Patent Publication Number: US-2021163917-A1

Title: Enzyme formulation and method for degradation

Description:
BACKGROUND OF THE INVENTION 
     Hydrocarbon contamination resulting from drilling and extraction of oil has become one of the major environmental problems. Accidental releases of petroleum products are of particular concern for human health since hydrocarbon components cause extensive damage to the environment and contaminate the soil. The microbially mediated breakdown of heavy weathered total petroleum hydrocarbons (TPH) has its limitations due to the degradation of only up to 4-ring aromatic compounds and 25-carbon saturated compounds. Moreover, the presence of polycyclic aromatic hydrocarbons (PAHs) with two or more fused benzene rings in linear, angular or cluster structural arrangements and low solubility poses an additional remediation challenge. 
     Therefore, it would be desirable to provide a new enzyme formulation and method for enhanced degradation of hydrocarbons. The enzyme formulation can be particularly useful for enhanced remediation of hydrocarbon contaminated soil matrices. 
     It would also be desirable to provide an enzyme formulation and method for the degradation of vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds. 
     SUMMARY OF THE INVENTION 
     An enzyme formulation comprises an encapsulated fungal enzyme which is effective for degrading at least one material selected from the group consisting of hydrocarbons, vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds. 
     A degradation method comprises treating one of the above-mentioned materials with an encapsulated fungal enzyme to degrade the material. 
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The ability of ligninolytic enzymes to degrade larger molecular compounds through the use of radical reactions brings potential to enhance the degradation of crude oil and other hydrocarbons. Since ligninolytic enzymes are extracellular, they are able to diffuse effectively to the highly immobile high molecular weight hydrocarbons and result in metabolites that are more bioavailable for further microbially induced breakdown. 
     The present invention includes ligninolytic enzymes which are encapsulated to stabilize the enzymes. The invention can further include formulating the stabilized enzymes for a specified application such as for the breakdown of TPHs. 
     The invention can further include the encapsulation of other species of fungal enzymes for use in breaking down other materials. 
     Example of Process for Producing Ligninolytic Enzymes: 
     Fungal species of, but not limited to,  Phanaerochete chrysosporium, Nematoloma forwardii  or  Trametes versicolor  are cultivated in flask and bioreactor cultures in standard conditions. Other examples of fungal species and known producers of ligninolytic enzymes include  Phanerachete  spp,  Tremetes  spp,  Phlebia  spp,  Cerena  spp,  Merulius  spp,  Pellinius  spp,  Cyatus  spp, and  Stereum  spp. The selected fungus secretes extracellularly into the growth medium a suite of ligninolytic enzymes: manganese peroxidase, lignin peroxidase and laccase. The enzyme activity can be analyzed using standard methods. 
     The use of purified enzyme results in the highest possible activity. Once the activities of manganese peroxidase and lignin peroxidase reach maximum, the extracellular liquids with growth medium are collected. The fungal mycelium is separated from the liquid components via sterile gauze and incinerated. The remainder of extracellular liquids is then filtered and purified using standard methods. Purified enzyme suite is then lyophilized and utilized for further stabilization. 
     Any suitable ligninolytic enzyme(s) can be used in the invention. The major groups of ligninolytic enzymes include lignin peroxidases, manganese peroxidases, versatile peroxidases, and laccases, examples of which are known to enzyme scientists. 
     Although the enzymes may be a suite of enzymes produced by a fungus, alternatively a single type of ligninolytic enzyme may be used. Further, the enzyme(s) are not necessarily produced by a fungus, but could instead be produced by a microorganism or other source. Although purified enzymes are preferred, in certain embodiments the enzymes may be used in nonpurified form. 
     Enzymes Stabilization: 
     The enzymes are stabilized by encapsulation. For example, the enzymes can be encapsulated in a protective shell. In certain embodiments, the type of encapsulation is microencapsulation. The shell can be any material that is effective to stabilize the enzymes. In certain embodiments, the shell is effective to cause slow release of the enzymes. For example, in one enzyme release experiment the encapsulated enzymes had a very low enzyme activity the first 7 days, and the enzyme activity gradually increased to a high activity between days 7 and 28. 
