阅读说明：本技术 包含至少一种微生物的组合物及其用途 (Composition comprising at least one microorganism and use thereof ) 是由 T·米勒 S·欣特迈尔 J·黑尔里格尔 S-C·马腾斯-克鲁克 I·哈斯 L·法尔克 S 于 2019-07-08 设计创作，主要内容包括：本发明涉及一种组合物,所述组合物包含能够在碱性培养基中形成磷酸盐或碳酸盐沉淀物的至少一种微生物和至少一种钙源,其中所述组合物特征在于其包含至少一种硅化合物,所述硅化合物具有至少一个Si原子、至少一个C原子和至少一个H原子,还涉及一种生产建筑产品的方法,其中相应的组合为被用于所述生产中。(The invention relates to a composition comprising at least one microorganism capable of forming a phosphate or carbonate precipitate in an alkaline medium and at least one calcium source, wherein the composition is characterized in that it comprises at least one silicon compound having at least one Si atom, at least one C atom and at least one H atom, and to a method for producing a building product, wherein the respective combination is used in said production.)

1. A composition comprising at least one microorganism capable of forming a phosphate or carbonate precipitate in an alkaline medium and optionally at least one source of calcium, characterized in that the composition comprises at least one silicon compound comprising at least one Si atom, at least one C atom and at least one H atom.

2. Composition according to claim 1, characterized in that the microorganism is selected from the group consisting of bacteria, freeze-dried bacteria and bacterial spores, preferably bacterial spores.

3. Composition according to claim 1 or 2, characterized in that the microorganism is selected from bacterial spores or bacteria of the genera Enterococcus (Enterococcus), nitrobacter (diaphromobacter), lysinibacillus (lysinibacillus), Planococcus (Planococcus), Bacillus (Bacillus), Proteus (Proteus) or sarcina (Sporosarcina), preferably from the group comprising the following microorganisms: bacillus cohnii (Bacillus cohnii), Bacillus megaterium (Bacillus megaterium), Bacillus pasteurii (Bacillus pasteurii), Bacillus alkalophilus (Bacillus pseudobacterius), Bacillus sphaericus (Bacillus sphaericus), Bacillus species (Bacillus spp.), Bacillus subtilis (Bacillus subtilis), Bacillus Proteus vulgaris (Bacillus vulgaris), Bacillus licheniformis (Bacillus licheniformis), Flavobacterium nitrobacter sp (Diphrobacter sp), Enterococcus faecalis (Enterococcus faecalis), Bacillus sphaericus (Lysinbacter sphaericus), Bacillus Proteus vulgaris and Bacillus sarcina (Sporosarcina pasteurii), particularly preferably Bacillus subtilis or Bacillus subtilis.

4. Composition according to at least one of the preceding claims, characterized in that the weight ratio of microorganisms capable of forming phosphate or carbonate precipitates in alkaline medium to silicon compounds comprising at least one Si atom, at least one C atom and at least one H atom is from 100:1 to 1:100, preferably from 10:1 to 1: 2.

5. Composition according to at least one of the preceding claims, characterized in that the mass fraction of microorganisms capable of forming phosphate or carbonate precipitates in an alkaline medium is from 0.0001 to 10% by weight, preferably from 0.001 to 5% by weight and particularly preferably from 0.002 to 3% by weight, based on the total mass of the composition.

6. Composition according to at least one of the preceding claims, characterized in that it contains at least one mineral building material, preferably cement.

7. Composition according to at least one of the preceding claims, characterized in that it contains at least one enrichment medium (growth medium) for enriching the microorganisms, preferably tryptic soy broth (casein-soy-peptone medium).

8. Composition according to at least one of the preceding claims, characterized in that the at least one silicon compound comprising at least one Si atom, at least one C atom and at least one H atom has hydrophobic character.

9. Composition according to at least one of the preceding claims, characterized in that the at least one silicon compound comprising at least one Si atom, at least one C atom and at least one H atom is chosen from silane compounds, siloxane compounds, silicone oils, silicon alkoxides, organosilane compounds or organosiloxane compounds, preferably from organosilane compounds.

