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WO1996035780A1 - Nouvelles compositions d'hydrogels destinees a l'utilisation dans des bioreacteurs - Google Patents

Nouvelles compositions d'hydrogels destinees a l'utilisation dans des bioreacteurs Download PDF

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Publication number
WO1996035780A1
WO1996035780A1 PCT/US1996/006765 US9606765W WO9635780A1 WO 1996035780 A1 WO1996035780 A1 WO 1996035780A1 US 9606765 W US9606765 W US 9606765W WO 9635780 A1 WO9635780 A1 WO 9635780A1
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WIPO (PCT)
Prior art keywords
gel
silica
gels
alginate
cells
Prior art date
Application number
PCT/US1996/006765
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English (en)
Inventor
Dolloff Bishop
Rakesh Govind
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United States Environmental Protection Agency
University Of Cincinnati
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Filing date
Publication date
Application filed by United States Environmental Protection Agency, University Of Cincinnati filed Critical United States Environmental Protection Agency
Publication of WO1996035780A1 publication Critical patent/WO1996035780A1/fr

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    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08LCOMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04Alginic acid; Derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14Enzymes or microbial cells immobilised on or in an inorganic carrier

Definitions

  • This invention relates to new hydrogel compositions which i are useful for establishing biological nitches for cells to 5 remove pollutants from the environment.
  • hydrogel beads as supports for bio ass has been known. Such beads are available commercially and are used as delivery systems for nutrients and biological organisms.
  • alginate gels have properties of both solids and liquids.
  • alginate gels can be formed from 1% alginate and 99% water, yet show characteristics of solids such as shape retention and resistance to mechanical stress.
  • a gel consists of physically immobilized water with some properties that are similar to
  • silica sol which can be obtained commercially as LUDOXTM.
  • Silica gel is made from a colloidal solution of the silica.
  • Alginate gels provide advantages over silica sol, since the preparative solutions can be handled at neutral pH. Hence, living cells are not damaged when the alginate gels form.
  • alginate gel alone containing bio ass is inappropriate for use in construction of biofilters.
  • the alginate gels dissolve as a result of loss of calcium ions, especially in the presence of nutrients containing buffering anions and are also biodegradable.
  • the beads of alginate gels lack structural strength needed in supports for biomass in biofilters.
  • K-Carrageenan is also used for making gel beads. While the beads of K-Carrageenan are less easily disrupted by salts and buffers, they are subject to biological degradation by active cells. Furthermore, polysaccharides exhibit low absorption of organic pollutants that are the object of biofilter purification systems.
  • Silica gels provide absorbent matrixes that exhibit high absorption capacity the contaminants of interest. For purposes of use in biofilters, they provide advantages of higher absorp- tion of contaminants, which allows fast partitioning of contaminant from air or water onto the gel matrix. Organic contaminants such as toluene and tetrachloroethylene can move readily through silica gels. Silica gels provide better retainment of extracellular enzymes and cells inside the gel matrix, but these gels do not permit cell growth inside the gel.
  • This invention provides compositions and gel support systems containing inclusions of alginate gels and bacteria encapsulated in silica gels.
  • New gel preparations provide suitable void space for growth and maintenance of the active cells, overcoming the disadvantage of prior art silica gels.
  • the gel compositions provide suitable conditions for survival and growth of active cells, are sufficiently stable under conditions of use in biofilters and are resistant to decomposi ⁇ tion or biodegradation by entrapped cells.
  • the gels do not dissolve in water.
  • the constructs containing gel compositions of the invention can be formulated so that they can be packed into a bed.
  • the silica portion of the gel can absorb contami- nants to sufficient extent whilst exhibiting low oxygen diffusivity.
  • hydrogel compositions which could encapsulate bacteria for simultaneous creation of oxic (oxygen-rich) and anoxic zones inside the hydrogel structure.
  • Hydrogels containing the oxic and anoxic zones created inside the hydrogel supports make it possible to mineralize chlorinated compounds such as trichloro- ethylene (TCE) and perchloroethylene (PCE) using suitable organic sources (electron donors such as formate) for anaerobic microbial dehalogenation.
  • Hydrogels with encapsulated bacteria can be used to mineralize chlorinated compounds present in air, ground water and soil.
  • hydrogels can also be used to I 5 promote survival among more fragile but highly useful laborato ⁇ ry-grown cultures that are especially adapted for biodegrada- tion of targeted environmental pollutants. Furthermore, some zero valent metals (used as alternate electron donors) can also be introduced into the anoxic zone of the hydrogel to partially
  • compositions of the invention can be formulated in such a manner that zero valent metals, such as iron and
  • Silica sol was obtained commercially as LUDOXTM colloidal silica SM grad, and was mixed with 1-3% sodium alginate solution to provide a composition containing 1% to 10% by weight of alginate to silica. The mixture was adjusted to a