     In certain embodiments, the shell consists of a cross-linked hydrogel. Crosslinking is a way of curing the hydrogel. The process of crosslinking initiates from the outer layer and progresses to the core; in this way the enzymes are forced to stay inside the hydrogel. Furthermore, the crosslinking reaction provides rigidity to the hydrogel. It is also believed that a crosslinker such as manganese will get involved into the enzyme catalytic cycle and replenish manganese as needed during the decontamination process. 
     The cross-linked hydrogel is exemplified by but not limited to calcium alginate, manganese alginate, zirconium alginate, calcium poly(aspartate), manganese poly(aspartate) and zirconium poly (aspartate). 
     More generally, a good match of the encapsulating (shell) material and the enzymes may be determined based on the concentration, and the valency of the cation. For example, higher concentration and higher valent cation (Zr+4 is more effective than Ca+2) increases the gelation time. 
     In another embodiment the enzymes are stabilized by encapsulation in a shell having two or more layers. For example, the protective shell may comprise two layers wherein the first layer interfaced with the enzyme is a cross-linked hydrogel, and the second layer interfaced with the first layer is a hydrophobic material such as an oleogel. The two layer shell is for swelling the contaminants in the outermost layer followed by oxidation reaction in the inner layer when they come in contact with the enzyme. The hydrophobic material can attract and be attracted to hydrocarbons. 
     The oleogel is exemplified by but not limited to poly(lauryl methacrylate), poly(stearyl methacrylate), poly(isoprene) and poly (butadiene). 
     In certain embodiments, the encapsulant has one or more of the following benefits: room temperature process, bio-based and biodegradable matrix, absorbs water, fast synthesis, VOC free/no solvents, variable particle size, and stable pH 4 to 6 range. 
     In certain embodiments, the shell works as a donor of one or more mediators for activation of enzymes within the capsule. For example, as described above, a manganese crosslinker can replenish manganese to activate the enzyme catalytic cycle. In certain embodiments, the shell is formulated to attract oil molecules or other material to be degraded. 
     The encapsulated enzymes can be produced in the form of beads or any other form suitable for a particular application. In certain embodiments, the beads have a diameter within a range of from about 1.5 mm to about 5 mm, and more particularly within a range of from about 2.8 mm to about 3.5 mm (“diameter” refers to maximum diameter). In several examples, ligninolytic enzymes have been encapsulated in an alginate shell to produce spherical beads having diameters of 1.9 mm, 2.8 mm and 3.0 mm. 
     Example 
     The process of stabilizing the enzyme is further exemplified in the following example. 10 mg of manganese peroxidase was rinsed into 10 g of alginate stock solution with 1 mL of deionized water. The suspension was mixed until dissolve with vial mixer and uniform. 10 grams of alginate mixture was then drawn up into a 10 mL syringe. Alginate drops containing manganese peroxidase were dropped using 0.3 mm gauge needle into the 50 mL calcium chloride solution using syringe pump (Cole-Palmer 78-0100C) with the retention time of 100 mL/h. Formation of encapsulated gel-like beads of alginate-enzyme complex was detected. The enzyme capsules were then left to settle at the bottom of the container and refrigerated at 8 C until use. 
     Formulating the Stabilized Enzyme for a Specified Application: 
     The invention further includes formulating the stabilized enzyme for specified application. In one example, the stabilized enzyme is formulated in a liquid or solid matrix. The matrix comprises a peroxide such as hydrogen peroxide or its derivatives and dispersing aid such as surfactants. 
     Possible Market and Product Applications: 
     Encapsulated fungal enzymes in the environmental setting for the purpose of degradation of total petroleum hydrocarbons may be of interest to oil and gas companies. Furthermore, since radical reaction pathway of fungal ligninolytic enzymes is highly unspecific, the application of the technology may be broad ranging from: degradation of heavily weathered petroleum hydrocarbons, petroleum hydrocarbons, jet fuel, Navy special fuel, polyfluorinated compounds (PFCs), dioxins; PCBs, herbicides, pesticides, munition constituents, lubricants, oils, detoxification of industrial effluents, and dye effluents. The enzyme formulation and method can be useful for the degradation of vulcanized rubber, synthetic rubber, natural rubber, vulcanized polymers and perfluorinated compounds such as perfluorooctanesulfonic acid and perfluorooctane sulfonate. 
     Evaluation of Fungal Enzyme Extracts to Catalyze Remediation of Heavily Weather Crude Oil Contaminated Soil: 
     Objective: To develop a method to treat heavily weathered crude contaminated soil to &lt;1% TPH encapsulated fungal enzymes. 
     Hypothesis: Fungal enzymes can non-selectively break down long-chain hydrocarbons into shorter chain hydrocarbons that can be further degraded by microorganisms. 
     
