DE10146375A1 - Biocomposite (Biocer) for biosorption of heavy metals comprises an inorganic gel containing immobilized dry-stable cellular products - Google Patents

Abstract

A biocomposite material (Biocer) especially for biosorption of heavy metals comprises an inorganic gel containing uniformly distributed dry-stable cellular products. An Independent claim is included for the production of the biocomposite materials via a silica nanosol.

Description

The invention relates to a composite material (biocomposite material, Biocer), which is an inorganic gel and biological dispersed therein Includes materials. The composite material is used as a biofilter in the Environmental technology for the removal of heavy metal ions from aqueous Suitable solutions.

• - hohe mechanische, thermische und photochemische Stabilität,
• - hohe Transparenz,
• - biologische Inertheit (d. h. keine Nahrungsquelle für Mikroorganismen), und
• - steuerbare Porosität und variabler Immobilisierungsgrad.

It is known that great efforts are currently being made to immobilize biomolecules (molecules of biological or naturally formed materials) in inorganic matrices, since, among other things, the following advantages result from the polymer matrices previously used:

• - high mechanical, thermal and photochemical stability,
• - high transparency,
• - biological inertness (ie not a food source for microorganisms), and
• - Controllable porosity and variable degree of immobilization.

Such biocomposite materials offer numerous new potential advantageous applications, e.g. B. for production biocompatible surfaces in medical technology, for biocatalysis, Biogenesis and for novel drug delivery systems.

In addition to being able to attach biomolecules or bacterial cells inorganic carriers such as silica gel, bentonite and the like. a. adsorptive to the Fix surface, such as in IN 171047 there is the possibility of direct embedding of biomolecules in an inorganic matrix by using the modern sol-gel technology (cf. C. J. Brinker, G. Scherer, "Sol-Gel Science: The Physics and Chemistry of Sol-Gel Processing ", Academic Press Inc., Boston 1990).

The procedure is shown in the following scheme (procedure for the formation of a biocomposite material (BM = biomolecule)):

In this way, for example, the advantageous embedding of enzymes or other proteins in inorganic matrices is possible (see, for example, US Pat. No. 5,200,334, US Pat. No. 5,300,564). Attempts have recently been made to encapsulate cell tissue in an organosilicone (US Pat. No. 5,693,513) or animal and antimicrobial cells in a gel produced from an organosilcon in a similar manner (US Pat. No. 5,739,020). For the encapsulation of microorganisms, several patents describe the combination of SiO ₂ gels with water-soluble polymers such as polyvinyl alcohol, gelatin (US 4 148 689) or alginates (US 4 797 358, WO 96/35780).

It is known that a number of microorganisms are very effective Reversibly bind metal ions, e.g. B. uranium, thorium Pseudomonas strains (US 4,780,238, GB 2,145,428). Other options exist in the microbiological reduction of the metal ions insoluble metals (EP 1 016 633, US 6 080 572). Applications arise, for. B. for uranium extraction from sea water (JP 63237779, US 4,263,403).

• a) durch den Trocknungsprozess im anorganischen Gel werden die Mikroorganismen praktisch zerquetscht und als Beschichtungen nach Rissbildung z. T. direkt zerrissen,
• b) die überlebenden Mikroorganismen verhungern nach kurzer Zeit infolge Nahrungsmittelmangel und werden dabei so deformiert, dass ihr Metallbindungsvermögen stark verringert wird. Das führt dazu, dass die Biofilter-Wirkung von lebenden Mikroorganismen in einer reinen SiO₂-Matrix nach kurzer Zeit drastisch sinkt und derartige Biokomposite aufgrund ihrer geringen Lagerstabilität und starken Feuchteabhängigkeit für praktische Anwendungen nicht geeignet sind.