  • the survival and growth of active cells is maximized by using the combination of silica sol and alginate.
  • silica sol and alginate Once the gel had formed, stainless steel mesh cylinders 2.5 mm diameter and 5.1 mm long (open at both ends) were pushed into the gel layer, thereby enclosing the gel inside the wire mesh.
  • the use of the stainless steel mesh resulted in silica gel/calcium alginate beads encased in mesh. Such beads had structural strength so that the beads could be packed into a bed without compaction.
  • the alginate/silica sol/biomass mixture may be extruded as the gel is forming to provide threads of biomass. Such treads may also be cut to form beads. However, such beads will lack the enclosing mesh structure that gives improved structural strength. It is also possible to pour the silica/alginate mixture into plates with dividers or other projections from the floor to provide a mold that would result in gel formations with passages extending through the gel plate. Such gel plates with suitable support structures such as mesh could be stacked. Materials and Methods:
  • a 40 ml bioreactor (1.9 cm inner diameter( consisting of a jacketed cylinder, as shown in Figure 4, was constructed from borosilicate glass. The reactor was packed randomly with the gel beads. Air at a controlled rate was passed through the reactor and nutrient solution was trickled down from the top of the bioreactor counter current to the air flow. The air was contaminated with contaminants such as toluene, TCE and PCE, using a syringe pump that injected the liquid contaminant into the air line through a septum. The concentration of the contaminant in the air stream was varied in the following range: Toluene: 0-100 ppmv, TCE 0-25 ppmv and PCE 0-25 ppmv. The reactor temperature was maintained at 25 ⁇ C by circulating water from a constant temperature bath through the jacket of the bioreactor. Nutrient solution was trickled down the bioreactor at a flow rate of 1 liter per day and the nutrient composition was as follows:
  • KH 2 P0 4 (85 mg/L)), K,HP0 4 (217.5 mg/L)), Na 2 HP0 4 .2H 2 0 (334 mg/L), NH 4 C1 (25 mg/L), MgS0 4 .7H 2 0 (22.5 mg/L), CaCl 2 (27.5 mg/L) and FeCl 3 .6H 2 0 (0.25 mg/L), MnS0 4 .H 2 0 (0.0399 mg/L), H 3 BO 3 (0.0572 mg/L), ZnS0 4 .7H 2 0 (0.0428 mg/L), (NH 4 )6Mo 7 0 24 (0.0347 mg/L), FeC j .EDTA (0.1 mg/L), and yeast extract (0.15 mg/L).
  • EXAMPLE 1 A mixture of toluene and trichloroethylene (TCE) was injected into the incoming air stream, and the inlet and outlet concentrations of toluene and TCE were measured using a gas chromatograph. Results shown in Table l were obtained at various inlet air flow rates, when the inlet toluene concentra ⁇ tion was maintained at 100 ppmv and the inlet TCE concentra ⁇ tions at 25 ppmv.
  • TCE trichloroethylene
  • Carbon and chlorine balances were made by monitoring the increase in carbon dioxide in the exit air and increase in chloride ion concentration in the exit nutrients, as analyzed
  • the anoxic zone was created due to oxygen consumption in the aerobic zone by the oxic degradation of the partially dehalogenated products as they diffused out from the anoxic zone.
  • EXAMPLE 4 The procedure for making gel beads was slightly modified to include colloidal zero valent metal, such as iron, inside the bead. This was achieved by mixing the active aerobic cells with colloidal zero valent iron and then mixing the solution with the mixture of silica sol and sodium alginate, as presented before. The composition of the sodium alginate and
  • silica sol was the same as in the earlier experiments.
  • the mixture of silica soil, sodium alginate, cells and colloidal iron was contacted with calcium chloride solution, the mixture gelled due to the formation of calcium alginate.
  • the silica sol gelled to form silica gel with a dispersion of calcium alginate, cells and
  • the mechanism of PCE degradation is partial dechlorination 25 by the colloidal iron with subsequent aerobic degradation of the partially dehalogenated products by the active cells in the gel bead. Oxygen is consumed by the active cells, and this prevents oxygen from passivating the iron surface by forming oxides. Anaerobic bacterial dechlorination did not occur in this instance, since no organis cource was presnet as electron donor. In the absence of formate (or an alternative organic electron source) to drive the anaerobic microbial dehalo ⁇ genation, partial dehalogenation by zero valent iron becomes the main mechanism for initial decomposition of PCE. Discussion:
  • VOCs volatile organic compounds
  • Air emissions repre ⁇ sented the largest source of toxics, comprising 39% of the 6.24 billion pounds of chemicals released into the environment in 1988. Release of VOCs occurs at chemical or processing indus ⁇ tries, at facilities of commercial and industrial solvent users, at waste water treatment plants, and at Superfund and other waste disposal sites.
  • chlorinated aliphatics are used extensively as industrial solvents for degreasing and cleaning applications.
  • the General Electric Aircraft Engine Plant located in Cincinnati, Ohio currently uses a blended CAH mixture that requires carbon adsorption to prevent CAH air emissions.
  • the dry cleaning industry routinely uses PCE that would be regulated under the Clean Air Act Amendments. Improved technologies are needed to manage the environmental contamination by chlorinated aliphatics.
  • VOCs Conventional technologies that have been used to control VOCs includes: (1) adsorption onto porous materials, such as activated carbon; (2) absorption into liquid solution; (3) catalytic oxidation or incineration; and (4) selective separation using membranes.