10. Composition according to at least one of the preceding claims, characterized in that it contains at least one silicon compound comprising at least one Si atom, at least one C atom and at least one H atom and conforming to formula (I), (IIa) or (IIb),

R-SiR¹ _xR² _z (I)，

wherein the content of the first and second substances,

r is a linear or branched alkyl group having 1 to 20C atoms,

R¹is a straight or branched alkyl group having 1 to 4C atoms,

r2 is a straight-chain or branched alkoxy radical or a hydroxyl radical having 1 to 4C atoms, where the radical R¹And R²Which may be the same or different from each other,

x is equal to 0, 1 or 2,

z is equal to 1, 2 or 3 and x + z is 3,

(R’)₃Si-O-[Si(R’)₂-O]_m-Si(R’)₃ (IIa)，

wherein the individual radicals R' independently of one another denote hydroxyl, alkoxy, preferably alkoxy having 1 to 6, preferably 1 to 4, carbon atoms, alkoxyalkoxy, preferably alkoxyalkoxy having 1 to 6, preferably 1 to 4, carbon atoms, alkyl, preferably alkyl having 1 to 20, preferably 1 to 10, carbon atoms, alkenyl, preferably alkenyl having 1 to 20, preferably 1 to 10, carbon atoms, cycloalkyl, preferably cycloalkyl having 1 to 20, preferably 1 to 10, carbon atoms, and/or aryl, preferably aryl having 1 to 20, preferably 1 to 10, carbon atoms,

m is an integer of 2 to 30,

n is an integer of 3 to 30,

with the proviso that sufficient of the radicals R' in the compounds of the formulae (IIa) and (IIb) are alkoxy radicals to ensure that the quotient of the molar ratios of Si to alkoxy radicals in the compounds of the formulae (IIa) and (IIb) is at least 0.3, in particular at least 0.5.

11. Composition according to at least one of the preceding claims, characterized in that the at least one silicon compound comprising at least one Si atom, at least one C atom and at least one H atom is chosen from CH₃Si(OCH₃)₃、CH₃Si(OC₂H₅)₃、C₂H₅Si(OC₂H₅)₃、i-C₃H₇Si(OC₂H₅)₃、C₂H₅Si(OCH₃)₃、i-C₃H₇Si(OCH₃)₃、n-C₃H₇Si(OCH₃)₃、n-C₃H₇Si(OC₂H₅)₃、i-C₃H₇Si(OCH₃)₃、n-C₄H₉Si(OCH₃)₃、n-C₄H₉Si(OC₂H₅)₃、i-C₄H₉Si(OCH₃)₃、n-C₄H₉Si(OC₂H₅)₃、n-C₅H₁₁Si(OCH₃)₃、n-C₅H₁₁Si(OC₂H₅)₃、i-C₅H₁₁Si(OCH₃)₃、i-C₅H₁₁Si(OC₂H₅)₃、n-C₆H₁₃Si(OCH₃)₃、n-C₆H₁₃Si(OC₂H₅)₃、i-C₆H₁₃Si(OCH₃)₃、i-C₆H₁₃Si(OC₂H₅)₃、n-C₈H₁₇Si(OCH₃)₃、n-C₈H₁₇Si(OC₂H₅)₃、i-C₈H₁₇Si(OCH₃)₃、i-C₈H₁₇Si(OC₂H₅)₃、n-C₁₀H₂₁Si(OCH₃)₃、n-C₁₀H₂₁Si(OC₂H₅)₃、i-C₁₀H₂₁Si(OCH₃)₃、i-C₁₀H₂₁Si(OC₂H₅)₃、n-C₁₆H₃₃Si(OCH₃)₃、n-C₁₆H₃₃Si(OC₂H₅)₃、i-C₁₆H₃₃Si(OCH₃)₃、i-C₁₆H₃₃Si(OC₂H₅)₃Or partial condensates of one or more of the compounds mentioned, orMixtures of the compounds mentioned, mixtures of the partial condensates or mixtures of the compounds with the partial condensates.

12. A method for producing building products, preferably based on mineral building materials, characterized in that a composition according to at least one of claims 1 to 11 is used during production.

13. The method according to claim 12, characterized in that the building product is a mortar, mortar-based component/product, reinforced concrete, (steel) concrete parts, concrete blocks, roof tiles, bricks or cellular concrete blocks.