So far, it has not been possible to effectively immobilize such metal-binding microorganisms using a porous sol-gel matrix. In the above-mentioned techniques, SiO _{2 was} combined with organic polymers as a carrier. The problem is that the microorganisms do not survive being embedded in the pure metal oxide gels without damage. This has at least two causes:

• a) by the drying process in the inorganic gel, the microorganisms are practically crushed and used as coatings after cracking z. T. directly torn,
• b) the surviving microorganisms starve after a short time due to lack of food and are deformed in such a way that their metal binding capacity is greatly reduced. This means that the biofilter effect of living microorganisms in a pure SiO ₂ matrix drops drastically after a short time and such biocomposites are not suitable for practical applications due to their low storage stability and strong moisture dependence.

It is the object of the invention to provide an improved one Specify composite material with biomolecules, especially as filter material for removing heavy metal ions from aqueous solutions suitable is. The composite material should be stable in storage and dry and have a high biofilter effect. It's the job too of the invention, a method for producing such Specify composite material.

This task is accomplished through a composite material and a process its manufacture with the features according to patent claims 1 or 9 solved. Advantageous embodiments of the invention result from the dependent claims.

The basic idea of the invention is in particular the use of Biocomponents that have a similarly high metal binding capacity like living microorganisms, but much more stable against mechanical and thermal influences as well as drying influences are. Surprisingly, the tasks according to the invention be solved by making a Biocomposite material that is suitable for the biosorption of heavy metals, drying-stable cell products, such as B. spores, cell wall Proteins (so-called S-layers) or killed cells in one inorganic silicon or metal oxide gel using the sol-gel process embeds.

One object of the invention is in particular a Biocomposite material (Biocer), which is an inorganic gel and homogeneous therein distributed, drying-stable cell products (e.g. spores, Protein structures, especially cell wall proteins, or killed Cells) contains. Among the drying stable cell products are generally understood substances or materials made from biological cells have been obtained. For example, by Nutrient deprivation or mechanical influence from living cells Cell parts separated or formed that are long-term stable are. The cell products consist of biomolecules.

The inventor succeeded for the first time in drying-stable Cell products, preferably from metal-binding microorganisms are derived through a porous sol-gel matrix immobilize, so that the biocomposites both as a coating of carriers or as bulk products (granules) new industrial Applications z. B. as filling material for biofilter columns result.

Metal oxides of elements of II.-V. Main and subgroup of the periodic table such as SiO ₂ , Al ₂ O ₃ , ZrO ₂ , TiO ₂ , B ₂ O ₃ , ZnO, CaO, P ₂ O ₅ or mixtures thereof, which are obtained by a sol-gel process , Acid or alkaline hydrolysis of the corresponding metal alkoxides according to the scheme shown above results in inorganic nanosols which can be gelled by neutralization, heating, fluoride addition or solvent evaporation to give solvent-containing lyogels and, after drying, can be converted to xerogels in the form of bulk products. Thin transparent xerogel films can also be produced by coating the nanosols on any support (e.g. glass, paper, foils, metal or textiles) and then drying them.

For the modification of the sol-gel layer properties, the hydrolysis process of the metal alkoxides can be carried out in the presence of mixed alkyl trialkoxysilanes R-Si (OR ') ₃ and / or dialkoxysilanes R ₂ -Si (OR') ₂ , whereby modified metal oxide gels are formed, which contain 0 to 2 parts by weight of R-SiO _3/2 and / or R ₂ = SiO based on 1 part by weight of metal oxide gel. R is an organic alkyl or aryl radical which may contain amino, hydroxyl, epoxy or alkoxy groups or is substituted by halogens. R 'is an alkyl radical, primarily with 1-4 carbon atoms. With this modification, e.g. B. the mechanical properties of the composites can be specifically improved.

For embedding the biocomponent in the inorganic gel matrix it is advantageous to remove the alcohol from the nanosols, which on the one hand as a solvent for homogenizing the Hydrolysis mixture (alkoxysilanes are water-insoluble), on the other hand as Hydrolysis product is present. You can do this by evaporating the Alcohol with adequate water at 20 ° C by passing through of an inert gas stream.

