The present invention relates to a new process for preparing cement and cement- based products, and in particular a construction material based on or comprising a calcium carbonate cement, wherein enzymatic processes are used to generate changes in pH which induce dissolution and subsequently precipitation, or recrystallization, of calcium carbonate, thereby to render it functional as a cement. The calcium carbonate cement

(precipitated/recrystallized calcium carbonate) is used to bind together a particulate starting material to form a construction material. Advantageously the pH changes may be generated through the metabolic activity of microorganisms, particularly bacteria. The invention also provides novel bacteria suitable for such use and construction materials obtained by the methods of the invention.

Cement is an important component in the construction industry. It is normally used as a binder in binding other materials together, and also as a component of mortar for masonry. The cement industry accounts for approximately 5 % of worldwide man-made carbon dioxide (C0 ₂ ) emissions, of which 50 % is from the chemical process of cement production itself (the conversion of calcium carbonate (CaC0 ₃ ) to calcium oxide (CaO)), and 40 % from burning fuel to obtain the high temperatures required for the process to take place. For example, Portland cement and magnesium cement are produced by heating crushed limestone at 1450°C and 750°C, respectively. These processes thus not only inherently emit a lot of C0 ₂ , but also involve high energy consumption and the associated further high levels of C0 ₂ emission.

Due to increased environmental awareness, numerous approaches have been introduced in recent years in order to minimize the environmental impact of the cement industry. One of the most effective methods of reducing emissions and energy use in cement production today is to replace a portion of the Portland cement with pozzolanic materials of natural (volcanic) or industrial (fly ash, blast furnace slag) origin. New cement materials such as energetically modified cement and bio-cement have also been introduced over the last few decades, in an effort to reduce the energy consumption and environmental pollution associated with cement production by the use of alternative routes.

Bio-cement is a material that is produced via a biological approach. To date, the most commonly reported system of bio-cement is the use of ureolytic bacteria which can produce a urease enzyme (US 8728365). The urease enzyme then hydrolyses urea provided in the system to form carbonate ions, which react with provided calcium ions to form insoluble calcium carbonate (CaC0 ₃ ). Cementation is achieved by the precipitation of CaC0 ₃ . This mechanism is known as microbial induced carbonate precipitation (MICP). Studies of MICP are abundant, and aim to use it for a variety of different applications, including stone conservation (Rodriguez-Navarro, C. et al., Applied and Environmental Microbiology, 2012. 78(1 1 ): pp. 4017-4029), crack remediation in concrete (Achal, V. et al., Construction and Building Materials, 2013. 48: pp. 1-5), cement mortar (Ghosh, P. et al., Cement and

Concrete Research, 2005. 35(10): pp. 1980-1983), soil stabilization (van Paassen, L.A. et al., Ecological Engineering, 2010. 36(2): pp. 168-175), self-healing concrete (Jonkers, H.M. and Schlangen, E., Concrete Repair, Rehabilitation and Retrofitting, 2009, pp. 215-220, Taylor & Francis Group, London), contaminant adsorption and immobilization (Fujita, Y. et al., Environmental Science & Technology, 2010. 44(19): pp. 7652-7658) and construction of subsurface barriers against pollutant spread or reservoir leakage (Cunningham, A.B. et al., Energy Procedia, 201 1. 4: pp. 5178-5185), etc. The great potential of bio-cement as a greener alternative to traditional cements is demonstrated by the use of the concept by several companies, for instance by Calcite Bioconcept (France), who use an MICP approach for the surface treatment of stonework, and by bioMASON Inc., which employs natural microorganisms and chemical processes to manufacture biological cement-based masonry building materials.

Though bio-cement is, in environmental terms, a great improvement on more traditional cements such as Portland cement, there remains the issue that calcium ions must be added to the mix for the formation of CaC0 ₃ . These are generally added in the form of a calcium chloride solution (US 8728365, US 8951786), although other calcium salts such as calcium nitrate may also, or additionally be used. Calcium chloride is generally obtained as a by-product of the Solvay process, which has the overall formula:

2 NaCI + CaC0 ₃ → Na ₂ C0 ₃ + CaCI ₂

Thus calcium is obtained initially from CaC0 ₃ , added to the bio-cement mix in the form of CaCI ₂ and then precipitates out as CaC0 ₃ again. The inventors of the present application have designed a significantly improved method of producing bio-cement whereby the calcium ions are obtained directly from CaC0 ₃ , by dissolving CaC0 ₃ in acid, based on the following reaction using lactic acid as an example (although another acid may be used in an analogous reaction):

CaC0 ₃ + lactic acid (Hlac)→ Ca ²⁺ + HC0 ₃ ^" + Lac ^"

The calcium (and carbonate) ions can then be used in a process akin to a traditional bio-cementation process: hydrolysis of urea yields carbonate in the following reaction:

(NH ₂ ) ₂ CO + 3 H ₂ 0→ 2 NH ₄ ⁺ + HC0 ₃ ^" + OH ^"

In the traditional process, hydrolysis of urea is used to generate carbonate ions for synthesis of the calcium carbonate. However, according to the present invention the urea hydrolysis reaction is used in a different way: this reaction causes an increase in pH, causing the CaC0 ₃ previously dissolved in acid (as described above) to re-precipitate, according to the reaction: Ca ²⁺ + HC0 ₃ ^" + OH ^" → CaC0 ₃ + H ₂ 0

solidifying the cement. Thus, the production of carbonate is not necessary as a result of this reaction, and other enzymatic reactions which generate, or result in an increase in pH, may alternatively be employed, such as for example enzymatic reactions which use, or consume, acid as a substrate. As is normal in the art, by performing such a reaction in the presence of a loose aggregate or particulate material, the precipitated CaC0 ₃ will bind the aggregate or particles together, yielding a composite material. Such a composite material is a construction material, such as a concrete or mortar.

Particularly advantageously, the inventors have isolated novel strains of bacteria from sites near a limestone quarry in Tromsdalen, Norway, which may be used in this process. A large number of such strains have been obtained and certain of these, whose species have not yet been identified or confirmed and may be previously unknown, have been deposited at the NCIMB (UK), which is located at the following address: Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA. Five strains have been deposited, known as AP-004, AP-006, AP-029, AP-044 and UP-009. These strains were all deposited by the applicant on 22 June 2016, and have the following accession numbers:

AP-004 - 42596;

AP-006 - 42597;

AP-029 - 42598;

AP-044 - 42599; and

UP-009 - 42600.

These strains are able to produce high levels of acid, particularly lactic acid, sufficient to dissolve CaC0 ₃ to yield calcium ions, and may thus be used in the first step of the process. These strains offer a distinct technical advantage with regard to acid production over previously-known strains, as no commercially-available strain which was screened was found to produce sufficient acid under appropriate conditions to be used in this method, unlike the newly-isolated strains listed above. The method of the application thus enables the bio-production of calcium ions directly from CaC0 ₃ , cutting out the need for the chemical production of CaCI ₂ (or other calcium salts) in the process. By cutting out this production step, the method of the application significantly reduces the environmental footprint of the cement. The Solvay Process, whereby CaCI ₂ is produced, is highly energetically costly, requiring the heating of CaC0 ₃ to temperatures of 950-1 100°C. By cutting out this step, the method of the invention significantly reduces the environmental impact of bio-cement.

Thus, in a first aspect, the invention provides a method of forming a construction material from CaC0 ₃ and a particulate starting material, said method comprising: (i) enzymatically generating an acid in a CaC0 ₃ -containing preparation to decrease the pH of said preparation, thereby dissolving at least a part of the CaC0 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 part of the dissolved CaC0 ₃ to precipitate and to bind together at least part of the particulate starting material and precipitated CaC0 _3, and optionally a part of any undissolved CaC0 _3, to form the construction material;

wherein the CaC0 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 CaC0 ₃ -containing preparation, or may be added during or after any one of steps (i) or (ii), or in step (iii).

In a second aspect, the invention provides a construction material formed by the method of the first embodiment.

In a third aspect, the invention provides a method of forming a cement from CaC0 ₃ , said method comprising:

(i) enzymatically generating an acid in a CaC0 ₃ -containing preparation to decrease the pH of said preparation, thereby dissolving at least a part of the CaC0 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 CaC0 ₃ to precipitate to form a cement, wherein the precipitated CaC0 ₃ is able to function as a cement to bind together particulate or other construction materials;

wherein where the method comprises step (ii) the CaC0 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 CaC0 ₃ -containing preparation, or may be added during or after any one of steps (i) or (ii), or in step (iii).

