WO1996035780A1

WO1996035780A1 - New hydrogel compositions for use in bioreactors

Info

Publication number: WO1996035780A1
Application number: PCT/US1996/006765
Authority: WO
Inventors: Dolloff Bishop; Rakesh Govind
Original assignee: United States Environmental Protection Agency; University Of Cincinnati
Priority date: 1995-05-12
Filing date: 1996-05-10
Publication date: 1996-11-14

Abstract

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 decomposition 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 contaminants to sufficient extent whilst exhibiting low oxygen diffusivity.

Description

APPLICATION FOR UNITED STATES PATENT NEW HYDROGEL COMPOSITIONS FOR USE IN BIOREACTORS

Field of the Invention;

This invention relates to new hydrogel compositions which i are useful for establishing biological nitches for cells to 5 remove pollutants from the environment. Background of the Invention;

The use of 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.

10 Gels have properties of both solids and liquids. For example, 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

15 those of a semipermeable membrane. Water can move in and out of the gel, depending on the external environment.

One example of an available material for gel formation is silica sol which can be obtained commercially as LUDOX™. Silica gel is made from a colloidal solution of the silica.

20 Gelation occurs as pH drops from 10 to about 7.5. The resulting gel is stable under neutral pH conditions. However, if colloidal silica at pH 10 is mixed with active cells, the cells may be damaged. More importantly, silica does not provide void space for cell growth.

{25 One strategy to overcome inhibition arising on account of pH has been to add active cells after the pH value of the colloidal silica has decreased to about 9. However, at pH 9 the silica sol begins to gel and the viscosity of the partially gelled material is sufficiently high to create difficulties in trying to mix the cells with the gelling material. Further¬ more, the mixing with the viscus gel causes shear forces that can result in disruption of cell walls. Alternative gelling materials have been used as carriers of nutrients and biological materials. Some of materials most widely used for gel formation are alginates. Chemically, the alginates consist of linear polymers of 1,4-linked beta-D- mannuronic acid and 1,4-linked alpha-L-guluonic acid. The polymers may be arranged in blocks of either one of the acids or in alternating blocks of mannuronic and guluonic acids.

U.S. patent 3,649,239 to Mitchell, which is incorporated herein in its entirety by reference, teaches use of alginate gels as delivery systems for slow release of fertilizer chemicals into the soil.

U.S. Patent 4,053,627 to Scher, which is incorporated herein in its entirety by reference, teaches use of alginate gel preparations as means of providing controlled release of juvenile hormones into the environment to control insect reproduction and for delivery of other materials for agricul¬ tural purposes.

U.S. Patent 4,668,512 to Lewis, et al, which is incorpo¬ rated herein in its entirety by reference, teaches use of algi¬ nate/fungal/bran compositions to make gel pellets which are used to inoculate agricultural fields.

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.

However, 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. Furthermore, 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.

Various types of organic molecule transformations have been possible using microorganisms. In some systems, mineral¬ ization generates carbon and energy for microbial growth whilst causing degradation of organic pollutants to carbon dioxide, biomass, water and, in some instances, ammonia. In some instances a processes known as co-metabolism occurs whereby microorganisms, in the presence of a growth substrate, effect changes in some recalcitrant substrates which do not, in their original form, promote growth. Co-metabolic transformations may thus lead to transformations of pollutants so that full mineralization may then occur as result of action of other organisms. However, these co-metabolic processes do not occur in prior art silica gels. Description of the Invention;

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.

It was a specific objective of this invention to provide 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. These 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

10 dehalogenate poly-chlorinated compounds. Partially dechlori- nated products are then aerobically biodegraded in the oxic zones of the hydrogel.

The compositions of the invention can be formulated in such a manner that zero valent metals, such as iron and

15 aluminum, which are useful in the process of dehalogenation of poly-chlorinated compounds in anaerobic conditions, can be incorporated into the gels. Hence, it is possible to combine aerobic degradation of dehalogenated products with partial dehalogenation by zero valent metals.