• 1. in Sporen durch verringertes Nahrungsangebot und Zusatz von Mangan-Salzen,
• 2. in S-Layer, d. h. in isolierte Zellwandproteine, die in den Zellhüllen für das Metallbindungsvermögen verantwortlich sind, durch mechanische Zerstörung der Zellwände und enzymatische Reinigung nach Engelhardt et al., J. Bacteriol. 168 (1): 309 (1986),
• 3. in abgetötete Zellen durch Zusatz von Zellgiften wie Natriumazid.

For the production of the biocomposite material according to the invention, which is suitable for the biosorption of heavy metal ions, preferably spores, cell wall proteins (so-called S-layers) or killed cells are used as drying-stable cell products. These are made from living microorganisms that have been isolated and cultivated from an environment containing heavy metals. Living Bacillus sphaericus JG-A12 cells, which were isolated and cultured from waste water from a uranium mine (Grube Haberland / Johanngeorgenstadt / Germany), proved to be particularly suitable for this. The living cells can be easily converted into storage and dry stable cell products:

• 1. in spores due to reduced food supply and the addition of manganese salts,
• 2. in S-layer, ie in isolated cell wall proteins, which are responsible for the metal binding capacity in the cell shells, by mechanical destruction of the cell walls and enzymatic cleaning according to Engelhardt et al., J. Bacteriol. 168 (1): 309 (1986),
• 3. in killed cells by adding cell poisons such as sodium azide.

The different biocomponents mentioned are called aqueous Suspension mixed with aqueous nanosols and either on Coated or gelled by neutralization, dried and ground. The proportion of cell products stable to drying can 0.1-50% by weight based on the dry biocomposite.

To increase the porosity and thus improve reactivity compared to heavy metal ions, the nanosol can be water-soluble Additives such as organic salts, polyols, polycarboxylic acids, urea Derivatives or carbohydrates can be added.

The additives mentioned are in the application of the biocomposites in aqueous solutions gradually washed out and increase by the thus additionally created pores the accessibility of the Heavy metal ions for the immobilized biocomponent. The share of Additives can be 5-50% by weight based on the dry biocomposite be.

• 1. Herstellung von alkoholischen SiO₂-Nanosolen durch saure oder alkalische Hydrolyse von Tetraalkoxysilanen, ggf. modifiziert durch Cohydrolyse mit Metallalkoholaten oder -halogeniden, Tri- oder Dialkoxysilanen,
• 2. ggf. Substitution des flüchtigen Alkohols durch Wasser im Nanosol durch Durchleiten eines inerten Gasstromes bei Temperaturen unter 20°C,
• 3. homogenes Mischen des rein wässrigen anorganischen Nanosols mit einer wässrigen Dispersion der trocknungsstabilen Zellprodukte,
• 4. Gelbildung durch Neutralisation, Erwärmen oder Fluorid-Zugabe und anschließende Zerkleinerung und Gefriertrocknung bzw. Luftrocknung des Gels.

The process for producing a biocomposite material is thus characterized by the following steps, cf. above scheme:

• 1. Production of alcoholic SiO ₂ nanosols by acidic or alkaline hydrolysis of tetraalkoxysilanes, optionally modified by cohydrolysis with metal alcoholates or halides, tri- or dialkoxysilanes,
• 2. if necessary, substitution of the volatile alcohol by water in the nanosol by passing an inert gas stream at temperatures below 20 ° C.,
• 3. homogeneous mixing of the purely aqueous inorganic nanosol with an aqueous dispersion of the drying-stable cell products,
• 4. Gel formation by neutralization, heating or fluoride addition and subsequent comminution and freeze drying or air drying of the gel.

To produce a layered biocomposite material after the homogeneous mixing of the purely aqueous inorganic Nanosols with an aqueous dispersion of the drying-stable Cell products coating a material surface, e.g. B. by Diving, spraying, watering. Then the educated Composite film dried in the air or at moderate temperatures.