Advantageously, in the methods of the invention the enzymes for the steps of enzymatically generating a decrease or increase of pH may be provided in the form of a microorganism or microbial extract.

Further aspects of the invention provide use of a cement prepared according to the method of the invention, as defined above, to connect construction materials, such as masonry units, together, and a method of connecting construction materials together, said method comprising applying a cement prepared according to the method of the invention, as defined above, to at least one construction material unit and placing it together with at least one other construction material unit.

In a still further aspect, the invention provides a bacterium suitable for use in in the methods of the invention, more particularly for the production of acid in the methods, said bacterium being:

(i) alkaliphilic;

(ii) able to produce acid under aerobic conditions at a pH of at least 8.5

(particularly in the presence of CaC0 ₃ , e.g. limestone, and more particularly also under low oxygen conditions); and

(iii) able to dissolve CaC0 ₃ to produce Ca ²⁺ ions, (particularly in the presence of glucose and CaC0 ₃ ).

In one preferred embodiment the bacterium is capable of forming spores. The invention also provides a bacterium as deposited at the NCI MB under accession number 42596, 42597, 42598, 42599, 42600, or a mutant or modified strain thereof.

In particular, such a mutant or modified strain will have the identifying characteristics of the parent bacterium from it is derived. Most notably, the mutant or modified strain will have the properties listed above. The mutant or modified strain may be a genetically engineered variant of a deposited bacterium, and/or it may have been obtained through mutagenesis (random or directed) or adaptation of the deposited bacterium.

In one preferred embodiment a bacterium of the invention is able to produce urease, particularly produce and secrete urease. Accordingly, in an embodiment of the invention, the bacterium is a modified strain of a bacterium as deposited at the NCI MB under accession number 42596, 42597, 42598, 42599, 42600 which has been genetically engineered to express a urease enzyme. Thus a bacterium of the deposited strains may be modified by the introduction of a nucleic acid molecule encoding a urease enzyme, such that the modified bacterium is capable of producing and secreting a urease enzyme. Nucleotide sequences encoding urease enzymes are known and widely available in the art.

As mentioned above, the methods of the present invention rely on a process of enzymatically-catalysed dissolution and re-precipitation of CaC0 ₃ to achieve cementation; CaC0 ₃ dissolved by the enzymatically-produced acid (i.e. in the pH decrease step) and subsequently allowed to re-precipitate by increasing pH is able to act as a cement, or binder, to cement, or bind, together particulate material with which it is in contact. In this way, the re- precipitated CaC0 ₃ forms the binder (cement) which together with the particulate starting material forms the construction material. The construction material can accordingly be seen as a hardened cement in the particulate material (e.g. the particulate starting material in the method above). This is achieved not only by precipitation of a new mineral phase (i.e. new grains or particles of CaC0 ₃ ) from the dissolved CaC0 ₃ , or by the transformation of naturally reactive materials, but also by the dissolution and re-crystallization of the surface of the CaC0 ₃ grains. Thus, the grain surfaces of the CaC0 ₃ are dissolved and the dissolved CaC0 ₃ is allowed to re-precipitate (re-crystallize), including on the grain surface. This process activates the surface of the CaC0 ₃ (i.e. the grain surface), such that it is able to function as a binder, or cement. Further, precipitated crystals or particles of CaC0 ₃ may form between the particles of the particulate starting material and may act as a cement to bind the particulate starting material and the CaC0 ₃ together. The precipitation may accordingly be alternatively defined as, or as including, recrystallization of CaC0 ₃ .

It will be understood from the above that a complete dissolution of the CaC0 ₃ is not required. There may be a localised dissolution and re-precipitation of the CaC0 ₃ , and more particularly a localised dissolution and re-precipitation of the surface of the CaC0 ₃ (or more particularly, of the surface of CaC0 ₃ grains or particles). A part of an individual CaC0 ₃ particle or grain may be dissolved. In step (iii) of the method of forming the construction material, the binding together of the particulate starting material and precipitated CaC0 ₃ may be defined as a step of consolidation, that is the CaC0 ₃ (and specifically the

precipitated CaC0 ₃ ) are consolidated together to form a composite material. The composite material is the construction material.

By using enzymatic reactions to cause (or induce) the changes in pH the processes of dissolution and precipitation may be controlled. Thus, enzymatic processes may be used to generate (i.e. to cause, or result in) changes in pH which may be controlled in time, or locally. In particular where microorganisms are used, microbial metabolic activity (i.e.

involving the enzymes) may serve to generate the required changes in pH in a controlled manner. The microorganism, contacted with the CaC0 ₃ in the CaC0 ₃ -containing preparation and provided with nutrients for growth and as a substrate for generation of acid (e.g. a sugar, such as glucose, from which a carboxylic acid can be produced) will grow and utilise the substrate to generate acid. This will reduce the pH and dissolve the CaC0 ₃ (step (i)). In particular the CaC0 ₃ may be dissolved locally, e.g. the surface of the CaC0 ₃ may be dissolved, in the vicinity of the microorganism producing the acid. When the substrate has been consumed or depleted, acid production will stop, or at least be markedly or significantly reduced, and other enzymatic reactions carried out by microorganisms (which may be the same or different to the acid-producing microorganism), e.g. hydrolysis of urea by urease, or another ammonia-generating reaction, or an acid-consuming reaction, cause an increase in pH to occur, causing the dissolved CaC0 ₃ to precipitate (step (iii)). A similar effect may be achieved using a microbial extract which contains the enzymes required for the enzymatic steps (e.g. a cell-free extract), or indeed a preparation comprising the enzymes and other reagents necessary for the enzymatic reactions. The use of a microorganism comprising enzymes for both steps (i) and (ii) advantageously means that the method may be carried out in a single step, providing all the reagents and starting materials together. Thus, the microorganism(s), CaC0 ₃ , starting particulate material, nutrients and substrates for the enzymatic reactions may be provided together in the CaC0 ₃ -containing preparation.

However, as will be described below, this is not necessary, and for example the CaC0 ₃ may be dissolved in a first step, and the DCCP produced in this step may subsequently be contacted with the reagents of steps (ii) and (iii), together (i.e. simultaneously), or sequentially. Optionally, there may be an intervening step of removing undissolved CaC0 ₃ (i.e. the undissolved CaC0 ₃ may be removed after step (i)) _,

By the term "construction material" as used herein is meant a composite material formed from cement and a particulate starting material as defined below (e.g. an aggregate). The cement is formed of precipitated CaC0 ₃ crystals, which upon precipitation from solution bind the particles of the particulate starting material together to form a hardened material. The construction material is thus the consolidated product of the method. The construction material may be, or may be like, mortar or concrete. The construction material may thus be used, for instance, in masonry, to bind together stones or bricks or suchlike. Alternatively, the construction material may be used in (or may be produced as) concrete-like masonry units, blocks, bricks, slabs etc. In certain embodiments, the construction material may be used in ground stabilisation or in fundaments. For instance, the construction material may be used in the foundations of a building or suchlike. The construction material may be used for any purpose for which a cement-containing composite material is commonly used. It will be seen, therefore, that the construction material may be a solid product, or component, and may be provided in unit form (in the sense of a unit being an individual item of a masonry or construction component), e.g. a brick, block, panel, tile, column, pillar, counter-top, pre-cast veneer or any pre-cast element used in building or manufacture etc. It can thus be any shaped solid construction component. Methods of shaping or forming construction material into such a solid or shaped form are well known in the art, and any of these could be used, for example forming the construction material in a mould, compression, drying etc.

A cement is a preparation which may be used to bind materials together, including notably particulate starting materials to prepare construction materials. Cement may be used to prepare a concrete-like material. However, it may also be used to bind together already- prepared construction materials (e.g. in unit form, such as blocks or bricks). In one embodiment cement may be used to prepare a mortar to bind construction materials together.

The particulate starting material used will determine many of the properties of the construction material, and thus may be chosen based on the desired properties of the construction material. For instance, the use of an aggregate consisting of a fine material, such as sand, may yield a construction material with properties similar to mortar or low- strength concrete; use of a coarser aggregate, such as gravel, may yield a construction material with properties similar to a stronger concrete. If necessary, the construction material may be reinforced, for instance using rebar or other reinforcements such as natural (e.g. cellulose) or synthetic fibres (e.g. glass fibres, rock wool, synthetic polymer fibres, etc.).