       Phanerochaete chrysosporium  
     
     Secrete a suite of oxidoreductases (manganese peroxidase, laccase and lignin peroxidase). 
     The cation radical of heme porphyrin reacts with an array of compounds and initiates non specific of recalcitrant environmental pollutants. (See  FIG. 2 ) 
     Enzyme Characteristics: 
     Non specific degradation of recalcitrant environmental pollutants e.g. TPH. 
     Dosage for treating recalcitrant contaminants—1 U/1.89 mg/kg PAH. 
     Encapsulation: 
     To provide reactive ingredient (enzyme) in an easily applicable form without the risk of introducing non native fungal species. 
     Examples of Encapsulation Technology: 
     Microencapsulation via spray drying with mixture of polymer and solvent (solid material) 
     Encapsulation into hydrogel particles using non aqueous dispersion process 
     Encapsulation using complex co-aservation 
     Encapsulation via electrospray 
     Selection of Encapsulant: 
     Match suitable encapsulation route with critical process metrics to gain high probability of success. 
     Ionotropic Alginate Gellation Benefits: 
     Room temperature process 
     Bio-based and biodegradable matrix 
     100% aqueous 
     Fast process 
     VOC free/No solvents 
     Variable particle size 
     Contaminated Samples: 
     Grand Calumet River Sediments 
     Contamination from multiple industries including oil refineries on the banks of the river 
     Contamination in place since 1970&#39;s 
     Contaminants include PCBs, heavy metals, crude oil, PAHs, heavily weathered petroleum hydrocarbons, petroleum hydrocarbons, vulcanized rubber, jet fuel, Navy special fuel, synthetic rubber, polyfluorinated compounds (PFCs), dioxanes; PCBs, herbicides, pesticides, and munition constituents. 
     
       
         
           
               
            
               
                   
               
               
                 Soil Characteristics 
               
            
           
           
               
               
            
               
                   
                 Concentration 
               
               
                   
                 (mg/kg dry weight) 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 TPH in Soil 
                   
               
               
                   
                  C6-C12 
                 ND 
               
               
                   
                 &gt;C12-C28 
                 1,530 
               
               
                   
                 &gt;C28-C35 
                 581 
               
               
                   
                 Total C6-C35 
                 2,110 
               
               
                   
                 PAHs in Soil 
               
               
                   
                 Acenaphthene 
                 0.231 
               
               
                   
                 Acenaphthylene 
                 1.45 
               
               
                   
                 Benzo[a]anthracene 
                 9.78 
               
               
                   
                 Chrysene 
                 31.6 
               
               
                   
                 Phenanthrene 
                 19.1 
               
               
                   
                 Fluoranthene 
                 15.31 
               
               
                   
                   
               
               
                   
                 Soil Moisture Content 17% 
               
            
           
         
       
     
     Metagenomics and Metaproteomics: 
     To understand the shift in microbial population as a result of application of fungal enzymes and degradation of TPH 
     To detect suite of microbial proteins directly involved in TPH degradation 
     Use data to optimize treatment 
     Application of Omic Technologies: 
     Biodegradation: Baseline and time/dose response characterization 
     Community structure (microbes) 
     Functional potential (genes) 
     Function (proteins) 
     Additional Work: 
     Optimize encapsulation conditions 
     Conduct experiment with encapsulated enzyme formulation 
     Analysis of metaproteome after treatment with encapsulated enzyme to compare protein composition 
     Application to Field Treatment: 
     Formulation of encapsulated enzyme with hydrogen peroxide embedded 
     Apply encapsulated enzyme into vadose zone soils using backhoe 
     Encapsulant is resistant to mechanical stress due to size 
     Reaction is expected to occur rapidly with the reduction in TPH seen within 28-30 days after application 
     Measure TPH concentration to determine when to reapply enzyme 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  illustrates the experimental setup for alginate encapsulation of enzyme. 
         FIG. 2  illustrates the reaction pathways of heme porphyrin with various reactants. 
         FIG. 3  illustrates the molecular structure of a tetrasaccharide monomer of the ionotropic alginate ion polymer. 
     
    
    
     EXPERIMENTATION 
     Laboratory Experiment Approach: 
     Dry Soil 
     Grow culture of  P. chrysosporium  and collect extracellular enzyme extract
 
Lyophilize enzyme extract and resuspend in 10 ML
 
Apply to soil microcosm and add hydrogen peroxide (reaction substrate)
 
     pH 4.5, Temp 25 C 
       
                            Treatments                                     Set Up   Soil   Enzyme   Peroxide                                                 1   ✓                   2   ✓   ✓           3   ✓   ✓   ✓                        
20 g soil
 
2 mL purified enzyme at 20 U/mL
 
100 μL 10 mM hydrogen peroxide added every other day
 
TPH and PAH measured after 7 days and 14 days
 
All treatments prepared in duplicates
 
     
       
         
           
               
            
               
                   
               
               
                 Results - Soil, Enzymes + H 2 O 2 , Day 7 
               
            
           
           
               
               
               
               
            
               
                   
                 Day 0 
                 Day 7 
                 Percent 
               
               
                   
                 (mg/kg dry weight) 
                 (mg/kg dry weight) 
                 Loss 
               
               
                   
                   
               
            
           
           
               
               
            
               
                 TPH in Soil 
                   
               
               
                  C6-C12 
                 ND 
               
               
                 &gt;C12-C28 
                 1,530 
               
               
                 &gt;C28-C35 
                 581 
               
               
                 Total C6-C35 
                 2,110 
               
            
           
           
               