20 Materials and Methods;

Silica sol was obtained commercially as LUDOX™ 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

,25 pH of between 7-8 using 5 normal HC1. Biomass containing active aerobic and anaerobic cells were added to the solution to provide a final solution containing 2% to 10% biomass. The mixture was stirred and then poured into a petri dish to a depth of 5 mm. Calcium chloride solution was poured on top of the mixture in the petri dish. The silica sol and sodium alginate mixture immediately gelled due to the diffusion of calcium forming calcium alginate on the outer surface. The gel was allowed to cure from 10 minutes to 24 hours. During the curing process, the pH of the silica sol decreases. The resulting gel is a combination of silica gel with pockets of calcium alginate and cells inside the silica gel.

The survival and growth of active cells is maximized by using the combination of 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.

Other methods of producing supports of silica gel with alginate gel containing viable organisms can be practiced. For example, 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^βC 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:

KH₂P0₄ (85 mg/L)), K,HP0₄ (217.5 mg/L)), Na₂HP0₄.2H₂0 (334 mg/L), NH₄C1 (25 mg/L), MgS0₄.7H₂0 (22.5 mg/L), CaCl₂ (27.5 mg/L) and FeCl₃.6H₂0 (0.25 mg/L), MnS0₄.H₂0 (0.0399 mg/L), H₃BO₃ (0.0572 mg/L), ZnS0₄.7H₂0 (0.0428 mg/L), (NH₄)6Mo₇0₂₄ (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.

TABLE 1: Percent degradation of toluene and TCE in the bio¬ reactor at various inlet air flow rates. Inlet concentrations were toluene 100 ppmv and TCE 25 ppmv. Nutrients were trickled down the bioreactor at a flow rate of 1 liter per day. Percent degradation for each contaminant is defined as follows: [ (inlet concentration - outlet concentration) (inlet concentration)] X 100.

Air flow rate (ml.min) % degradation Of % degradation of toluene TCE

1 100 63.8

10 100 31.2

18 60 20.8

30 40 18.7 40 32 17.6

This example demonstrates co-metabolic degredation of TCE with toluene as the co-metabolite. EXAMPLE 2:

Studies were conducted with only TCE entering the bioreactor at an inlet concentration of 25 ppmv. The nutrient composition was changed as follows: KH₂P0₄ (85 mg/L)),

(217.5 mg/L)), Na₂HP0₄.2H₂0 (334 mg/L), NH₄C1 (25 mg/L), MgS0₄.7H₂0 (22.5 mg/L), CaCl₂ (27.5 mg/L) and FeCl₃.6H₂0 (0.25 mg/L), MnS0₄.H₂0 (0.0399 mg/L), H₃BO₃ (0.0572 mg/L), ZnS0₄.7H₂0 (0.0428 mg/L) , (NH₄)6Mo₇0₂₄ (0.0347 mg/L) , FeCl₃.EDTA (0.1 mg/L) , yeast extract (0.15 mg/L) and formate (50 mg/L). Results obtained are shown in Table 2. Table 2: Percent degradation of TCE in the bioreactor at various air flow rates. Inlet TCE concentration was 25 ppmv and nutrient flow rate was 1 liter/day.

5 Air flow rate (ml/min) % degradation TCE i

35 100

L 40 67.2

50 40.7

10 60 22.1

65 10.8

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

15 by an ion chromatograph. The chlorine balance was developed at steady-state conditions within an error band of 15% of the calculated increase in chloride ion concentration.

By adjusting the nutrients, a degradation pathway is developed for anaerobic microbial dehalogenation in the anoxic

20 zone followed by oxic biodegradation of the anoxic degradation products in the outer aerobic zone of the gel bead. 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.

25 A mathematical model was developed to describe the diffusion of TCE and oxygen and consumption of oxygen due to aerobic degradation of the dehalogenated products. In the liquid film at the outer surface in the gel bead constructed as described herein, the oxygen concentration is about 8 mg/L

30 due to presence of air outside the bead. As oxygen diffuses inside the gel bead, it is consumed due to aerobic degradation of the dehalogenated products diffusing outward. At some point in the interior of the gel bead, oxygen is completely consumed producing an anoxic zone in the interior portion of the gel bead. It is in this anoxic zone that dehalogenation of TCE occurs. The formate in the nutrient medium is rapidly absorbed by the gel bead and provides the organic carbon source (an electron donor) needed for partial dehalogenation of TCE in the anoxic zone. Other potential carbon sources are acetate, butyrate and other carboxylic acids. EXAMPLE 3:

A study was conducted in the manner of Example 2 using PCE instead of TCE at inlet concentration of 25 ppmv. Results are shown in Table 3.

