 was tested using AP-004 and AP-029. DCSM (20 ml) was mixed with 40 g chalk powder in a plastic container (120 ml) closed with a screw cap. The suspension was inoculated with 0.5 ml washed cell concentrate made by centrifuging down the cells in 50 ml shake flask culture cultured in DM1, washing the cells once with sterile water and re-suspending them in 5 ml sterile water. The inoculated suspensions were incubated without shaking or stirring at room temperature and 30 C. During the next 3-5 days pH decreased to below pH 6 (FIG. 11).
Re-Alkalization of Chalk Suspensions with S. pasteurii
After acidification of the chalk suspensions with acid-producing bacteria, to the chalk suspensions were added 2.0 ml DC, 100 l TMS1-pasteurii and a 10 times concentrated washed culture of S. pasteurii cultured in SDM. This was added within an hour of the pH measurement on day 5, and resulted in a rapid increase in pH to around pH 9 (FIG. 11). The pH in the suspension remained high for several weeks (Table 3).

Conclusions

[0166] A defined mineral medium with glucose as the only C-source for the acidification of chalk suspensions has been designed. The medium is suitable for use with the two isolated acid-producing strains AP-004 and AP-029. When the acidic chalk suspensions (pH 5-6) were re-alkalized with S. pasteurii+urea, pH remained stable at around pH 9 for weeks, and no secondary acidification phase was observed. This is in contrast to the previously employed complex medium, where a second, and unwanted, acidification phase occurred.

TABLE-US-00003 TABLE 3 Days after addition of S. pasteurii + urea 1 4 6 9 25 Sample pH AP 004 Room temp. (22-23 C.), No citrate 9.18 9.15 9.11 9.11 9.06 AP 004 Room temp. (22-23 C.), Low citrate (0.2 g/L) 9.26 9.22 9.17 9.18 9.02 AP 004 Room temp. (22-23 C.), High citrate (1.0 g/L) 9.19 9.14 9.10 9.14 9.02 AP 004 30 C., No citrate 8.81 8.88 8.77 9.04 8.96 AP 004 30 C., Low citrate (0.2 g/L) 8.75 8.76 8.77 8.74 9.00 AP 004 30 C., High citrate (1.0 g/L) 8.68 8.74 8.80 8.68 9.15 AP 029 Room temp. (22-23 C.), No citrate 8.66 9.09 9.10 9.11 8.60 AP 029 Room temp. (22-23 C.), Low citrate (0.2 g/L) 8.58 9.03 9.08 9.07 8.79 AP 029 Room temp. (22-23 C.), High citrate (1.0 g/L) 8.60 8.96 8.90 9.01 8.88 AP 029 30 C., No citrate 8.73 8.78 8.87 8.66 9.30 AP 029 30 C., Low citrate (0.2 g/L) 8.54 8.64 8.74 8.50 9.04 AP 029 30 C., High citrate (1.0 g/L) 8.52 8.63 8.75 8.52 9.23

Example 5: Consolidation of Sand Grains by AP-004 and S. pasteurii Strains

Materials and Methods

Materials

[0167] Sand with 50-70 mesh particle size from Sigma-Aldrich was used in the consolidation experiment. Crushed limestone used in this study is an industrial grade chalk powder, which is obtained from Franzefoss Miljkalk AS, with a density of 2.7 kg/dm.sup.3 and particle size ranging from 1-200 m.

Limestone Dissolution

[0168] AP-004 bacteria were cultured in RM at pH 9.5, and used as the inoculum. 40 g of crushed limestone was added into 1 L of RM (pH 9.5) with 30 ml of AP-004 inoculum. The mixture was stirred using a magnetic stirrer, and the pH and Ca.sup.2+ ion concentration were monitored using a pH/ISE meter. The mixture was stirred for 2-3 days, until the point where no significant changes in the pH and Ca.sup.2+ ion concentration were observed. Next, the remaining limestone particles and bacteria were filtered out using a 0.22 m vacuum filtration set. Then, the filtrate was used as the calcium source for the CaCO.sub.3 precipitation. The filtrate is referred to herein as the dissolved chalk solution (DCS).

Preparation of Reagent

[0169] Freeze-dried S. pasteurii (SP) bacterial powder was cultured in CM overnight. The SP bacterial inoculum was diluted 10 times using fresh CM before use. Then, the diluted SP bacterial solution was mixed with the DCS at a ratio of 1:1. The mixture was used as the reagent in the consolidation experiment.

Consolidation Experiment

[0170] A simple setup as shown in FIG. 12 was used in the consolidation experiment. First, a 20 ml syringe was filled with sand. Filter paper was placed both on the top and the bottom of the syringe to prevent grain loss during the experiment. Then, 6 ml of reagent was added from the top of the syringe (inlet). The reagent was drawn downwards to fill up the syringe using another syringe that was attached at the outlet. The reagent was kept in the syringe for at least 3 h before fresh reagent was added. This process was repeated 20 times.

Results

[0171] In this work, a pH/ISE meter was used to keep track of the limestone dissolution process. FIG. 13 shows the changes of pH and Ca.sup.2+ ion concentration of the mixture. Limestone dissolution is confirmed through the reduction in pH and increase in Ca.sup.2+ ion concentration of the mixture. DCS with a final pH ranging from pH 5.5-pH 6.5, and a final Ca.sup.2+ ion concentration ranging from 0.022-0.035 mol/l, was used in the consolidation experiment.

[0172] Through the consolidation experiment, two consolidated sand samples were successfully produced, which are shown in FIG. 14. Most of the sand was well cemented, and the blocks retained their structures even when immersed in water. This proves the feasibility of utilizing DCS produced by AP-004 and SP strains to achieve cementation.

[0173] The consolidated sand samples were viewed under an SEM. From FIGS. 15A and 16A it can be clearly seen that there are CaCO.sub.3 crystals precipitated within the pore spaces between sand grains. These crystals act as a cement to bind the sand grains together. Different morphologies of precipitated CaCO.sub.3 crystals are observed, as shown in FIGS. 15B, 16B and 16C. Most formed a layered structure, in which layers of CaCO.sub.3 crystals stack on top of each other. Spherical crystals were also detected in the second consolidated sample (FIG. 16C). Bacteria are also seen on the surface of sand grains or the precipitated CaCO.sub.3 (FIGS. 15C and 16A).

[0174] Elements present in the consolidated sample were identified using EDS. EDS scans from the selected crystals, presented in FIG. 17, show a high amount of calcium (Ca), which is very likely to be CaCO.sub.3. The gold peak seen in the EDS spectrums is due to gold coating of samples before SEM imaging.

Conclusions

[0175] Sand grains were successfully consolidated through our two-step process: dissolution using AP-004 bacteria and recrystallization using an SP strain. SEM images showed the binding of sand grains by precipitated crystals.