  • Biofiltration involves microbial degradation of the VOCs in air. As compared to non-biological options, Leson and Winer (1991) indicate that biofiltration is cheap, reliable and represents a more natural approach for control of VOCs.
  • Current biofilters use either natural bioactive materials, such as soil, peat or compost, or immobilizing activated sludge bacteria on inert pellets of activated carbon or porous ceramic material.
  • the biofilm consists of mainly aerobic bacteria, which reduces their effectiveness for treatment of polychlorinated VOCs.
  • TCE requires a co- metabolite, such as toluene or phenol to degrade under aerobic conditions while PCE is recalcitrant under aerobic conditions.
  • encapsulated biomass in gel beads allows biofiltration of chlorinated VOCs, as shown in the experimental studies.
  • encapsulated biomass in gel beads is capable of efficient ⁇ ly degrading all the contaminants, rather than only the aerobically degradable compounds, as occurs in conventional biofiltration.
  • the encapsulated biomass gel technology is simple to operate and highly reliable, since it involves no moving parts in the reactor itself.
  • the high degradation rates achieved in hydrogel pellets allows the CAHs to be destroyed in small reactors with minimal operating costs associated with pumping the mineral nutrients.
  • Preliminary economic evaluation of the proposed process using hydrogel pellets indicates that bioremediation of TCE contaminated air in the applicable concentration range ( ⁇ 40 ppmv) has lower annual costs than competitive technologies such as carbon adsorption and catalytic oxidation.
  • Estimated savings of about 50% using the biological treatment system compared to the carbon adsorption system can be achieved.
  • Chlorinated solvents consisting primarily of chlorinated aliphatic hydrocarbons (CAHs) , have been used widely for degreasing of aircraft engines, automobile parts, electronic components, and clothing. Due to water solubilities exceeding drinking water standards and densities higher than water, CAHs migrate downward through soils contaminating ground water and penetrate deeply into aquifers forming dense non aqueous phase liquids (DNAPLs) on aquifer bottoms.
  • CAHs chlorinated aliphatic hydrocarbons
  • the major chlorinated solvents used in the past are carbon tetrachloride (CT) , tetrachloroethene (PCE) , trichloroethene (TCE) , and 1,1,1-trichloroethane (TCA) .
  • CT carbon tetrachloride
  • PCE tetrachloroethene
  • TCE trichloroethene
  • TCA 1,1,1-trichloroethane
  • CAHs chloroform
  • MC methylene chloride
  • cis-and trans-l,2-dichoroethene cis-DCE, trans-DCE
  • 1,1-dichloroethene 1,1-dichloroethene
  • vinyl chloride VC
  • DCA 1,1-dichloroethane
  • CA chloroethane
  • DOE Department of Energy
  • DOE Department of Energy
  • the DOE Hanford site has massive contamination of soil and ground water with carbon tetrachloride (CT) with the ground water plume extending over ⁇ 5 60 square miles.
  • C carbon tetrachloride
  • CAHs At concentrations below their inhibition level, most of the CAHs are aerobically degradable. Some CAHs, such as TCE require co-metabolites or specialized organisms for aerobic degradation. Co-metabolism is a complicated process compared
  • PCE is aerobically
  • CAHs are transformable under anaerobic conditions using formate or another suitable organic source such as acetate, butyrate, etc, as the organic carbon (electron donor) source. Rates of anaerobic transformations are greater for highly chlorinated CAHs compared to the less
  • Anaerobic/aerobic treatment using hydrogel pellets offers a multimedia, multi- pollutant and multi-industry technology.
  • the hydrogel pellets can be used to treat surface soil contamination by CAHs by combining soil vacuum extraction with biofiltration, enhanced bioventing of vadose zone contamination, air stripping of ground water followed by biofiltration and in-situ treatment of ground water using a bio-cassette approach.
  • the proposed technology can handle contamination by several pollutants (aerobically degradable compounds as well as mixture of CAHs) and can treat multimedia contamination through suitable combination of existing separation technologies with the proposed technology.
  • Viable-cell immobilization in a hydrogel offers many advantages: (1) enhanced biological stability, since the viable cells are protected from environ ⁇ mental contamination by other microorganisms, fungi; (2) high biomass concentration; (3) reduced oxygen permeation, thereby allowing the creation of an anoxic zone, even though the reactor may have air present or high levels of dissolved oxygen ; (4) reduced biomass growth rates; and (5) advantageous partition effects of contaminant between air/water and hydrogel phase.
  • the hydrogel pellets can also be mixed with contaminated soil or soil slurries, thereby allowing the diffusion of the contaminants into the gel bead and consequent mineralization of the contaminant through a synchronous anoxic/oxic pathway.
  • Contaminants that do not degrade under aerobic conditions, such as DDT, PCB's are particularly amenable for this type of approach using the gel beads.
  • Gel encapsulation can also be used for microbial cultures that do not attach to surfaces.
  • SRBs sulfate reducing bacteria
  • Gel encapsulation can also be used for microbial cultures that do not attach to surfaces.
  • SRBs sulfate reducing bacteria
  • SRBs can be encapsulated in gels using the procedure given in this application and used in fixed or expanded beds for bioreduction of sulfate in acid mine drainage or produced by absorption of sulfur dioxide from stack gases.