Thus, depending on the desired characteristics of the construction material, any suitable particulate material may be used as the particulate starting material, and in particular any aggregate material (namely an aggregate material used in the construction industry, for instance soil, sand, sawdust, asphalt, a rock aggregate such as crushed stone, gravel or slag, or sintered clay or shale). The material may be natural or synthetic. Recycled materials may be used, e.g. recycled building materials, including e.g. recycled concrete. If the particulate material is crushed stone, the type of stone used is not particularly limited, so long as it does not dissolve, or substantially dissolve in the acid used to form the DCCP. Thus a CaC0 ₃ -based stone, such as limestone, is not used as the particulate starting material, although it is not precluded that it may be included in the method (for example where a DCCP is prepared in a first step and added to a particulate material in step (ii), CaCO¾ e.g. a CaC0 ₃ .based stone, or similar may be added to or included with the particulate material). In one preferred embodiment of the invention, the particulate starting material is sand, in which case any size or type of sand may be used.

When soil, sand or any other material which forms a constituent of the ground is used as the particulate starting material, the soil, sand or suchlike may be removed from the ground or may remain in the ground. In other words, in certain embodiments, the

construction material may be formed within the ground, for the purpose of stabilising it. In this embodiment, the DCCP of the method, comprising the enzymes (e.g. microorganism(s)) and reagents necessary for step (iii) of the method is effectively poured into the ground wherein the resultant cement binds together loose particulates, consolidating and thus stabilising the ground.

The size of the particulate material is not critical and may be varied within wide limits, according to choice, for example depending on the nature of the desired construction material. In some embodiments large particles may be used, e.g. aggregates comprising particles of cm or mm size, e.g. gravels, stones, crushed rocks etc. Thus the size range of particles may in a representative range be from 1 μηη, or 10 μηη, to 5 cm, or more. In some embodiments the particulate starting material may consist of, or at least essentially consist of, small particles, in particular particles on a μηη scale. In other words, the particles may be between, or in the range of 1 μηη and 1 mm (1000 μηη) in size, for instance between, or in the range of, 100 and 1000 μηι or 100 and 500 μπι, for example 200 and 400 μπι, or 200 and 300 μηι. Sand may for example comprise particles in this size range. Any commercially- available sand product may be used, for example sand typically used in cement

manufacture. Large size particles may however be, for instance, between 1 mm and 2 mm in size, or have a wider range in size, for instance between 100 μηη and 2 mm. By size here is meant the length of the longest dimension of the particle; if the particle is a sphere, its size is its diameter.

The CaC0 ₃ which is used in the method of the invention may be provided in any form. Thus, it may be any rock or stone comprising or based on CaC0 ₃ . For instance, it may be in any crystal or polymorphic form: calcite, aragonite or vaterite. The CaC0 ₃ may be obtained from any suitable source rock, i.e. a rock which is predominantly formed from, or at least contains, CaC0 ₃ . In particular, it may be any carbonate rock composed predominantly of calcite or aragonite (this includes rocks where CaC0 ₃ is the main ingredient. Useful source rocks of the invention include marble and various forms of limestone, such as tufa and travertine, and chalk. Chalk may be viewed as a type of limestone, typically as a soft and white form of limestone, composed of the calcite form of CaC0 ₃ , but geologically speaking it is a distinct type of carbonate rock, specifically a sedimentary calcium-bearing rock. In engineering terms, the term chalk is also used to refer to a crushed limestone.

Preferably, the CaC0 ₃ used in the method is in the form of limestone or chalk. A CaC0 ₃ - containing preparation is any preparation or composition containing the CaC0 ₃ , i.e.

containing a CaC0 ₃ material. Thus, it may be a suspension of the CaC0 ₃ in a medium, generally an aqueous medium, suitable for carrying out the enzymatic steps (i) and (iii). The preparation may contain one or more of the other reagents and components for carrying out the method.

When CaC0 ₃ is dissolved in acid, the dissolution occurs at the surface of the CaC0 ₃ , where the CaC0 ₃ is in contact with the acid. The rate of dissolution of CaC0 ₃ will therefore be increased if the surface area of the CaC0 ₃ exposed to the acid is increased, particularly if the ratio of the surface area of the CaC0 ₃ to its volume is increased. The CaC0 ₃ dissolved in acid may therefore be in the form of a particulate material, including in particular a powder. A CaC0 ₃ powder may be obtained by for instance crushing and/or grinding CaC0 ₃ or a CaC0 ₃ -containing rock.

Preferably the CaC0 ₃ powder consists of, or at least essentially consists of, particles no more than 1 mm and no less than 1 μηη in size. In other words, the particles may be between, or in the range of, 1 μηη and 1 mm in size, for instance between, or in the range of, 1 μηη and 900 μηι, 1 μηη and 800 μηι, 1 μηη and 700 μηι, 1 μηη and 600 μηι, 1 μηη and 500 μηη, 1 μηη and 400 μηη, 1 μηη and 300 μηη, 1 μηη and 200 μηη or 1 μηη and 100 μηη. As for the particulate material described above, by size here is meant the length of the longest dimension of the particle. By enzymatically generating an acid in a CaC0 ₃ -containing preparation, the pH of the preparation is decreased, dissolving the CaC0 ₃ . As noted above, by CaC0 ₃ -containing preparation is meant a material which comprises CaC0 ₃ (as described above) and may further comprise other substances. For instance, the CaC0 ₃ -containing preparation may comprise the particulate starting material, and/or one or more of the reagents for

enzymatically increasing the pH of the material in step (iii) of the method, e.g. urea.

The acid used to dissolve CaC0 ₃ in the method of the invention is generated enzymatically. That is to say, a non-acid substrate is converted into the acid using enzymes. Suitable substrates are discussed further below. The identity of the acid is otherwise not particularly limited, provided that it is suitable for use with the particulate starting material (i.e. it will not dissolve or chemically react with the particulate starting material) and will not denature the enzyme(s) used to generate an increase in pH later in the method.

Preferably, the enzymatically generated acid is a carboxylic acid. The carboxylic acid may be a monocarboxylic acid (with one carboxyl group), a dicarboxylic acid (with two carboxyl groups), a tricarboxylic acid (with three carboxyl groups) or a polycarboxylic acid (with more than three carboxyl groups), provided it is generated enzymatically. Similarly, a straight-chain or branched-chain carboxylic acid may be used, as may a cyclic or aromatic carboxylic acid, provided it is generated enzymatically. Carboxylic acids may conveniently be enzymatically generated by a process of fermentation. Preferred carboxylic acids which may be enzymatically generated in the method of the invention include acetic acid, formic acid, propionic acid, butyric acid, gluconic acid, succinic acid and lactic acid. For example, lactic acid may be produced by fermentation of glucose, a process whereby glucose is firstly converted to pyruvate by glycolysis, and the pyruvate is then converted to lactic acid by lactate dehydrogenase. The skilled individual will be aware of many other carboxylic acids that may be enzymatically produced, and how these other carboxylic acids may be enzymatically produced. Combinations of acids may be used, for example lactic acid and acetic acid. In particular, where a microorganism is used more than one acid may be produced by the organism. In some embodiments the microorganism may be selected or engineered to produce only one, or predominantly only one, carboxylic acid, for example lactic acid. Isolates have been obtained which produce only lactic acid.

The enzymatic generation of an acid may be performed using a microorganism, or using a microbial extract which contains enzymes (an enzyme-containing microbial extract). When a microorganism is used to perform the enzymatic generation of the acid, the microorganism is present or included in the CaC0 ₃ -containing preparation or is added to it to initiate acid generation. Nutrients for use by the microorganism (i.e. which it can use for growth, e.g. carbon source, nitrogen source, amino acids, nucleotides etc.) may also be included in the preparation. The nutrients may be included in the preparation in the form of a growth or culture medium. Such media include e.g. LB medium, TY medium etc., though a defined medium may be preferable such that the metabolism of the microorganism can be more closely controlled. As described in more detail below, a substrate is required for the enzymatic reactions, including for the production of acid. A carbon source, e.g. a sugar such as glucose, provided for growth may also serve as substrate for acid generation, or separate carbon sources and substrates may be added.

Thus, supplements for use by the microorganisms as substrates for acid production may also be included in or added to the CaC0 ₃ -containing mixture. The substrate for acid production may also be a nutrient. In particular, the substrate may be a sugar. Any suitable sugar (i.e. any sugar that can be enzymatically converted into an acid (particularly a carboxylic acid, preferably lactic acid or acetic acid)) may be used. Preferably a six-carbon sugar (i.e. a hexose) is used, such as glucose, galactose or fructose. A disaccharide of six- carbon sugars, such as sucrose or lactose, may also be a suitable substrate, provided that the microorganism performing the conversion of the substrate into acid is able to hydrolyse the disaccharide into its constituent monosaccharides. In one preferred embodiment the substrate for acid production is the monosaccharide glucose.