            
               
                 PAHs in Soil 
               
            
           
           
               
               
               
               
            
               
                 Acenaphthene 
                 0.231 
                   
                   
               
               
                 Acenaphthylene 
                 1.45 
               
               
                 Benzo[a]anthracene 
                 9.78 
               
               
                 Chrysene 
                 31.6 
               
               
                 Phenanthrene 
                 19.1 
               
               
                 Fluoranthene 
                 15.31 
               
               
                   
               
            
           
         
       
     
     ADDITIONAL EXPERIMENTS 
     1. Selection of Fungal Strains for Production of High Concentrations of Enzymes: Manganese Peroxidase and Laccase. 
     Exp. 1 . Phanerachaete chrysosporium  1309 , Lenzites betulina  141 , Trametes versicolor  159 , Trametes cervina  33 , Trametes ochraceae  1009 , Trametes pubescens  11 , Stereum hirsutum  42 , Trametes zonatus  540 , Trametes hirsuta  119 , Phlebia radiata  312.
 
Composition of synthetic medium, (g/l): KH 2 PO 4 —1.0; MgSO 4 —0.5; CaCl 2 —0.1; FeSO 4 ×7H 2 O—0.005; peptone—2.0; yeast extract—2.0; glycerol—10.0; veratryl alcohol—0.3, pH 5.0.
 
                                                        Laccase, U 1 −1         pH                                 Cultivation days                                                         4   6   8   11   4   6   8   11                 P. chrysosporium  1309   0   0   0   0   5.0   6.4   6.5   6.5         L. betulina  141   0   0   0   0   6.0   6.5   7.1   7.0         T. versicolor  159   2352   890   613   0   6.2   7.3   6.7   6.4         T. cervina  33   0   0   0   0   6.2   5.8   5.6   6.7         T. ochraceae  1009   121   252   231   111   6.0   5.9   6.1   6.0         T. pubescens  11   0   8   3   0   5.9   5.0   5.7   6.0         S. hirsutum  42   3   0   0   0   5.0   5.0   5.0   5.0         T. zonatus  540   4200   3276   2394   2100   5.8   6.2   6.8   6.6         T. hirsuta  119   256   143   336   806   4.8   4.3   6.0   5.5         P. radiata  312   17   8   10   15   6.1   5.8   5.8   5.3                                     MnP, U 1 −1  (610 nm)   MnP, U 1 −1  (270 nm)                         Cultivation days                                                     4   6   8   11   4   6   8   11                 P. chrysosporium  1309   0   0   0   0   0   103   43   0         L. betulina  141   0   0   0   0   0   0   0   9         T. versicolor  159   77   890   16   0   99   60   21   0         T. cervina  33   59   0   559   254   21   125   168   236         T. ochraceae  1009   48   252   205   87   176   280   267   232         T. pubescens  11   0   8   0   0   0   0   0   0         S. hirsutum  42   0   0   0   0   0   9   9   13         T. zonatus  540   86   3276   144   100   146   112   99   86         T. hirsuta  119   0   143   0   0   13   9   9   17         P. radiata  312   0   8   0   0   0   9   0   0                                             LiP, U 1 −1                     Cultivation days                                             4   6   8   11                         P. chrysosporium  1309   2   6   3   3             L. betulina  141   1   2   2   4             T. versicolor  159   9   4   2   2             T. cervina  33   1   1   3   6             T. ochraceae  1009   4   6   19   19             T. pubescens  11   2   1   8   2             S. hirsutum  42   0   0   1   1             T. zonatus  540   2   7   14   25             T. hirsuta  119   6   5   1   3             P. radiata  312   9   13   5   8                        
Composition of medium, (g/l): KH 2 PO 4 —1.0; MgSO 4 —0.5; CaCl 2 —0.1; FeSO 4 ×7H 2 O—0.005; peptone—1.0; yeast extract—2.0; veratryl alcohol—0.3; MP—40.0. pH 5.0.
 