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Abstract

La présente invention concerne des compositions et des systèmes de supports de gels comportant des inclusions de gels d'alginates et de bactéries enrobées dans des gels de silice. De nouvelles préparations de gels donnent un espace vide approprié pour la croissance et le maintien des cellules actives, surmontant l'inconvénient des gels de silice correspondant à l'état de la technique. Ces compositions de gels donnent des conditions appropriées de survie et de croissance de cellules actives, elles sont suffisamment stables dans les conditions d'utilisation dans les filtres biologiques et elles résistent à la décomposition ou à la biodégradation par les cellules piégées. Ces gels ne sont pas solubles dans l'eau. Les structures contenant des compositions de gels selon l'invention peuvent être formulées de telle manière qu'elles puissent être logées dans un lit. La partie du gel constituée par de la silice peut absorber des contaminants dans une mesure suffisante tout en faisant preuve d'une faible diffusibilité de l'oxygène.
PCT/US1996/006765 1995-05-12 1996-05-10 Nouvelles compositions d'hydrogels destinees a l'utilisation dans des bioreacteurs WO1996035780A1 (fr)

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US08/439,973 1995-05-12

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100445377C (zh) * 2006-12-21 2008-12-24 天津大学 仿生制备用于固定化β—葡萄糖醛酸苷酶的二氧化硅—海藻酸微囊的方法
GB2452552A (en) * 2007-09-08 2009-03-11 Univ Sheffield Hallam Corrosion-inhibiting sol-gel coating
DE102009037768A1 (de) 2009-08-17 2011-02-24 Gesellschaft zur Förderung von Medizin-, Bio- und Umwelttechnologien e.V. Bioaktives Kompositmaterial
US9226992B2 (en) * 2005-08-17 2016-01-05 Orthox Limited Implantable cartilaginous tissue repair device

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148689A (en) * 1976-05-14 1979-04-10 Sanraku-Ocean Co., Ltd. Immobilization of microorganisms in a hydrophilic complex gel
US4659664A (en) * 1985-05-10 1987-04-21 Excel-Mineral Company, Inc. Structures containing immobilized microbial cells
US4797358A (en) * 1983-12-05 1989-01-10 Kikkoman Corporation Microorganism or enzyme immobilization with a mixture of alginate and silica sol

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4148689A (en) * 1976-05-14 1979-04-10 Sanraku-Ocean Co., Ltd. Immobilization of microorganisms in a hydrophilic complex gel
US4797358A (en) * 1983-12-05 1989-01-10 Kikkoman Corporation Microorganism or enzyme immobilization with a mixture of alginate and silica sol
US4659664A (en) * 1985-05-10 1987-04-21 Excel-Mineral Company, Inc. Structures containing immobilized microbial cells

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9226992B2 (en) * 2005-08-17 2016-01-05 Orthox Limited Implantable cartilaginous tissue repair device
CN100445377C (zh) * 2006-12-21 2008-12-24 天津大学 仿生制备用于固定化β—葡萄糖醛酸苷酶的二氧化硅—海藻酸微囊的方法
GB2452552A (en) * 2007-09-08 2009-03-11 Univ Sheffield Hallam Corrosion-inhibiting sol-gel coating
DE102009037768A1 (de) 2009-08-17 2011-02-24 Gesellschaft zur Förderung von Medizin-, Bio- und Umwelttechnologien e.V. Bioaktives Kompositmaterial

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