However, any convenient or desirable carbohydrate or carbohydrate source may be used as the substrate, particularly any source of readily metabolizable, or utilisable, sugars, and this may depend on the enzymes/microorganisms used. Suitable carbohydrates, preferably easy utilizable sugars, may be derived from hydrolysis (e.g. enzymatic hydrolysis) of biomass or other waste or recycled materials, for example cellulose or cellulosic materials (such as from e.g. lignocellulosic biomass). Such materials may be available as sidestreams from agriculture and forestry.

It is also included that microorganisms used in the methods of the invention perform such hydrolysis steps themselves and produce acid from the resulting sugars. Thus, the substrate for acid production may be a direct substrate (such as a sugar) or it may be an indirect substrate, which may itself be metabolised, or enzymatically converted, into a substrate for acid production.

When a microorganism is used to perform the enzymatic generation of acid, the microorganism produces enzymes which catalyse a metabolic pathway whereby the substrate is converted into acid. When a microorganism is used to perform the enzymatic generation of acid, this step (i.e. the generation of acid) may preferably be performed under aerobic conditions. As will be known to the skilled individual, many microorganisms produce carboxylic acids as end products of anaerobic metabolism. It falls under the scope of the invention that the enzymatic production of acid takes place using a microorganism under anaerobic conditions (in which case either a facultative anaerobe or an obligate anaerobe may be used in the process). However, as mentioned above, it is a preferred embodiment of the invention that the enzymatic generation of acid is performed using a microorganism under aerobic conditions. The microorganism used to generate acid under aerobic conditions may be an obligate aerobe or a facultative anaerobe, so long as it produces acid under aerobic conditions.

By aerobic conditions, according to the invention, is meant conditions in which oxygen is present at a level sufficient that a facultative anaerobe (i.e. an organism able to grow both in the presence and absence of molecular oxygen (0 ₂ )) can perform aerobic respiration. For instance, aerobic conditions may be normal atmospheric conditions, i.e. conditions in which the reaction atmosphere contains approximately the same proportion of oxygen as is found in the earth's atmosphere, i.e. approximately 21 % v/v. Normal atmospheric conditions may contain from 15 to 25 % oxygen, such as from 18 to 24 % or from 20 to 22 % oxygen. When the bacterium is cultured in liquid medium under aerobic conditions, the liquid may contain approximately as much dissolved oxygen as an aqueous solution exposed to normal atmospheric conditions (e.g. 1-10 mg/L dissolved oxygen). However, conditions may be considered aerobic even if the amount of dissolved oxygen is less than this, so long as it meets the requirement detailed above (i.e. that sufficient dissolved oxygen is present that a facultative anaerobe can perform aerobic respiration).

The enzymatic generation of the acid may alternatively be performed using an enzyme-containing microbial extract. In this embodiment, a microbial culture is grown, the constituent microbes of which express the enzymes which perform the catalytic steps of a metabolic pathway whereby the substrate is converted into acid. From such a culture an enzyme-containing microbial extract may be obtained. Once a certain level of growth is reached, the microorganisms in the microbial culture are isolated from the medium, e.g. by centrifugation, and lysed (e.g. by sonication or high pressure homogenization, though a skilled individual will be aware of various other methods by which a cell extract can be obtained). The lysate obtained is an enzyme-containing microbial extract, which may be applied to the CaC0 ₃ -containing preparation. In this embodiment, a substrate for acid generation (as described above) will also need to be added to or included in the CaC0 ₃ - containing preparation, which the enzymes in the enzyme-containing microbial extract can convert to acid, but no other nutrients would of course be required. It is more preferable that a microorganism is used to perform the enzymatic generation of acid than that an enzyme- containing microbial extract is used.

The microorganism used to perform the enzymatic generation of acid, or the microorganism from which the enzyme-containing microbial extract is derived, may be a prokaryote, such as a bacterium (by which is meant a eubacterium) or an archaeon, or it may be a eukaryote, such as a fungus, particularly a yeast. Preferably though, the microorganism is a bacterium, most preferably a lactic acid-producing bacterium or an acetic acid-producing bacterium. Such a bacterium may be Gram-positive or Gram-negative.

An individual skilled in the art will be readily able to identify whether a microorganism produces acid. For instance, acid production by a microorganism can be identified by growth of the microorganism on RM-9.5 medium agar (see Examples) at an alkaline pH. Acid- producing microorganisms will alter the colour of the alkaline medium from blue to yellow. The identity of the acid produced can then be identified by e.g. HPLC.

When a microorganism is used to perform the enzymatic generation of acid, the microorganism must be able to produce high levels of acid (preferably lactic acid or acetic acid), in order to dissolve CaC0 ₃ ; they must be able to grow under conditions of high pH and high ionic strength, reflecting the conditions associated with high levels of dissolved limestone. Preferably, the microorganism can also grow and produce acid under conditions of low oxygen availability, and/or are able to sporulate (for ease of handling). Most preferably, the microorganism is able to produce a carboxylic acid (preferably lactic acid or acetic acid) to a concentration of at least 30 mM, in order to dissolve CaC0 ₃ .

Microorganisms able to produce even higher carboxylic acid concentrations are preferred. The concentration of acid produced can be calculated from quantifying analysis by e.g. HPLC.

The inventors of this application were unable to identify any commercially-available species or strains of microorganism having all of the above characteristics, and therefore suitable for use in this aspect of the invention. However, the inventors were able to isolate novel, previously unknown bacterial strains from sites close to a limestone quarry in central Norway, which met the above-described requirements. A large number of such isolates were obtained. Accordingly, the inventors have shown that when an appropriate site or source of microorganisms is selected, that is a site where CaC0 ₃ is present, suitable microorganisms, particularly bacteria, may be isolated, which are suitable for use in the methods of the invention. These strains are believed to be of previously unknown species, but are all related to species of the genus Bacillus. Representative strains have been deposited at the NCI MB with accession numbers as indicated above. In preferred embodiments of the invention, one or more of these strains is used to perform the enzymatic generation of acid, preferably lactic acid and/or acetic acid, by fermentation of a sugar, preferably glucose. Alternatively, an enzyme-containing microbial extract may be derived from one of these strains and used to perform the enzymatic generation of acid.

The enzymatically generated acid dissolves at least part of the CaC0 ₃ in the CaC0 ₃ - containing preparation. That is to say, at least some of the CaC0 ₃ in the preparation is dissolved. It may be that all of the CaC0 ₃ is dissolved in the acid, though this is not necessary, so long as sufficient CaC0 ₃ is dissolved that the Ca ²⁺ concentration in the resultant dissolved calcium carbonate preparation (DCCP) is high enough that sufficient CaC0 ₃ may be eventually precipitated out of the DCCP to bind together the aggregate (the particulate starting material). In particular, where chalk particles are used as the CaC0 ₃ - containing material, the particles may be only partially dissolved, leaving small chalk particles in the DCCP. Although this is not a necessary requirement, the DCCP may in one embodiment be a Ca ²⁺ -satu rated solution, which as herein defined requires that at least 15 g of CaC0 ₃ be dissolved per litre of acid. In such an embodiment sufficient substrate is added to or included in the CaC0 ₃ -containing preparation that the acid is produced to a sufficient concentration or in a sufficient amount to dissolve at least 15 g CaC0 ₃ per litre of acid. Such an amount of substrate may be readily identified by trial and error. However, as indicated above, such a high concentration of Ca ²⁺ ions is not necessary to perform the method of the invention and lower concentrations may be used (thus the amount of acid produced may be less than that required to produce a saturated solution). The amount of acid produced is not critical, as long as it sufficient to dissolve at least part of the CaC0 ₃ in the CaC0 ₃ -containing preparation, to allow cementation to occur. For example the concentration of acid in the DCCP may be much lower, e.g. about 0.02 to 0.05 M.

The DCCP that is obtained is then combined with the particulate starting material. The particulate starting material may be present in the CaC0 ₃ -containing preparation prior to the enzymatic generation of acid. In this instance, the DCCP and the particulate starting material are combined simply by the production of the DCCP by the dissolution of CaC0 ₃ in the acid, i.e. the combining process is passive and does not require an active step of combining performed by an individual. Alternatively, the particulate starting material may not be present in the CaC0 ₃ -containing preparation prior to the generation of acid therein, in which case the DCCP and the particulate starting material must be actively combined following DCCP formation.