                                                        Laccase, U 1 −1         pH                                 Cultivation days                                                                 4   6   8   11   4   6   8   11                         P. chrysosporium  1309   0   0   0   0   4.2   5.8   6.8   7.4             L. betulina  141   0   0   0   0   5.8   6.0   6.4   6.5             T. versicolor  159   106   143   235   0   5.1   6.2   6.1   6.0             T. cervina  33   0   0   0   0   5.2   5.4   6.2   6.3             T. ochraceae  1009   5544   5432   1596   722   3.2   5.0   5.2   5.6             T. pubescens  11   0   0   0   0   5.3   5.0   5.4   4.7             S. hirsutum  42   168   223   164   69   4.0   4.9   4.7   5.0             T. zonatus  540   8400   7896   5796   2520   3.8   5.2   5.2   5.7             T. hirsuta  119   1276   353   67   22   4.0   4.6   4.3   4.0             P. radiata  312   0   0   0   0   5.0   5.1   5.6   4.2                                         MnP, U 1 −1  (610 nm)   MnP, U 1 −1  (270 nm)                         Cultivation days                                                     4   6   8   11   4   6   8   11                 P. chrysosporium  1309   0   0   0   0   0   0   0   0         L. betulina  141   0   0   0   0   17   0   0   13         T. versicolor  159   0   0   0   0   9   9   0   9         T. cervina  33   46   966   846   742   0   615   512   396         T. ochraceae  1009   100   164   171   104   374   318   310   387         T. pubescens  11   0   0   0   0   0   0   0   0         S. hirsutum  42   0   0   0   0   17   17   0   0         T. zonatus  540   129   103   97   99   129   215   159   172         T. hirsuta  119   0   0   0   0   30   9   26   0         P. radiata  312   0   0   0   0   0   0   0   4                                             LiP, U 1 −1                     Cultivation days                                             4   6   8   11                         P. chrysosporium  1309   192   0   7   0             L. betulina  141   0   0   0   11             T. versicolor  159   0   15   37   15             T. cervina  33   0   0   0   0             T. ochraceae  1009   24   8   14   76             T. pubescens  11   0   0   0   3             S. hirsutum  42   0   2   1   38             T. zonatus  540   27   16   20   55             T. hirsuta  119   21   18   14   22             P. radiata  312   0   0   0   11                        
Exp. 2 . Cerrena unicolor  300 , Cerrena unicolor  301 , Cerrena unicolor  302 , Cerrena unicolor  303 , Cerrena unicolor  305 , Coriolopsis gallica  142 , Merulius tremelosus  206 , Pellinus tuberculosus  121 , Pellinus tuberculosus  131 , Cyatus striatus  978.
 
Composition of synthetic medium, (g/l): KH 2 PO 4 —1.0; MgSO 4 —0.5; CaCl 2 —0.1; FeSO 4 ×7H 2 O—0.005; peptone—2.0; yeast extract—2.0; glycerol—10.0; veratrylalcohol—0.3, pH 5.0.
 
                                                        Laccase, U 1 −1         pH                                 Cultivation days                                                                 5   7   9   12   5   7   9   12                         C. unicolor  300   336   468   286   798   6.0   6.1   6.2   6.1             C. unicolor  301   77   134   69   185   5.0   5.2   5.3   5.8             C. unicolor  302   172   840   1260   7644   5.8   5.8   6.0   6.0             C. unicolor  303   76   151   133   407   5.3   5.8   5.4   5.8             C. unicolor  305   1025   420   176   210   5.5   5.5   5.5   5.7             C. gallica  142   105   332   470   504   5.3   5.7   5.6   5.7             M. tremelosus  206   605   504   181   66   4.9   4.3   4.5   4.5             P. tuberculosus  121   4   20   0   17   5.8   6.2   6.1   6.1             P. tuberculosus  131   0   0   2   8   6.0   6.2   6.0   6.0             C. striatus  978   4   0   0   20   6.1   6.1   6.1   5.8                                         MnP, U 1 −1  (610 nm)   MnP, U 1 −1  (270 nm)                         Cultivation days                                                     5   7   9   12   5   7   9   12                 C. unicolor  300   437   79   67   42   645   17   0   0         C. unicolor  301   156   221   552   81   206   482   507   155         C. unicolor  302   34   40   41   70   52   43   26   0         C. unicolor  303   394   734   874   55   507   1015   576   95         C. unicolor  305   206   225   101   49   284   507   215   146         C. gallica  142   29   59   22   3   26   17   34   30         M. tremelosus  206   4   0   0   0   0   0   9   0         P. tuberculosus  121   22   56   152   160   0   0   0   0         P. tuberculosus  131   119   201   308   128   0   0   0   0         C. striatus  978   44   5   0   0   0   0   0   0                                             LiP, U 1 −1                     Cultivation days                                             5   7   9   12                         C. unicolor  300   1   2   3   0             C. unicolor  301   0   2   2   15             C. unicolor  302   4   3   2   0             C. unicolor  303   5   18   0   9             C. unicolor  305   1   4   1   30             C. gallica  142   9   12   12   3             M. tremelosus  206   0   0   0   0             P. tuberculosus  121   0   0   0   0             P. tuberculosus  131   0   0   0   0             C. striatus  978   0   0   4   0                        
Composition of medium, (g/l): KH 2 PO 4 —1.0; MgSO 4 —0.5; CaCl 2 —0.1; FeSO 4 ×7H 2 O—0.005; peptone—1.0; yeast extract—2.0; veratryl alcohol—0.3; glycerol—10.0; MP—20.0. pH 5.0.
 