14. The method according to claim 12 or 13, characterized in that the composition is applied before the building product or building structure is completed.

15. The method according to claim 12 or 13, characterized in that the composition is applied after completion of the building product or building structure.

Examples

Example 1: testing of the compatibility of microorganisms with hydrophobing and shrinkage-reducing Agents

The compatibility of Bacillus subtilis (DSM 10) and Bacillus alkalophilus (DSM8715) with hydrophobicizing and shrinkage reducing agents was investigated.

A mixture of 3g meat extract, 5g casein peptone and 1000mL distilled water was used as medium, which was adjusted to pH 7 using HCl/NaOH against Bacillus subtilis (DSM 10) and sodium sesquicarbonate against Bacillus alkalophilus (DSM8715) to pH 7.

First, preculture was carried out for each of the two strains: for this purpose, the inoculum dose of spores was placed in each case in a culture tube with 8mL of the corresponding medium and placed overnight in a laboratory shaker at 30 ℃ and 200 revolutions per minute.

Furthermore, prepared to have a concentration of 500g/L eachWS405 (hydrophobicizer) and neopentyl glycol (shrinkage-reducing agent) in aqueous stock solution at a concentration of 280 g/L.

For the main culture, two 6-well spot plates were each filled with 8mL of medium. Then, 10 μ L of the first preculture was added to each well of the first plate and 10 μ L of the second preculture was added to each well of the second plate. Will be provided withWS405 stock aqueous solution was added to three wells of two plates in an amount such thatWS405 concentration is 5g/L, 20g/L or 30 g/L. The neopentyl glycol stock solution was added to the other three wells of both plates in an amount such that the concentration of neopentyl glycol was 0.7g/L, 7g/L or 14 g/L.

The main culture was then placed in a laboratory shaker at 30 ℃ and 200 rpm for 4 days. The observation of the change in turbidity is used to determine whether the microorganism is growing in the presence of the hydrophobic and/or shrinkage reducing agent.

It was found that neither growth of any organism was impaired by the addition of the hydrophobizing agent or shrinkage reducing agent at the concentrations described.

The spores of the strain Bacillus subtilis DSM 32315 were investigated on agar plates with neopentyl glycol (7g/L) andcompatibility of WS405(20 g/L). A mixture of 3g meat extract, 5g casein peptone and 1000mL distilled water was used as medium, which was adjusted to pH 7 using HCl/NaOH. Colony formation was observed in all cases. This indicates that the additive does not affect the growth of the strain.

Example 2: preparation of test specimens

Test specimens were prepared using the formulation for producing a standard mortar having a mortar composition according to EN 480-1. For this purpose, 450 g were mixed using a mortar mixer from Hobart (Hobart)Classic CEM I52.5N cement and 1350 grams of CEN standard sand according to EN 196-1 were homogenized to provide a dry mixture.

The homogenized dry mixture was added to the mortar mixer at a slow mixing speed (set 1) over 30 seconds. 450 g of water are then added over 30 seconds and the entire mortar mixture is stirred for a further 60 seconds with a slow setting. The amount of water is chosen so that the weight ratio of water to cement is 1 to 2.

The mortar was then stirred at high speed (setting 2) for 60 seconds. The total mixing time was 3 minutes 30 seconds.

The steel molds (4 cm. times.4 cm. times.16 cm) for the three prisms were filled in each case to an overflow of 0.5 to 1.0cm using a box attachment and then compacted on a vibrating table at a frequency of 50Hz for 120 seconds. The mortar in the mold was then smoothed and covered with a glass plate. After 48 hours, the prisms were carefully demolded, labeled and stored under standard climatic conditions until testing was performed after 28 days.

Example 3: test the repair Effect of the compositions

A number of test specimens from example 2 were broken in the middle and treated at the fracture edge with either the composition of the prior art (S) or with the composition of the invention (E), which however lacks a hydrophobizing agent, and the broken specimens were subsequently bonded together again.

Treatment with the liquid remediation system, ER7 (prior art product), was performed such that 90 grams of component a in 500mL of water (40 ℃ temperature of water) was converted to solution a and 50 grams of component B in 250mL of water (40 ℃ temperature of water) was converted to solution B according to the instructions for use. The fractured edges were then sprayed twice with solution a and then once with solution B according to the instructions for use.