The DCCP/particulate starting material mixture is then hardened (or consolidated) by causing the dissolved CaC0 ₃ in the mixture to precipitate, yielding CaC0 ₃ crystals that bind together the particulate starting material to form a composite construction material. The CaC0 ₃ in the mixture is caused to precipitate by an increase in the pH of the mixture. This increase in the pH of the mixture is enzymatically generated.

Like the enzymatic generation of acid earlier in the method, this enzymatic generation of an increase in pH may be performed using a microorganism or an enzyme- containing microbial extract. Any reagent(s) may be used in this pH-increase step, so long as they may be used as substrate(s) for an enzyme-catalysed reaction which results in a pH increase. In a preferred embodiment of the invention, urea is hydrolysed by urease, which produces ammonium and thus drives an increase in pH. Alternatively, other ammonia- generating enzymatic reactions may be employed, or other reactions which result in a pH increase, such as for example reactions which utilise acid as a substrate. It is commonly known that many microorganisms utilize carboxylic acids as carbon source, including strains that are acid producers, and once the substrate for acid production (e.g. the sugar) is depleted, the microorganism continues using the produced acid as alternative C source.

All reagents used for this step of increasing the pH of the mixture may be present in the CaC0 ₃ preparation prior to step (i). For instance, where urea hydrolysis is used to increase the pH of the mixture, the urea may be present in the CaC0 ₃ -containing

preparation prior to the initiation of acid generation.

Alternatively, the reagents for step (iii) (e.g. urea) may only be added after step (i) has been performed. In other words, the DCCP is formed first, combined with the particulate starting material and then the reagents for step (iii) are added. In this instance, the reagents for step (iii) may be added before, during or after step (ii), in other words at any point after the DCCP is formed. In certain embodiments, when the reagents for step (iii) are not present in the CaC0 ₃ -containing preparation during step (i), if the particulate starting material is also not present in the CaC0 ₃ -containing during step (i), undissolved CaC0 ₃ can be removed from the DCCP by e.g. filtration, prior to the combination of the DCCP with the particulate starting material and the reagents of step (iii). However, in certain preferred embodiments of the invention, undissolved CaC0 ₃ is not removed from the DCCP, and instead acts as part of the aggregate which is bound together by the CaC0 ₃ cement, when the CaC0 ₃ precipitates in step (iii) and thus hardens the material. This is inherently the case when the CaC0 ₃ -containing preparation comprises the particulate starting material and/or the reagents for step (iii) prior to and/or during the production of the DCCP.

As described above, the enzymatic generation of an increase in pH may be performed using a microorganism or an enzyme-containing microbial extract, as may be the enzymatic generation of an acid which causes a pH decrease in step (i). However, it may be that neither of these steps is performed using a microorganism or an enzyme-containing microbial extract, instead being performed using (an) isolated and purified or commercially acquired enzyme(s). For instance, if the enzyme-generated increase in pH is caused by hydrolysis of urea by urease, this hydrolysis may be performed using e.g. commercially acquired Jack bean (Canavalia ensiformis) urease (Sigma-Aldrich U7752). However, preferably at least one of the enzymatic generation of an acid which causes a pH decrease in step (i) and the enzymatic generation of an increase in pH in step (iii) is performed using a microorganism or an enzyme-containing microbial extract, and most preferably both of these enzyme-mediated steps are performed using a microorganism or an enzyme-containing microbial extract. The enzymatic generation of an acid which causes a pH decrease in step (i) and the enzymatic generation of an increase in pH in step (iii) may both be performed using a bacterium or an enzyme-containing bacterial extract. Thus, in a preferred embodiment of the invention, the enzyme-generated increase in pH of step (iii) is performed using a microorganism (preferably a bacterium) which produces urease, or an enzyme-containing microbial extract (preferably an enzyme-containing bacterial extract) which is derived from a urease-producing bacterium, and which therefore contains urease, to hydrolyse urea, thus increasing the pH of the mixture.

It will be straightforward for the skilled person to determine whether a microorganism produces urease. For instance, microorganisms which produce urease can be identified by growing strains on Christensen urea medium agar at neutral pH (see Examples). Strains which produce urea will cause the medium to change colour from yellow to red.

In a particularly preferred embodiment of the invention, the generation of acid which causes the pH decrease in step (i) and the enzyme-generated pH increase in step (iii) are both performed using the same microorganism. In such an embodiment, the microorganism first produces a carboxylic acid by e.g. fermentation of a sugar, reducing the pH of the mixture and dissolving at least part of the CaC0 ₃ in the CaC0 ₃ -preparation. Then, when the sugar has been used up (i.e. all or substantially all of the sugar has been converted to acid), the microorganism will start to produce urease, which hydrolyses urea, forming ammonium and thus increasing the pH of the solution.

In one embodiment the urea may be present in the CaC0 ₃ -containing preparation in step (i) of the method. In certain such embodiments urease production by a microorganism may be controlled, e.g. induced. For example it may be induced upon sugar depletion, or upon generation of sufficient acid. Alternatively, the urea may not be present in the CaC0 ₃ - containing preparation in step (i) of the method. In this case, the urea may be added to the DCCP after it has been formed. In such a case, the addition of urea to the microorganism- containing DCCP may stimulate the microorganisms to produce urease. It is not necessary for all the substrate for acid production to be consumed, as long a sufficient acid is generated to dissolve sufficient CaC0 ₃ for cementation to occur.

As previously described, no commercially-available microbial strain was identified by the inventors as suitable for use in the enzymatic generation of acid to dissolve CaC0 ₃ in step (i) of this invention, but it was found possible to isolate suitable strains from a limestone- containing environment. Five such representative suitable strains, isolated from sites close to a limestone quarry in Norway, have been deposited at the NCI MB. Of these strains, one (UP-009, closely related to Bacillus weihenstephanensis) was also able to produce urease.

Thus, in a preferred embodiment, the bacterium of strain UP-009, with the NCIMB accession number 42600, is used both to generate acid to dissolve the CaC0 ₃ in the CaC0 ₃ -containing preparation of step (i), and to generate an increase in the pH of the mixture in step (iii) by the use of urease to hydrolyse urea. Alternatively, one of the other deposited lactic acid-producing strains of bacteria (as defined above) may be engineered to produce urease, such that the strain can be used both in the production of lactic acid and for the production of urease. Methods for the engineering of bacterial species to produce exogenous proteins, including the introduction of metabolic pathways (i.e. genes encoding enzymes to perform one or more reactions in concert) are well known in the art of microbiology. Further, commercial urease-producing strains may also be used, e.g.

Sporosarcina pasteurii DSM33, and as further described below.

In other embodiments, microorganisms (preferably bacteria) or enzyme-containing microbial extracts (preferably enzyme-containing bacterial extracts) are used both to generate the acid which causes the pH decrease in step (i) and to perform the enzyme- generated pH increase in step (iii), but different microorganisms/enzyme-containing microbial extracts are used for each purpose. In other words, a first microorganism or enzyme-containing microbial extract is used to generate the acid which decreases the pH in step (i) of the method, and a second microorganism or enzyme-containing microbial extract is used to perform the enzymatic generation of an increase in pH in step (iii). When the enzymatic generation of an increase in pH in step (iii) is achieved by hydrolysis of urea by urease, the second microorganism may be any urease-producing microorganism. Its identity is not, therefore, particularly limited. Suitable species of microorganism can be found, for instance, in US 8,182,604, US 8,728,365 and US 8,951 ,786, and include e.g. Sporosarcina pasteurii, Sporosarcina ureae, Proteus vulgaris, Bacillus sphaericus, Myxococcus xanthus, Proteus mirabilis and Helicobacter pylori. Alternatively, the microorganism may be a strain which does not natively produce urease but has been engineered to do so. It will be straightforward for the skilled individual to engineer a microorganism to render it urease- producing.

In certain embodiments of the method, the pH increase of step (iii) is not generated by the enzymatic processing of an additional reagent. Rather, the pH increase of step (iii) is generated by the metabolic removal of acid by a microorganism. In other words a microorganism, which may or may not be the same microorganism as was used to generate the acid in step (i), metabolises the acid and thus removes it from the mixture, causing the pH of the mixture to increase. As indicated above, it is known that microorganisms may degrade acids that they have themselves produced.