     
       
         
           
               
               
               
               
               
             
               
                   
                   
               
             
            
               
                   
                 Laccase, U 1 −1   
                   
                 pH 
                   
               
            
           
           
               
               
               
            
               
                   
                 Cultivation days 
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
            
               
                   
                   
                 5 
                 7 
                 9 
                 12 
                 5 
                 7 
                 9 
                 12 
               
               
                   
                   
               
               
                   
                   C. unicolor  300 
                 7392 
                 6888 
                 3654 
                 9576 
                 5.5 
                 5.6 
                 5.6 
                 5.8 
               
               
                   
                   C. unicolor  301 
                 4508 
                 6552 
                 2394 
                 4620 
                 4.7 
                 4.8 
                 5.0 
                 5.0 
               
               
                   
                   C. unicolor  302 
                 4620 
                 7560 
                 5516 
                 12432 
                 4.6 
                 5.7 
                 5.8 
                 6.0 
               
               
                   
                   C. unicolor  303 
                 2520 
                 4340 
                 2520 
                 2016 
                 4.3 
                 5.0 
                 5.0 
                 5.2 
               
               
                   
                   C. unicolor  305 
                 4620 
                 5992 
                 3276 
                 5460 
                 4.9 
                 4.9 
                 5.0 
                 5.0 
               
               
                   
                   C. gallica  142 
                 3528 
                 2898 
                 5292 
                 2688 
                 4.8 
                 5.7 
                 5.2 
                 5.2 
               
               
                   
                   M. tremelosus  206 
                 2982 
                 3318 
                 3570 
                 3864 
                 4.4 
                 4.5 
                 4.5 
                 4.5 
               
               
                   
                   P. tuberculosus  121 
                 13 
                 42 
                 34 
                 121 
                 4.7 
                 5.6 
                 5.5 
                 5.7 
               
               
                   
                   P. tuberculosus  131 
                 10 
                 8 
                 7 
                 17 
                 4.4 
                 5.3 
                 5,5 
                 5.5 
               
               
                   
                   C. striatus  978 
                 500 
                 622 
                 672 
                 1596 
                 4.7 
                 4.6 
                 4.6 
                 4.6 
               
               
                   
                   
               
            
           
           
               
               
               
            
               
                   
                 MnP, U 1 −1  (610 nm) 
                 MnP, U 1 −1  (270 nm) 
               
            
           
           
               
               
            
               
                   
                 Cultivation days 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 5 
                 7 
                 9 
                 12 
                 5 
                 7 
                 9 
                 12 
               
               
                   
               
               
                   C. unicolor  300 
                 423 
                 92 
                 30 
                 14 
                 980 
                 120 
                 34 
                 0 
               
               
                   C. unicolor  301 
                 953 
                 1122 
                 760 
                 808 
                 2242 
                 2761 
                 705 
                 1152 
               
               
                   C. unicolor  302 
                 16 
                 24 
                 13 
                 24 
                 0 
                 17 
                 9 
                 0 
               
               
                   C. unicolor  303 
                 962 
                 1072 
                 313 
                 22 
                 2219 
                 1376 
                 237 
                 56 
               
               
                   C. unicolor  305 
                 843 
                 935 
                 911 
                 513 
                 1213 
                 1084 
                 714 
                 731 
               
               
                   C. gallica  142 
                 74 
                 57 
                 108 
                 111 
                 52 
                 69 
                 52 
                 60 
               
               
                   M. tremelosus  206 
                 11 
                 25 
                 58 
                 0 
                 0 
                 17 
                 34 
                 0 
               
               
                   P. tuberculosus  121 
                 701 
                 898 
                 591 
                 124 
                 0 
                 22 
                 0 
                 0 
               
               
                   P. tuberculosus  131 
                 846 
                 363 
                 400 
                 347 
                 26 
                 26 
                 86 
                 26 
               
               
                   C. striatus  978 
                 0 
                 0 
                 4 
                 0 
                 17 
                 0 
                 38 
                 0 
               
               
                   
               
            
           
           
               
               
               
               
            
               
                   
                   
                 LiP, U 1 −1   
                   
               
               
                   
                   
                 Cultivation days 
               
            
           
           
               
               
               
               
               
               
            
               
                   
                   
                 5 
                 7 
                 9 
                 12 
               
               
                   
                   
               
               
                   
                   C. unicolor  300 
                 47 
                 98 
                 196 
                 0 
               
               
                   
                   C. unicolor  301 
                 1 
                 37 
                 67 
                 110 
               
               
                   
                   C. unicolor  302 
                 6 
                 74 
                 103 
                 0 
               
               
                   
                   C. unicolor  303 
                 24 
                 35 
                 59 
                 5 
               
               
                   
                   C. unicolor  305 
                 47 
                 73 
                 25 
                 70 
               
               
                   
                   C. gallica  142 
                 3 
                 121 
                 107 
                 6 
               
               
                   