• - hohes Biosorptionsvermögen gegenüber Schwermetallionen wie Uran, Kupfer (vgl. Beispiele),
• - beliebig lange Lagerung der Biokomposite bei unterschiedlichen Trocknungsbedingungen und Luftfeuchten ohne Verlust der Bioaktivität,
• - Gewährleistung einer hohen Reversibilität der Metalladsorption, da die Metallionen durch Waschen mit 0.5 M Citronensäure vollständig vom Biocer entfernt werden können,
• - vielfältige Applikationsmöglichkeiten in Form von bulk-Produkt mit einstellbarer Korngröße sowie als gut haftende Beschichtung mit einstellbarer Schichtdicke.

The process provides biocomposite materials that are suitable for the biosorption of heavy metals and have the following advantages over the prior art:

• - high biosorption capacity against heavy metal ions such as uranium, copper (see examples),
• - Storage of the biocomposites for as long as required under different drying conditions and air humidity without loss of bioactivity,
• - Ensuring a high reversibility of the metal adsorption, since the metal ions can be completely removed from the biocer by washing with 0.5 M citric acid,
• - Various application options in the form of bulk product with adjustable grain size and as a well-adhering coating with adjustable layer thickness.

The biocomposite material according to the invention can therefore, for. B. advantageous as a biofilter in environmental technology for the removal of Heavy metal ions from aqueous solutions can be used.

The invention has the following advantages in particular. It is possible to use the sol-gel immobilization of cell products Production of biofilters to use, for. B. the removal of Heavy metal ions from natural water or industrial waste water serve. In contrast to the embedding of living cells Composite formation with cell products technologically easier to handle. The cell products can be made available in a targeted and reproducible manner and how to use a chemical substance. The This makes the filter effect of the composite material reproducible adjustable. A particular advantage arises when using spores in the composite material, because spores, for example from bacteria are formed, on the one hand extremely durable in the dry state and on the other hand can be reactivated by adding moisture. A filter with the composite material according to the invention enables thus an independent activation of the filter function Start of use.

Further details and advantages of the invention will become apparent from the Description of the accompanying embodiments and drawings seen. Show it:

Fig. 1 is an illustration of the metal binding capacity of a composite material according to the invention compared with uranium and copper (100% corresponds to 2 mg of copper or 7.5 mg uranium), and

Fig. 2 is an illustration of the increase in the reactivity of a composite material according to the invention by freeze-drying of the Biokomposites or by the addition of sorbitol.

embodiments Preparation of the acidic aqueous nanosol B

100 ml of tetraethoxysilane, 400 ml of ethanol and 200 ml of 0.01 M hydrochloric acid are stirred at room temperature for 20 h. The acidic Nanosol A (approx. 4.2% SiO ₂ in 70% ethanol) is formed.

450 ml of water are successively added to 600 ml of the alcohol-containing nanosol A thus obtained, while passing a strong air stream, and the air stream is passed through until a final volume of 600 ml is reached. A purely aqueous SiO ₂ nanosol B (solid content approx. 4.2 SiO ₂ % in water, pH = 3-4, average particle diameter approx. 6 nm) is obtained which is stable for several days when stored below 5 ° C.

Production of biocomposites

Aqueous suspensions of spores, S-layer and killed cells from Bacillus sphaericus JG-A12, isolated and cultured from Uranium mine wastewater is used in different concentrations the Nanosol B mixed with mechanical stirring and after Neutralization with 0.1 n sodium hydroxide solution by dip coating (Drawing speed 30 cm / min) Coatings made on glass. to Manufacture of bulk products is done after the sol has gelled formed aqueous gel stored at 4 ° C for 3 days, then crushed, freeze-dried or at room temperature until Weight constant air-dried and classified to one Particle size of 355-500 µm.

Biosorption of the heavy metal ions uranium and copper

• a) 17.23 mg (2.8 × 10¹⁰) Sporen
• b) 36.4 mg S-layers
• c) 36.4 mg (2.6 × 10¹⁰) abgetötete B. sphaericus Zellen.

The investigations were carried out with 200 mg of the screened biocomposites, which contain the following concentrations:

• a) 17.23 mg (2.8 × 10 ¹⁰ ) spores
• b) 36.4 mg S-layers
• c) 36.4 mg (2.6 × 10 ¹⁰ ) killed B. sphaericus cells.