The steps of the method may be conducted in any convenient way, depending for example on the nature of the construction material it is intended to produce. Thus the various ingredients (reaction components) may simply be contacted (e.g. added together) and allowed to react. Thus, microorganism(s), CaC0 ₃ , starting particulate material, nutrients and substrates for one or both of the enzymatic reactions may be provided together, optionally mixed, and the reactions of dissolution and precipitation of the CaC0 ₃ may be allowed to occur, e.g. without any (further) mixing. Alternatively, the reaction mixture may be mixed during the dissolution step (step (i)), but without mixing during precipitation and consolidation (step (iii)). The reactions may also be performed in a wide temperature range; including at room or ambient temperature, including outdoor temperatures, for example at construction sites. Heating of the reaction mixture(s) is not required, and "incubation" of the microorganisms may simply take place by standing, or holding, of the reaction mixtures for a period of time. Thus, for example, a temperature range of 10 to 40 °C may be used, or possibly even lower temperatures.

The step of generating an increase in the pH of the mixture in step (iii), thereby causing CaC0 ₃ to precipitate and to bind together the particulate material (i.e. to

consolidate), may be performed in a mould, thus allowing the size and shape of the resultant construction material to be determined. In some embodiments, depending on when the materials in the mixture are combined (as described above), the entirety of the method (i.e. steps (i), (ii) and (iii)) may be performed in the mould. The consolidation, or hardening, of the construction material may be performed over a period of several hours, e.g. at least 5 hours, at least 10 hours, at least 20 hours, at least 50 hours or at least 100 hours. Optimal timespans for the method may be determined by the skilled person.

The relative amounts of CaC0 ₃ , particulate starting material and, where relevant, urea used in the method may be varied considerably. As described above, in one

embodiment the DCCP may be a saturated Ca ²⁺ solution, containing at least 15 g/L CaC0 ₃ , preferably at least 20, 25 or 30 g/L CaC0 ₃ . However, in other embodiments the DCCP may contain lower concentrations, or amounts, of CaC0 _3. In some representative embodiments, the particulate starting material may be combined with the DCCP at a concentration of between, or in the range of, 1.5 kg/L and 4 kg/L, preferably between, or in the range of, 2 kg/L and 3 kg/L, and urea may be combined with the DCCP (e.g. included or added to the CaC0 ₃ -containing preparation) to give a resultant concentration preferably between, or in the range of, 50 mM and 500 mM, most preferably between, or in the range of, 100 mM and 200 mM. In other embodiments, lower concentrations may be used.

In order to aid or improve the hardening of the above-described mixture into a composite material, it may be advantageous for the mixture to be compacted prior to initiation of the hardening (consolidation) (precipitation and binding). For instance, the

CaC0 ₃ -containing preparation, and any other reagents pre-mixed with it prior to step (i), may be compacted in a mould prior to its combination with the DCCP. Following hardening, it may also be advantageous to wash by-products and suchlike out of the construction material using water, and/or to dry the material by heating, e.g. in an oven, for between, or in the range of, e.g. 1 and 5 hours at a temperature e.g. between, or in the range of, 50 and 80°C.

The invention also provides a construction material formed by the method of the invention, as described above. Such a material consists of aggregate particles, e.g. sand or stone such as gravel, bound together by precipitated CaC0 ₃ crystals. It is these crystals which form the cement. As described above, the construction material may also comprise particles of undissolved CaC0 ₃ (e.g. limestone or chalk), which when present is also bound together by precipitated CaC0 ₃ crystals. The construction material will also comprise within it the anions of the acid used to dissolve CaC0 ₃ in the first step of the method. When a carboxylic acid is used for this purpose, the anions are the organic carboxylate ions, e.g. if lactic acid is used lactate molecules will be present in the construction material.

The invention further provides a method of forming a cement from CaC0 ₃ , said method comprising:

(i) enzymatically generating an acid in a CaC0 ₃ -containing preparation to decrease the pH of said preparation, thereby dissolving at least a part of the CaC0 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 CaC0 ₃ to precipitate to form a cement, wherein the precipitated CaC0 ₃ is able to function as a cement to bind together particulate or other construction materials;

wherein where the method comprises step (ii) the CaC0 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 CaC0 ₃ -containing preparation, or may be added during or after any one of steps (i) or (ii), or in step (iii).

In this method, the enzymatic generation of an acid, the acid, the CaC0 ₃ -containing preparation, the particulate starting material, the DCCP and the enzymatic generation of an increase in pH are all as defined above.

The CaC0 ₃ cement may be used to bind together construction materials, particularly masonry units such as bricks, slabs, blocks, concrete units etc. The DCCP may be combined with a particulate material, preferably sand, to yield a mortar or mortar-like material which may be used to bind together construction materials. Alternatively, the CaC0 ₃ cement may be used alone to bind together the construction materials, i.e. without containing a particulate starting material or aggregate. By "bind together" is meant that the bound construction materials are stuck together and cannot be easily separated, such as when bricks are bound together by mortar in bricklaying. In particularly preferred

embodiments, the construction materials are made using the method of the first aspect of the invention (i.e. they consist of a construction material of the invention). In a further aspect, the invention provides a method of connecting construction materials together, said method comprising applying a cement prepared according to the paragraphs immediately above to at least one construction material unit and placing it together with at least one other construction material unit. In this embodiment the cement effectively functions as a mortar. The construction material units may be bricks, slabs, blocks, etc. Most preferably, the construction material units consist of a construction material made according to the method of the first aspect of the invention (i.e. the construction material units consist of a construction material of the invention).

The method of connecting construction materials together may be used in any form of construction. For instance, it may be used in bricklaying, paving, roofing, road building, etc. When construction materials are connected according to this aspect of the invention, they are bound together as described above.

The invention further provides a bacterium suitable for use in the methods of the invention, particularly in the production of acid in the methods of the invention, said bacterium being:

(i) alkaliphilic;

(ii) able to produce acid under aerobic conditions at a pH of at least 8.5

(particularly in the presence of CaC0 ₃ , e.g. limestone, and more particularly also under low oxygen conditions); and

(iii) able to dissolve CaC0 ₃ to produce Ca ²⁺ ions, (particularly in the presence of glucose and CaC0 ₃ powder)

In particular the bacterium may be a bacterium (i.e. a species or strain) of the genus Bacillus or Jeotgalibacillus, or any other Bacillus-related species, as long as it has the properties indicated above.

The ability to produce acid and to dissolve CaC0 ₃ to produce Ca ²⁺ ions may be determined as described further below. In particular, the bacterium may be able to dissolve CaC0 ₃ to produce Ca ²⁺ ions at a temperature range of e.g. 10 to 40°C or 10 to 35°C, more particularly 15 to 30°C, or more generally at a temperature below 30°C, or at around 20°C. Further, the bacterium may be able to dissolve CaC0 ₃ to produce Ca ²⁺ ions when provided with glucose and CaC0 ₃ and allowed to stand, without mixing. Thus, the bacteria may be able to produce acid and dissolve the CaC0 ₃ when incubated, or allowed to grow, in the presence of glucose and CaC0 ₃ , but also simply when contacted with, or provided with glucose and CaC0 ₃ (i.e. without necessarily been placed under growth conditions, or without incubation with heating and/or aeration).

Preferably, the bacterium is capable of forming spores.

A spore is a metabolically inactive, dormant form of a bacterium. Such spores are easy to handle as they require no nutrients and are largely resistant to changes in temperature and atmospheric conditions. The bacterium capable of forming spores is in particular capable of forming endospores. Such bacteria particularly include members of the phylum "Firmicutes", such as Clostridia and Bacilli. Bacteria capable of forming spores may be identified by e.g. culturing the bacteria in nutrient-poor medium, heating the culture at high temperature for a period of time sufficient to kill vegetative cells (e.g. at 80°C to 100°C for 10 to 30 mins) and then plating the heated culture onto suitable growth medium (which may for example be a rich or a defined or minimal medium). Resultant bacterial colonies can be assumed to be of spore-forming bacteria, the colonies derived from germination of spores on the growth medium. The skilled individual will be aware of other methods of spore isolation, such as culture incubation in ethanol or suchlike to kill vegetative cells.

By alkaliphilic is meant an organism (in this case a bacterium) capable of growth under alkaline conditions. Such a bacterium is able to grow at a pH of at least 8, preferably at least 8.5 or at least 9. The bacterium is also able to produce acid at a pH of at least 8.5, preferably at least 9. In particular, the bacterium is able to produce acid in the presence of CaC0 ₃ , the presence of which may cause a high pH in a mixture. The acid produced by the bacterium is preferably a carboxylic acid, such as acetic acid, formic acid, propionic acid, butyric acid, gluconic acid, succinic acid or lactic acid. Such an acid may be produced by fermentation, preferably fermentation of a sugar, particularly a hexose sugar such as glucose, fructose or galactose.