Air flow rate (ml/min) % degradation PCE

10 100

15 86.7 20 72.4

30 41.8

50 12.8

Chloride ion balances were obtained at steady-state to prove that complete mineralization of PCE had occurred. Each experiment had to be conducted for over 5 days to achieve a stable exit concentration of chloride ion in the exit nutri¬ ents. No other by-products were observed in the exit gas phase at the above operating conditions. 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. When i 5 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. Then as the beads were cured, the silica sol gelled to form silica gel with a dispersion of calcium alginate, cells and

10 colloidal iron. These gel beads were then randomly packed in a 40 ml bioreactor, as before, and air contaminated with PCE was passed through the reactor. Nutrient solution, as in Example 1, was trickled down the bioreactor at a flow rate of 1 liter per day. Results obtained are shown in Table 4.

15

Table 4: Percent degradation of PCE in the bioreactor at various air flow rates. Inlet PCE concentration was 25 ppmv.

Air flow rate (ml/min) % degradation PCE

20 10 100

20 100

30 96

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 icrobial 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:

In recent years, control of volatile organic compounds (VOCs) has received increased attention. 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. In addition, chlorinated aliphatics are used extensively as industrial solvents for degreasing and cleaning applications. For example, 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.

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. In all of these biofilters, 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.

The use of encapsulated biomass in gel beads allows biofiltration of chlorinated VOCs, as shown in the experimental studies. In many cases, where mixtures of non-chlorinated VOCs and polychlorinated compounds are present as contaminants in air, 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.

Ground water contamination is quite widespread around Superfund sites. In many cases, the contaminants are chlori¬ nated solvents, such as carbon tetrachloride, TCE or PCE. 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. The major chlorinated solvents used in the past are carbon tetrachloride (CT) , tetrachloroethene (PCE) , trichloroethene (TCE) , and 1,1,1-trichloroethane (TCA) . These compounds are biotransformed to form a variety of CAHs, including chloroform (CF) , methylene chloride (MC) , cis-and trans-l,2-dichoroethene (cis-DCE, trans-DCE) , 1,1-dichloroethene (1,1-DCE), vinyl chloride (VC) , 1,1-dichloroethane (DCA) , and chloroethane (CA) . Thus these solvents and their natural transformation products are important widespread soil and ground water contaminants. Ground water toxicity problems associated with CAHs occur at over 358 major hazardous waste sites and many minor sites across the nation. As examples. Department of Energy (DOE) site at Savannah River has 2.1 million pounds of TCE contami¬ nating soils and ground water. The DOE Hanford site has massive contamination of soil and ground water with carbon tetrachloride (CT) with the ground water plume extending over _ψ 5 60 square miles.

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

10 to the usual biological treatment processes. Full-scale field applications of co-metabolic destruction of CAHs are greatly limited by the availability, cost, and potentially adverse environmental impacts of the secondary substrates needed for induction of co-metabolic activity. PCE is aerobically

15 recalcitrant. All of the 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

20 chlorinated CAHs. One of the transformation products, vinyl chloride is more toxic than the parent compounds but is rapidly degraded under aerobic conditions.

Combining anaerobic transformations with aerobic degrada¬ tion of the transformation products is a significant environ-

,25 mental bioremediation technology that has widespread applica¬ tions for treatment of CAHs in soils and ground water. 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. Hence, 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.

Specialized bacterial cultures grown in the laboratory have usually been found to be not competitive with indigenous cultures. By entrapping the specialized cultures in gels, as taught in this application, these bacterial cultures can be provided protection from indigenous cultures. This would allow the specialized culture to function in the environmental system without being affected by other cultures.

Gel encapsulation can also be used for microbial cultures that do not attach to surfaces. For example, 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.

Claims

1. A composition of matter comprising a solution containing silica sol and alginate wherein the ratio of alginate to silica in said solution is, by weight, 1-10 parts algi¬ nate to 90-99 parts silica.

2. A composition of claim 1 containing, additionally, 1% to 10% biomass.

3. A composition of matter comprising a gel containing silica and alginate wherein the ratio of alginate to silica in said gel is 1-10 parts alginate to 90-99 parts silica.

4. A composition of claim 3 containing microorganisms.

5. A composition of claim 3 in the form of a bead.

6. A composition of claim 3 in the form of a plate of gel.

7. A composition of claim 3 in a thread.