                   M. tremelosus  206 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   P. tuberculosus  121 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   P. tuberculosus  131 
                 47 
                 0 
                 0 
                 2 
               
               
                   
                   C. striatus  978 
                 7 
                 0 
                 0 
                 0 
               
               
                   
                   
               
            
           
         
       
     
     2. Alginate Encapsulation of Enzyme Cocktail 
     Mycorernedation via Encapsulation and Controlled Release of Ligninolytic Enzymes from Alginate microparticles 
     The goal of this work is to develop the use of alginate encapsulation approaches for ligninolytic enzymes for the stabilization and controlled release in soils contaminated with target hydrocarbons. The ideal result will be the identification of the materials and methods yielding alginate microparticles meeting the following: 
     Small enough size that they can be dispersed in aqueous medium and sprayed onto soil 
     High active until loading (Units/mass of dispersion) 
     Long term stability 
     Demonstrated ability to degrade hydrocarbons in contaminated soil. 
     Task 1—Investigate the Effect of 1VIn2+ on Enzyme Activity in Alginate Beads 
     We will test the effect of the inclusion of Mn2+ on the encapsulation of three ligninolytic enzymes: lignin peroxidase (LiP), manganese-dependent peroxidase (MnP) and laccase. We will evaluate the capsules&#39; size and enzyme loading. A promising formulation will be selected for investigation into methods to reduce the size. 
     Task 2—Investigate Methods of Reducing the Alginate Bead Size 
     Methods to be investigated are microemulsion and extrusion techniques. These methods will be evaluated based on particle size, dispersability, and potential sprayability. In addition, we will evaluate the enzyme loading in each microcapsule (units/mass capsule), The most promising method(s) will be chosen for testing long term stability and efficacy in contaminated soil. 
     Task 3—Stability and Efficacy 
     One or more promising methods will be chosen for final stability and efficacy tests. These will be tested against unencapsulated control enzymes. Results will be gauged on both the ability of the capsule to improve enzyme stability as well as ability to degrade the target hydrocarbons in contaminated soil. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Experimental Test Matrix for Tasks 1 and 2. Alginate concentration will be constant 
               
               
                 at 20 mg/mL (2% w/v) based on prior results and published data. Enzyme loading 
               
               
                 will be chosen based on desired active units per mass of alginate. 
               
            
           
           
               
               
            
               
                   
                 Processing Variables 
               
            
           
           
               
               
               
               
               
            
               
                   
                 Compositional Variables Cross 
                 Method 
                 For 
                   
               
               
                   
                 Link Density/Amendment 
                 Extrusion, 
                 Excursion 
                 For Emulsion 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                   
                 Run 
                 C BaCl2   
                 C CaCl2   
                 C MnCl2   
                 Spray, 
                 Flow Rate 
                 Stir Rate 
                 C tween80   
               
               
                 Task 
                 (#) 
                 (mM) 
                 (mM) 
                 (mg/mL) 
                 Emulsion 
                 (mL/hr) 
                 (RPM) 
                 (mg/mL) 
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 1 
                 1 
                 0 
                 10 
                 100 
                 Extrusion 
                 10 
                   
                   
               
               
                   
                 2 
                 1 
                 0 
                 100 
                 Extrusion 
                 10 
               
               
                 2 
                 3 
                 TBD 
                 TBD 
                 100 
                 Extrusion 
                   
                 200 
                 2 
               
               
                   
                 4 
                 TBD 
                 TBD 
                 100 
                 Extrusion 
                   
                 400 
                 2 
               
               
                   
                 41 
                 TBD 
                 TBD 
                 100 
                 Extrusion 
                   
                 200 
                 4 
               
               
                   
               
            
           
         
       
     
     Encapsulation Experiments Continued 
     Background/Executive Summary 
     In the first round of alginate experiments, we saw that the conditions were not able to yield discrete alginate particles. The beads did not solidify and most of the collection bath became brown indicating that enzyme was not efficiently encapsulated. We hypothesized that the concentration of the crosslinking divalent ions were too low, and that this was resulting a weak encapsulating hydrogel matrix. To test this, we amended our test matrix to test three combinations of crosslinking divalent ions, each with higher concentrations of CaCl2 and BaCl2. The concentration of MnCl2 was kept constant at 100 mM since this is already high, and because the Mn2+ ion place a role in the enzyme activity in addition to crosslinking the alginate. The result confirmed our hypothesis and increasing the CaCl2 and BaCl2 concentrations yielded much more robust, and spherical beads. However, the collection bath still showed some brown color. We will run enzyme activity tests to quantify the units per bead. This will be done by dissolving a bead in 55 mM sodium citrate and running an assay on the solution. We will also run assays on the collection bath solutions. 
     Approach 
     Materials 
     A stock solution of Alginate in DI water was prepared at 40 mg/mL and dissolved by heating in an autoclave. Other stock solutions were prepared accordingly. ABTS (10 mg/mL), CaCl2 (200 mM), MnCl2 (200 mM) and BaCl2 (10 mM) in DI water. Enzyme (MnP from C, unicolor 300) was used as received. This was a vicious dark brown liquid with the following estimated enzyme concentrations: laccase (437 U/mL), MnPA270 (265 U/mL), yielding a total enzyme concentration of 840 U/mL. 
     Procedure 
     