More particularly, the bacterium of the invention is able to produce acid at a pH of at least 8.5 under aerobic conditions. Aerobic conditions according to the invention are defined above. Furthermore, the skilled person will understand the term aerobic conditions in relation to bacterial culturing. Fundamentally, aerobic conditions are the opposite of anaerobic conditions. At a basic level, if, in order for a bacterium to produce acid, the amount of oxygen in the bacterial growth conditions must be reduced relative to atmospheric conditions, the bacterium can be understood as being unable to produce acid under aerobic conditions. Thus, according to the invention, a bacterium able to produce acid under aerobic conditions is able to produce acid under atmospheric conditions or under conditions whereby the amount of oxygen present has not been reduced by human intervention.

Though bacteria of the invention are able to produce acid under aerobic conditions, this does not preclude them being also able to produce acid under low oxygen conditions or anaerobic conditions also. The bacterium of the invention may thus be an obligate aerobe or a facultative anaerobe, but may not be an obligate anaerobe. The bacterium may be able to produce acid under anaerobic conditions and aerobic conditions. Preferably, the bacterium of the invention is able to produce acid under low-oxygen conditions.

By "low-oxygen conditions" is meant conditions with less oxygen than under normal growth conditions. This means not only less oxygen than under normal atmospheric conditions, but also less dissolved oxygen than would ordinarily be present in aqueous solution. However, it does not mean that there is no oxygen present, i.e. low-oxygen conditions are aerobic conditions. By anaerobic conditions is meant conditions under which no or essentially no oxygen is present. Such conditions may be identified using e.g. a redox indicator, such as resazurin. A bacterium able to grow and produce acid under anaerobic conditions may thus be identified by testing its ability to grow and produce acid under such conditions (e.g. in an anaerobic cabinet or chamber). A bacterium able to grow and produce acid under low-oxygen conditions may be identified by testing its ability to grow and produce acid under conditions of reduced oxygen, or simply in a culture which is not properly aerated (e.g. in a liquid culture grown in a container, such as a flask or bottle, which is largely full of liquid (such that minimal environmental oxygen is present), sealed and is incubated without shaking). The skilled individual will be able to identify conditions whereby a culture is not properly aerated without difficulty, e.g. by simply growing a culture without the conditions usually used to ensure proper aeration of a bacterial culture.

Alkaliphilic, acid-producing bacterial strains can be identified as follows:

Strains are plated onto agar made up with RM-9.5 medium (1.0 g/L yeast extract (Oxoid), 3.0 g/L peptone (Oxoid), 10.0 g/L glucose, 5.0 g/L NaCI, 2.5 ml/L thymol blue solution (1 % w/v), 15 g/L agar; pH 9.5 (the pH was adjusted to 9.5 using 1.2 g/L Na ₂ C0 ₃ )). Strains able to grow on this medium are alkaliphilic, and acid production can be identified by a change in the colour of the medium from blue to yellow, caused by the associated decrease in pH. However, the skilled individual will be aware of other methods by which alkaliphilic, acid- producing bacteria may be identified. As described, the above test may be performed under aerobic conditions (including low-oxygen conditions) and anaerobic conditions to identify bacteria able to produce acid under the respective growth condition.

The bacterium is able to dissolve CaC0 ₃ to produce Ca ²⁺ ions. This dissolution of

CaC0 ₃ is achieved by the acid produced by the bacterium. The bacterium is thus able to dissolve CaC0 ₃ particularly under conditions where it will produce acid, such as when a fermentable sugar (e.g. glucose) is present. The dissolution of CaC0 ₃ occurs when a mineral consisting of CaC0 ₃ , or which at least comprises CaC0 ₃ at its surface, is contacted by an acid. Maximal dissolution of CaC0 ₃ can thus be achieved by maximising the CaC0 ₃ surface area which is in contact with the acid. For any given amount of CaC0 ₃ this can thus be achieved by providing it in a particulate form, e.g. a powder.

Accordingly, an assay for CaC0 ₃ dissolution may be performed as follows: CaC0 ₃ powder is mixed with RM-9.5 medium containing 30 g/L glucose and 10 g/L CaC0 ₃ to which a volume of bacterial culture has been added (for example 15 g CaC03 powder in a 50 ml reaction tube to which a mixture of 120 μΙ bacterial culture and 6ml of medium containing the glucose is added). The mixture is incubated standing and covered with parafilm at room temperature and monitored for acid and Ca ²⁺ production using pH and Ca ²⁺ electrodes present in the culture vessel (e.g. 50 ml tube) over a period of time, e.g. from 6 to 24 hours, e.g. from 8, 10, 12, 14 or 16 hours to 18, 20, 21 , 22 or 24 hours.

In certain embodiments of the invention, the bacterium is strain AP-004, which has the NCIMB accession number 42596, strain AP-006, which has the NCIMB accession number 42597, strain AP-029, which has the NCIMB accession number 42598, strain AP-044, which has the NCIMB accession number 42599, or strain UP-009, which has the NCIMB accession number 42600. As described above, these strains are all spore-forming, alkaliphilic, acid-producing strains, which are able to grow and produce acid under low oxygen conditions and which are able to dissolve CaC0 ₃ to produce Ca ²⁺ ions using the acid they produce. The bacterium may alternatively be a derivative of one of these strains, e.g. a mutant of one of these strains, or a modified variant or derivative of one of these strains, e.g. which carries one or more non-native plasmids.

In a most preferred embodiment, the bacterium is further able to produce urease. Such a bacterium may be identified, for instance, by growth on Christensen urea medium (1.0 g/L peptone, 10 g/L glucose, 5.0 g/L NaCI, 2.0 g/L KH ₂ P0 ₄ , 20.0 g/L urea, 0.012 g/L phenol red, 15 g/L agar; pH 6.5). Bacterial production of urease causes an increase in pH which changes the colour of the medium from yellow to red. The skilled individual will be aware of other methods of detecting urease production by a bacterium. In a preferred embodiment, the bacterium is strain UP-009, which has the NCIMB accession number 42600. As described above, UP-009 is a urease-positive, acid-producing bacterium.

Alternatively, the bacterium may be a derivative of UP-009, such as a mutant strain or a strain thereof carrying a plasmid. In another embodiment, the bacterium may be an acid- producing bacterium as described above (such as AP-029, AP-044, AP-004 or AP-006) which has been engineered so as to produce urease, or a derivative of such a strain (e.g. a mutant or plasmid-carrying variant thereof).

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

Figure 1 shows changes of pH and Ca ²⁺ ion concentration over dissolution time.

Figure 2 shows a consolidated sample of sand20 biocement.

Figure 3 shows SEM images of sand20 biocement.

Figure 4 shows an SEM image of sand40 biocement.

Figure 5 shows SEM images of (a) 90%sand20 and (b) 50%sand20.

Figure 6 shows elemental mapping of sand20 sample using EDS on SEM.

Figure 7 XRD patterns of sand, limestone and the sand20 biocement sample.

Figure 8 shows the compressive strength of consolidated samples produced using different numbers of reagent injections and having different limestone content. Figure 9 shows pH (A) and Ca 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.

Figure 10 shows the results of two chalk dissolution experiments using B. safanensis strain AP-004. Changes in the pH and Ca 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.

Figure 1 1 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.

Figure 12 shows a picture of the setup for the consolidation experiments.

Figure 13 shows the change over time in pH and Ca 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.

Figure 14 shows two consolidated sand samples made using the method of the invention. The scales shown are in centimetres.

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

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

Figure 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

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 Milj0kalk AS (Norway), with a density of 2.7 kg/dm ³ and particle size ranging from 1-200 μηη. Preparation of Reagent

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 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 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 ²⁺ ion concentration was measured through atomic absorption spectroscopy (Perkin Elmer AAnalyst 400, USA).

Preparation of Bio-cement

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.

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 1 : Processing conditions and material designation

Grains Number of

Sample code Urease

Sand (%) Limestone (%) 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)

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

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-Κα radiation with a wavelength of 0.154 nm.

Mechanical Test

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.

CaC0 ₃ Content Measurement

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 (HCI) at 30°C and stirred with a magnetic stirrer.