The treatment with the composition according to the invention was carried out such that first 15 g of tryptic soy broth and 50 g of spores of Bacillus subtilis DSM 32315 were stirred in 500mL of water and the solution was sprayed onto the broken edges.

After bonding, the test specimens were fixed with teflon tape. The test specimens were stored in a water bath at room temperature. The test specimens were immersed in a water bath to a depth of 0.5cm and the cracks were not below the water level. The cracks were sprayed with water periodically at 2 day intervals.

Crack repair was observed for both test specimens even after a period of 1 day (fig. 5a, 5b, 6a and 6 b). In each case, a vertical downward force of at least 1.23N can be applied on the repaired crack. This force corresponds to the mass of the lower part of the test specimen multiplied by 9.81m/s²The acceleration of gravity of (1). For reference, one test sample was brushed with a special tryptic soy broth. After the same time has elapsed, there is noRepair of cracks was observed.

Example 4: production of test specimens with repair additives

The preparation of the test specimens was carried out as described in example 2. However, the aqueous portion of the added composition is considered to be the forming part of the mixing water and will therefore be considered, so all mixtures used to prepare the test specimens are prepared with the same water to cement ratio to ensure comparability of the results. The appearance of the materials and mortar mixtures used during processing is reported in table 1 a.

Table 1 a: mortar mixture (without water portion) and appearance thereof

Examples

Cement

Standard sand

LSR

32315A spore

TSB

WS 405

WA CIT

Appearance of the product

4a

450g

1350g

13.5g

4.5g

-

-

Normal viscosity

4b

450g

1350g

13.5g

4.5g

18g

Normal viscosity

4c

450g

1350g

13.5g

4.5g

18g

Normal viscosity

To evaluate the hydrophobic effect of the silane additive (silicon compound containing at least one Si atom, at least one C atom and at least one H atom), the decrease in the water absorption of the capillary was determined over a period of 24 hours and 14 days. Test sample 4a (biomass only) was used as reference.

The dry mass of each test specimen was determined before the onset of water absorption. Each test specimen was then stored vertically with a bottom surface of 40mm x 40mm in a suitable container of 3mm constant water depth. Suitable blocks or liners (glass inserts or beads) should be used to ensure that water enters the submerged bottom surface unobstructed. The individual test specimens must not come into contact with one another and the container should be closed during the test. After a specified time interval, the quality of the individual test samples should be determined and noted in the test protocol. To remove the adhering water from the test specimen, a light wipe was applied with a dry cloth (test setup similar to EN 480-5, but with other measurement durations, and no three determinations). The percentage reduction in water absorption was determined by the following method:

ref is reference example (4 a); ex as an example (4b/4c)

The results after 24 hours are shown in table 1b and the results after 14 days are shown in table 1 c.

TABLE 1b reduction of capillary Water absorption after 24 hours

UWS-underwater storage; WA-Water absorption

TABLE 1c reduction in capillary Water absorption after 14 days

UWS-underwater storage; WA-Water absorption

As is apparent from tables 1b and 1C, the addition of a silicon compound (hydrophobizing agent) containing at least one Si atom, at least one C atom and at least one H atom significantly reduced the water absorption of the test specimens.

The test specimen was then divided into two parts, again placed on top of each other at the fracture edge (plated on top of one other), and then stored upright in a bowl of water (water fill height about 5mm) for 69 days so that the crack was immersed in the water on one side.

At 200 x magnification, a side view of a test specimen containing a crack shows that the crack was repaired (filled) 18 days after the block from example 4a was bisected (fig. 1). At 100 x magnification, the top view of the fracture surface shows that calcium carbonate is formed in the crack 69 days after the block from example 4a is bisected (fig. 2). A 100-fold magnification of the side view of the test specimen from example 4c shows that calcium carbonate is formed in the crack 69 days after the block from example 4a is bisected.

Example 5: effect of microbial concentration and calcium Source

The objective was to determine the quality of the microorganisms and the effect of the additional calcium source on the flexural strength and water absorption of the test specimens. To this end, test specimens with different combination options of biomass, tryptic soy broth, calcium source and hydrophobic agent (WS405) were employed.