         
         
           
             1. To a 20 mL glass scintillation vial, add:
           a. 3.3 mL Alginate Stock (40 mg/mL; via 10 mL B-D syringe and 18 gauge hypodermic   b. 2.9 mL enzyme (MnP from C unicolor 300; via 10 mL B-D syringe and 18 gauge hypodermic   c. 0.132 mL of ABTS (10 mg/mL in DI water; via volumetric pipette)   d. 0.289 mL of DI water (via volumetric pipette)   
         
             2. This resulted in pre-alginate solution with the following concentrations:
           a. 365 U/mL total enzyme (composed of the following enzymes)   b. 190 U/mL Laccase   c. 115 U/mL MnPA610   d. 60 U/mL MnPA610   e. 0.2 mg/mL ABTS   f. 20 mg/mL Alginate   
         
             3. This solution was dispensed into collection baths with various concentrations of crosslinking ions (shown in Table 1 below), including MnCl2, CaCL2. For each run, 1 mL of pre-alginate solution was dispensed (at 10 mL/hr) through a 22 gauge stainless steel, blunt tipped needle into 50 mL of crosslinking solution in the collection bath. As the droplets hit the solution, they immediately solidified and sank to the bottom of the dish. The dish was rotated by hand to avoid accumulation of beads in one place in the dish. Note, that throughout the dispensing step, the collection bath gradually adopted a light brown color, indicating that some of the enzyme was diffusing from the beads into the collection bath. 
             4. After 1 mL was dispensed, the dish was left to sit for at least 30 minutes to allow the crosslinking to complete. Then the liquid was pipetted off the stored as the decantate. The dry beads were imaged using a camera (images were later analyzed for particle diameter using ImageJ software). These were then resuspended in 1 mL of DI water and stored in the refrigerator. 
           
         
       
    
     Results 
     The two of the runs (samples 3 and 4) yielded discrete beads that were able to be measured using ImageJ. If we assume that all of the enzyme in the pre-alginate solution was encapsulated in the beads (i.e. 100% encapsulation efficiency), and we estimate the bead volume from the measured diameters, we can estimate the enzyme concentration per bead (U/bead). This is shown in the Table below. Images and particle size distributions are shown in Figure below. 
     
       
         
           
               
             
               
                 TABLE 1 
               
             
            
               
                   
               
               
                 Bead size and Estimated Enzyme loadings for successful runs (3 and 4). 
               
            
           
           
               
               
               
               
               
               
               
            
               
                   
                   
                   
                   
                   
                 Bead 
                   
               
               
                 Date 
                 Run 
                 C BaCl2   
                 C CaCl2   
                 C MnCl2   
                 Diameter 
                 Enzyme Loading (U/bead) 
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 (y/m/d) 
                 (#) 
                 (mM) 
                 (mM) 
                 (mg/mL) 
                 (mm) 
                 Laccase 
                 MnP 270   
                 MnP 610   
               
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
            
               
                 2015 Mar. 18 
                 1 
                 10 
                 100 
                 0 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 2015 Mar. 18 
                 2 
                 0 
                 100 
                 1 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                 2015 Mar. 23 
                 3 
                 50 
                 100 
                 2.5 
                 2.8 ± 0.1 
                 2.18 
                 1.32 
                 0.690 
               
               
                 2015 Mar. 23 
                 4 
                 100 
                 100 
                 0 
                 3.0 ± 0.1 
                 2.69 
                 1.63 
                 0.848 
               
               
                 2015 Mar. 23 
                 5 
                 0 
                 100 
                 5 
                 NA 
                 NA 
                 NA 
                 NA 
               
               
                   
               
            
           
         
       
     
     It looks like sample 3 had better encapsulation efficiency than 4. Although it could be improved. Most of the enzyme is being lost in the bath. There are three things we could try to improve the encapsulation efficiency.
         (1) Test higher concentrations of crosslinkers (CaCl2, and BaCl2)   (2) Store the beads dry and then disperse them in water when we&#39;re ready to test   (3) Reduce the amount of time that the beads are sitting in the bath before collection       

     Note: Encapsulation efficiency is based on enzyme activity assays. Thus, it is possible that there is some de-activation enzyme encapsulated that was not detected by the assay. This would mean that the actual encapsulation efficiency of the enzyme was higher by some unknown amount, and that this was effect by enzyme de-activation in the process.