Changes in pH and Ca ²⁺ ion concentration were monitored by pH meter and ISE until constant values were reached, in order to ensure a full dissolution of the CaC0 ₃ crystals. The remaining solids were filtered out of the solutions using filter paper, washed several times with distilled water, dried and re-weighed. CaC0 ₃ content was determined using the following equation:

_ _{r n} , ~. Weiqht o f samples before acid diqest ion

CaCOr, content (wt %) = — -— -—

Weight of samples after acid digesti ion

(1 ) Porosity Measurement

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

During the preparation of CRS, changes of pH and Ca ²⁺ ion concentration were monitored throughout the dissolution process as shown in Figure 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 ⁺ ) ions from the lactic acid during the dissolution process, as described in the following equation: CaC0 ₃ + Lactic acid (HLac)→ Ca ²⁺ + HC0 ₃ ^" + Lac ^"

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 ²⁺ ions in the solution was measured as a function of time, as illustrated in Figure 1. The Ca 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 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 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 ²⁺ ions, causing the chelated calcium to no longer be detected by ISE. During the dissolution of limestone, Ca ²⁺ ion concentration increased in the solution, but at the same time chelation of calcium occurred. When the solution reached saturation point, the Ca ²⁺ ion concentration in the solution became constant. Meanwhile, more and more Ca ²⁺ ions were chelated by lactate and could thus no longer be detected by ISE. Hence, the measured Ca 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 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 ion concentration of 0.065 mol/l.

Morphological Studies

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

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.

Figure 3 shows SEM images of the consolidated sand sample, sand20. From Figure 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 Figures 3B and 3C, with a diameter ranging from 20-100 μηη. From a close- up of the spherical crystal (Figure 3D) at higher magnification, it can be observed that the spheres appeared to be spherical aggregates of smaller sub-units of around 1-5 μηη. Similar crystal morphology was also observed in the sand40 samples, as shown in Figure 4. The XRD results (which will be discussed further later) confirm that these precipitated crystals are calcite, the most stable polymorph of CaC0 ₃ . Other researchers have previously reported similar CaC0 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.

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 (Figure 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 Figure 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

Elements present in the sand20 sample were identified using EDS mapping. Distribution of different elements in the framed area is illustrated in Figure 6. Big grains that contained silicone (Si) and oxygen (O) correspond to sand (quartz, Si0 ₂ ). The spherical crystal aggregates contained calcium (Ca), carbon (C) and O and are expected to be CaC0 ₃ . This is further confirmed by using XRD analysis. Figure 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 2Θ of 29.3°, which is known to be calcite (104). A similar calcite peak is also observed in the sand20 sample (marked as ^* in Figure 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

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.

Results from the uniaxial compression test are presented in Figure 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 CaC0 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 CaC0 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 CaC0 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

Soil samples were collected near an open quarry for CaC0 ₃ in Tromsdalen, a side-valley to Verdalen, Nord-Trandelag, 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 NaCI, 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 ₂ C0 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 Protocol - Benita Brink:

httpi//www.microbelibrarv.orq/librarv/laboratorv+test/322 3-urease est-protocol; 1.0 g/L peptone (Oxoid), 10 g/L glucose, 5.0 g/L NaCI, 2.0 g/L KH ₂ P0 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.

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

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 ₂ S0 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

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 μΙ 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 CaC0 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.

For limestone dissolution experiments, 15 g of CaC0 ₃ powder was mixed with 6 mL RM-9.5 medium containing 30 g/L glucose and 10 g/L CaC0 ₃ in 50 mL tubes, to which 120 μΙ bacterial culture was added. A respective setup without bacterial culture added was used as a negative control. Calibrated pH and Ca 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

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.

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 CaC0 ₃ powder in the medium and at low oxygenation. In addition, isolates were taxonomically classified using 16S rRNA gene sequence analysis.

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.

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 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.

Limestone Dissolution Experiments

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 ²⁺ from the limestone powder. Both pH and Ca ²⁺

concentration evolution profiles were significantly above background level, determined by a negative control reaction, containing medium only (Figure 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 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 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 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

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 CaC0 ₃ (rich medium (RM): glucose ^■ H ₂ 0 33 g/L, NaCI 5 g/L, Na ₂ C0 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 CaC0 ₃ added after pH adjustment) in a beaker, and mixed well using a magnetic stirrer.

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 ²⁺ ion concentration were monitored using pH/ISE electrodes.

Results and Discussion

The results of two experiments are presented. The pHs of the solutions decreased and the Ca 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 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

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.

A chemically defined medium based on pure chemical compounds such as glucose, mineral salts (NH ₄ CI, K ₂ HP0 ₄ , MgS0 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

Four acid-producing (AP) isolates were selected for the study. They have been assigned to species as follows: AP-004 - Bacillus sp. closely related to B. pumilus and B. safanensis; AP-006 - Bacillus sp. closely related to B. simplex; AP-029 - B. pumilus; and AP-044 - B. Iicheniformis. 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

Defined Medium 1 (DM1 ): Taurine 12.5 g/L; glucose- H ₂ 0 1 1.0 g/L; NH ₄ CI 2.3 g/L; trisodium citrate ^■ 2H ₂ 0 1.0 g/L; K ₂ HP0 ₄ , 0.70 g/L; MgS0 ₄ ^■ 7H ₂ 0 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 ₂ 0 1 1.0 g/L; NH ₄ CI 2.3 g/L;

K ₂ HP0 ₄ 0.70 g/L; MgS0 ₄ ^■ 7H ₂ 0 0.40 g/L; TMS1 5 ml/L; WVS 10 ml/L; trisodium citrate ^■ 2 H ₂ 0 0 g/L, 0.2 g/L or 1.0 g/L. One part medium was mixed with two parts chalk (CaC0 ₃ ).

Semi-Defined pasteurii Medium (SDM): Urea 20 g/L; taurine 6.25 g/L; glucose ^■ H ₂ 0 5.5 g/L; NH ₄ CI 2.3 g/L; trisodium citrate ^■ 2H ₂ 0 1.0 g/L; vitamin-reduced casamino acids 1.0 g/L; K ₂ HP0 ₄ 0.70 g/L; MgS0 ₄ ^■ 7H ₂ 0 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; NaCI 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 ₂ 0 55 g/L; urea 20 g/L; vitamin-reduced casamino acids 10 g/L; TMS 1-pasfei/n/ 5 ml/L.

WVS: Folic acid 20 mg/L; pyridoxine ^■ HCI 10 mg/L; thiamine ^■ HCI 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 : FeS0 ₄ ^■ 7H ₂ 0 5.0 g/L; ZnS0 ₄ ^■ 7H ₂ 0 0.44 g/L; CuS0 ₄ ^■ 5H ₂ 0 0.39 g/L; MnCI ₂ ^■ 2H ₂ 0 0.15 g/L; CoS0 ₄ ^■ 7H ₂ 0 20 mg/L; Na ₂ Mo0 ₄ ^■ 2H ₂ 0 10 mg/L; cone. HCI 50 ml/L.

TMS'\ -pasteurii: As above for TMS1 with additional 0.5 g/L NiCI ₂ ^■ 6H ₂ 0. Experimental Procedures

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.

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.

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

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 CaCI 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.

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 (N0 ₃ ^" ) instead of ammonium (NH ₄ ⁺ ) 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

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. 1 1 ). Re-Al alization 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 μΙ TMS1 -pasfei/r// 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. 1 1 ). The pH in the suspension remained high for several weeks (Table 3).

Conclusions

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.

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

Materials and Methods

Materials

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 Milj0kalk AS, with a density of 2.7 kg/dm ³ and particle size ranging from 1 -200 μηη.

Limestone Dissolution

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 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 ²⁺ ion concentration were observed. Next, the remaining limestone particles and bacteria were filtered out using a 0.22 μηη vacuum filtration set.

Then, the filtrate was used as the calcium source for the CaC0 ₃ precipitation. The filtrate is referred to herein as the dissolved chalk solution (DCS).

Preparation of Reagent

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

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

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

Through the consolidation experiment, two consolidated sand samples were successfully produced, which are shown in Figure 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.

The consolidated sand samples were viewed under an SEM. From Figures 15A and 16A it can be clearly seen that there are CaC0 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 CaC0 ₃ crystals are observed, as shown in Figures

15B, 16B and 16C. Most formed a layered structure, in which layers of CaC0 ₃ crystals stack on top of each other. Spherical crystals were also detected in the second consolidated sample (Figure 16C). Bacteria are also seen on the surface of sand grains or the

precipitated CaC0 ₃ (Figures 15C and 16A).

Elements present in the consolidated sample were identified using EDS. EDS scans from the selected crystals, presented in Figure 17, show a high amount of calcium (Ca), which is very likely to be CaC0 ₃ . The gold peak seen in the EDS spectrums is due to gold coating of samples before SEM imaging. Conclusions

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.