The preparation of the test specimens was carried out as described in example 2. However, the components and concentrations reported in table 2a were used. The calcium source used is calcium lactate hydrate. Example 5i is a reference sample.

For simpler metering of the microorganisms, 0.68 g of 32315 spores was first diluted with 50mL of tap water to produce a spore mixture having a concentration of 0.0136g (spore 32315)/mL, respectively.

Table 2 a: formulations used for preparing test specimens

Examples

Cement

Standard sand

Water (W)

Spore solution

TSB

Calcium source

WS405

5a

450g

1350g

224.0g

1mL

4.5g

0g

0g

5b

450g

1350g

222.9g

1mL

4.5g

0g

2.25g

5c

450g

1350g

224.0g

1mL

4.5g

3.15g

0g

5d

450g

1350g

222.9g

1mL

4.5g

3.15g

2.25g

5e

450g

1350g

224.9g

0.1mL

4.5g

0g

0g

5f

450g

1350g

223.8g

0.1mL

4.5g

0g

2.25g

5g

450g

1350g

224.9g

0.1mL

4.5g

3.15g

0g

5h

450g

1350g

223.8g

0.1mL

4.5g

3.15g

2.25g

5i

450g

1350g

225.0g

0mL

0g

0g

0g

After the test specimens have been stored for 28 days at 23 ℃ and 50% relative humidity (standard climatic conditions), the flexural tensile strength and the reduction in water absorption after 24 hours of the test specimens are measured. To determine the water absorption after 24 hours, the test specimens were stored upright in a water bath. They were immersed in water to a depth of about 5 cm. After 24 hours, the amount of water absorbed by the test specimen was determined gravimetrically. The results are shown in table 2 b.

Table 2 b: test results

Examples	SCrack (crack)	Reduction of Water absorption	Grade
				5a	836.6N	-22.5％	-
5b	3016.3N	65.3％	++
				5c	294.6N	-36.4％	--
5d	2547.2N	65.7％	+
				5e	565.9N	-40.8％	--
5f	2808.6N	68.2％	++
				5g	727.3N	6.8％	-
5h	2553N	69.1％	+
				5i	3849.4N	0.0％

The results shown in table 2b clearly show that significantly higher strengths can be obtained with the addition of TSB, microorganisms and hydrophobing agent than without the addition of hydrophobing agent. Furthermore, the water absorption was increased (negative% value) compared to the untreated test sample without addition of the hydrophobizing agent but with addition of the TSB and spore solutions. Further addition of a calcium source appears to result in a slight improvement in the reduction of water absorption but also in a slight reduction of flexural strength.

Example 6: effect of surface treatment

The purpose of this experiment was to investigate the effect of surface treatment with a solution of hydrophobing agent, spores, tryptic soy broth, calcium lactate and water.

For this purpose, a mixture of distilled water and optionally WS405, spores, TSB and/or Ca-Lactat H is used₂Formulation of O commercial concrete cubes from Rocholl GmbH were treated. The compositions of the formulations employed in examples 6a to 6e are reported in table 3 a. Example 6e is a reference sample.

Table 3 a: formulation used in example 6

The cubes were first dipped into the corresponding formulations until 200g/m were reached²About the amount of (a). The actual amount of formulation applied was determined gravimetrically and is also reported in table 3 a. After 14 days, the aspiration was determined using the castten tube testThe water rate decreases. For this, the water absorption was determined after 24 hours and was referred to the water absorption of reference sample 6 e. The results are reported in table 3 b.

Table 3 b: reduction of water absorption without cracks

The cubes (including 6e) were then fractured and the fractured surfaces brushed with the corresponding formulation at the amounts reported in table 3 c. The cubes were then placed one on top of the other again at the fracture edge, fixed with teflon tape and then stored in a bowl of water (water fill height about 5mm) for 14 days so that the crack was immersed in water on one side. The reduction in water absorption was determined as follows: the cubes were dried and weighed. They were then stored under water for 24 hours. The reduction in water absorption is determined from the mass difference before and after underwater storage according to the following formula:

reduction in water absorption (% mass after/mass before)/mass before/[ (mass after/mass reference before)/mass before/mass _ reference 100

The results of the water absorption reduction are shown in table 3c below.