To determine the metal binding capacity, the biocomposites were treated with 35 ml of a solution 0.9% NaClO ₄ , pH 4.5 with 9 × 10 ^-4 M UO ₂ (NO ₃ ) ₂ × 6 H ₂ O or CuCl ₂ at 30 ° C. for 48 h. shaken. The remaining metal ion concentration in the solution was determined by ICP-MS (Inductive coupled plasma mass spectroscopy). The results are shown in Fig. 1.

• 1. Zell-Komposite besitzen die höchste Metallbindungskapazität.
• 2. Die Bindungskapazität für Uran ist verglichen mit Kupfer beträchtlich größer, da aufgrund der natürlichen Herkunft der Biokomponenten deren spezifische Proteinstruktur vorrangig auf die Uran-Biosorption ausgerichtet ist.
• 3. Die Biosorption wird durch die Erhöhung der Porosität der Matrix, z. B. durch Gefriertrocknung oder Zusatz von 20 Gew.-% Sorbitol (bezogen auf Oxidmatrix) beträchtlich beschleunigt, wie aus Fig. 2 ersichtlich ist.

One recognises:

• 1. Cell composites have the highest metal binding capacity.
• 2. The binding capacity for uranium is considerably greater than that of copper, because due to the natural origin of the biocomponents, their specific protein structure is primarily geared towards uranium biosorption.
• 3. The biosorption is increased by increasing the porosity of the matrix, e.g. B. accelerated considerably by freeze drying or addition of 20 wt .-% sorbitol (based on oxide matrix), as can be seen from Fig. 2.

Claims (14)

1. Biocomposite material, in particular for the biosorption of Heavy metals, which an inorganic gel and homogeneously distributed therein, contains dry cell products. 2. Biocomposite material according to claim 1, wherein the inorganic Gel oxides from elements of II.-V. Main and sub group of the Periodic table or mixtures thereof. 3. Biocomposite material according to claim 1 or 2, in which the inorganic gel by hydrolysis products of trialkoxysilanes and / or Dialkoxysilanes is modified. 4. Biocomposite material according to one of claims 1 to 3, in which the dry stable cell products spores, cell wall proteins and / or comprise killed cells. 5. Biocomposite material according to one of claims 1 to 4, in which the dry stable cell products from living cells are formed, which are isolated from an environment containing heavy metals and be cultivated. 6. Biocomposite material according to one of claims 1 to 5, in which the proportion of cell products stable to drying 0.1-50% by weight based on the dry biocomposite. 7. Biocomposite material according to one of claims 1 to 6, which for Increase in porosity and reactivity towards Heavy metal ions water-soluble additives, especially organic salts, Polyols, polycarboxylic acids, urea derivatives or carbohydrates, contains. 8. Biocomposite material according to claim 7, in which the proportion of Additions 5-50 wt .-% based on the dry biocomposite is. 9. A method for producing a biocomposite material according to one of claims 1 to 8, comprising the following steps: 1. Production of an alcoholic SiO ₂ nanosol by acidic or alkaline hydrolysis of tetraalkoxysilanes, 2. substitution of the alcohol by water in the nanosol, 3. homogeneous mixing of the purely aqueous inorganic nanosol with an aqueous dispersion of the drying-stable cell products, and 4. Gel formation through layer formation, neutralization, heating and / or fluoride addition. 10. The method according to claim 9, in which in step (1) the SiO ₂ nanosol is modified by cohydrolysis with metal alcoholates or halides, tri- or dialkoxysilanes. 11. The method according to any one of claims 9 or 10, in which Step (2) the substitution of the alcohol by water in the Nanosol by passing an inert gas stream at temperatures below 20 ° C. 12. The method according to any one of claims 9 to 11, in which Step (4) a coating of a material surface, especially by dipping, spraying or watering, and a Drying the composite film formed. 13. The method according to any one of claims 9 to 12, in which after Step (4) comminution, freeze drying and / or the gel is air dried. 14. Use of a biocomposite material according to one of the Claims 1 to 8 as a biofilter in environmental technology for removal of heavy metal ions from aqueous solutions.