Table 3 c: reduction of water absorption after fracture

Examples	The dosage is [ g/m ]2]	Reduction of water absorption [% ]]
			6a	201.3	94.9
6b	199.3	91.6
			6c	210.7	93.2
6d	202.7	93.8
			6e	-	4.2*

Water absorption in% after 24 hours,

(subsequent mass-previous mass)/previous mass 100 ═ absolute water absorption

As is evident from table 3c, a reduction in the water absorption is observable even after the test specimens (cube) break and subsequent treatment with the composition of the present invention, compared to untreated test specimens.

The castellated tube test was performed after 8 weeks of storage. For this purpose, a tube is connected over the crack. The side stored below the water surface during these 8 periods is used. For example 6d, no water uptake was observed over a measurement duration of 0.5 hours. This means that cracks in the formulation have closed in the absence of calcium lactate.

Example 7: encapsulation with polyvinyl alcohol (PVA)

In this experiment, the stability of the uncoated spores and PVA coated spores of the strain bacillus subtilis DSM 32315 was analyzed during concrete mixing.

Coating of bacillus subtilis spores with polyvinyl alcohol:

coating/packagingThe apparatus used for the sealing is a Huttlin coater (Bosch) equipped with a fluidized bed attachment. To achieve coating/encapsulation, the biomass is first loaded into a huttlin coater, sprayed with an aqueous PVA solution and then dried. The biomass used was a mixture of 50% by weight of Bacillus subtilis DSM 32315 spores and 50% by weight of lime. The PVA solution used was 5% by weight of Kuraray4-88 PVA and 5% by weight of Kuraray8018 solution of PVA in water. Thus, the total concentration of PVA was 10 wt.%, based on the total mass of the solution. To prepare the PVA solution, Kuraray was first prepared4-88 PVA and Kuraray8018 the mixture of PVA is sprinkled into cold water with stirring and heated in a water bath to 90 to 95 ℃ until completely dissolved, and the solution is then cooled with stirring to avoid the formation of a crust. Subsequently, the biomass is first charged into a fluidized bed unit, heated and fluidized with a temperature controlled nitrogen stream. Once the fluidized bed reached the desired temperature, the PVA solution was added via a peristaltic pump. The relevant process set-up is summarized in table 4.

Table 4: arrangement of a fluidized bed process

Parameter(s)	Unit of	Value of
			N2Temperature of	℃	60-65
Bed temperature	℃	45-48
			N2Flow rate of	m3/h	20
Jet air pressure	Bar	0.5
			Microclimate	Mbar	150
Speed of pump	％	5
			Spraying rate of PVA	g/h	92±23

In coating/encapsulation of biomass, 750 grams of aqueous PVA solution (10 wt% PVA) was applied to 500 grams of biomass. This corresponds to a PVA proportion of 13% by weight, based on the total mass of the dry product.

Determination of stability:

to determine stability, equal amounts of coated spores (78 grams per 50 liter of concrete batch, corresponding to 0.5 wt% based on cement) and uncoated spores (46 grams per 50 liter of concrete batch, corresponding to 0.29 wt% based on cement) were placed in a cement mixer along with growth medium (92 grams TSB), the amounts adjusted to the spore concentration in the feed CFU/g. After dry mixing for 1 minute, a sample was taken and the appropriate amount of water (7.2kg) was then added to the concrete batch. Samples were taken again after a total of 3 minutes, 20 minutes, 60 minutes and 120 minutes. The collected samples were immediately and in duplicate diluted to about 1:100 in water, shaken and then aliquoted and stored at-20 ℃ until further processing.

To determine the spore number of the sample, the sample was thawed and diluted in polysorbate peptone salt solution (pH-7) in a serial dilution method so that countable colony counts were expected on TSA agar plates after the sample was removed and incubated at 37 ℃.

Table 5: stability of coated and uncoated DSM 32315 spores during concrete mixing

The results reported in table 5 show that spores in the concrete batch may not have been evenly distributed after one minute, thus initially resulting in a lower than expected spore count. After three minutes of mixing, the spore count approaches the expected spore count, both in batches containing coated spores and in batches containing uncoated spores. During mixing for up to 2 hours, it is evident from the spore count data that no more than one log step (step) of loss of coated or uncoated spores occurred.