Abstract:
The present invention is directed to methods of modifying the plant development process comprising of exposing a plant or plant part to volatiles biosynthesized by one or more bacteria or enzymes. Specifically, the embodiment uses one or more bacteria selected from the plant growth promoting bacteria group consisting of  Rhodococcus  spp.,  Pseudomonas  spp., or  Xanthobacter  spp., or a mixture thereof. A closed apparatus, FIG.  1 A, containing a tri-phasic system is used to expose the bacteria to hydrocarbons, iron, cyanide, and/or ammonium compounds; the method induces the biocatalyst to biosynthesize volatile compound(s) that deter ethylene production in climacteric plants or fruit resulting in the biocatalyst ability to delay fruit ripening.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    Not Applicable 
       FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
       [0002]    Not Applicable 
       SEQUENCE LISTING OR A COMPUTER PROGRAM 
       [0003]    Not Applicable 
       FIELD OF INVENTION 
       [0004]    The present invention is in the field of post-harvest biotechnology. The invention generally relates to the method of exposing plant or plant parts to one or more bacteria that release volatile compounds, specifically to the bacteria ability to deter ethylene production and delay fruit ripening in climacteric plants or fruit. 
         [0005]    The projected rise in global population raises concerns about the effectiveness of current agricultural techniques to meet the continuously growing demands for food, (Trostle and Seeley, 2013). Plant growth promoting bacteria (PGPB) may be a low cost and efficient tool to increase agricultural productivity and deter post-harvest loss. PGPB have also been used as biological fertilizers, biological pesticides, and biocatalyst to prevent post-harvest loss, (Ahemad and Kibret, 2014; Bashan 1998; Pierce et al., 2011; Perry, 2011). Several species of PGPB including  Azobacter, Bacillus, Azosprillum, Acetobacter, Pseudomonas , and  Rhodococcus  species these bacteria have been used for centuries as bacterial inoculants to modify plant development and improve nutrient uptake for plant cells, (Bashan, 1998; Vacheron et al., 2013). Ethylene production regulates plant development in climacteric fruit. Controlling ethylene concentrations can deter the plant development process and retard fruit ripening, (Binder et al., 2004; Burg, 1973; Trobacher, 2009; Victor et al., 2012; Yip and Yang, 1988; Yang and Oetiker, 1998). PGPB modify plant development by degrading ethylene, degrading ethylene precursors, and/or producing compounds that inhibit auxin production in plant cells, (Allen and Ensign, 1998; Arshad et al., 2007; DeBont and Albers, 1976; Dhungana et al., 2007; Elsgaard, 1998; Glick, 2012; Kloepper et al., 1991; Penrose and Glick, 2003). Plant growth promoting bacteria can also use dehydratase and nitrile degrading enzymes to biosynthesize cyanohydrins or auxins that manipulate the plant development process, (Egamberdieva, 2012; Hayat et al., 2010; Nomura et al., 2012; Kato et al., 2004). 
         [0006]    A previous method to induce bacteria to delay fruit ripening was developed by Pierce et al., 2011 and U.S. patent 2013/0274, 102 (2013). Bacteria were induced to delay fruit ripening after prolonged exposure to cobalt, urea, and/or asparagine. The cell paste was immobilized on cellulose material and placed on or near fruit to delay climacteric ripening. This method was arduous and laborious; the induction process lasted for a 13-14 day period. This method would be very costly to translate into actual manufacturing and industrial applications. The multiple culture steps, expensive equipment, and expensive materials, including urea and glucose, would drive-up production cost and reduce profits. 
         [0007]    The new method I developed exposes PGPB to hydrocarbons, iron, and ammonium compounds to enhance the bacteria ability to delay fruit ripening. My method requires fewer media transfers and samples are ready in half the time. The new method translates into reduced production and labor cost and increased reproducibility of results, improving the inventions relevance for industrial and manufacturing applications. Biocatalyst exposed to inducers were able to modify the plant development process and delay the effects of fruit ripening, including changes in hue, texture, firmness, reduced presence of brown spotting, and reduced fungal infection. 
       BRIEF SUMMARY OF INVENTION 
       [0008]    In accordance with one embodiment, the new method is directed to the use of short chain hydrocarbons, including but not limited to propylene (propene) and/or ethylene to induce PGPB. The additional necessary media components include heavy metals, including but not limited to iron, and ammonium compounds to induce biocatalyst ability to modify plant development in climacteric fruit. 
         [0009]    The PGPB are referred to as a biocatalyst comprising of one or more bacteria, particularly  Rhodococcus  or  Norcardia  spp., or a mixture thereof. The biocatalyst, comprising of PGPB include but is not limited to  Acetobacter, Azobacter, Azosprillum, Bacillus, Brevibacterium, Norcardia, Pseudomonas, Rhodococcus , or  Xanthobacter  spp. that can catalyze the hydrocarbon into volatile compounds that deter ethylene production in climacteric plants or fruit. 
         [0010]    The present new invention method occurs wherein the biocatalyst are cultivated in a closed tri-phasic system apparatus described herein. This invention further provides an apparatus used to facilitate the induction of PGPB to produce volatile compounds required to modify the plant development process and delay ripening in climacteric plants or fruit. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0011]    The invention was described above in broad terms, reference will now address the accompanying drawing, this drawing is not necessarily drawn to scale, and wherein: 
           [0012]      FIG. 1A  is a perspective front view of the apparatus of the past invention; the apparatus contains a tri-phasic system comprised of a solid, liquid, and gaseous phase labeled  9 ,  23 , and  24  respectively. 
           [0013]      FIG. 1B  is a detailed operational view of the proportion indicated by the section lines  2 - 3  in  FIG. 1 , a closed system apparatus of the present invention; the apparatus contains a tri-phasic system. The biocatalyst is maintained in the solid and liquid phase of the system as defined herein below, and comprises one or more of the microorganism of the invention. 
       
    
    
     DETAILED DESCRIPTION 
     FIGS.  1 A and  1 B—First Embodiment 
       [0014]    The inventor has discovered a method to induce plant growth promoting bacteria to perform as a biocatalyst that are capable of converting short chained hydrocarbons into volatile compounds that manipulate the plant development process and delay fruit ripening. This detailed description should not be considered a means of limiting this invention to a particular embodiment. The description contains the word “comprising”, or grammatical variations of the word, it is understood to imply inclusion rather than limitations. The one or more bacteria used in the methods and apparatuses of the invention may at times be more generally referred to herein as the “biocatalyst.” The hydrocarbons are aliphatic gaseous compounds, including but not limited to propylene (propene) and/or ethylene compound or mixture thereof. The volatile compounds produced from the reaction, include but are not limited to nitriles and/or cyanohydrin mixture or combination thereof. The bacteria also uptake additional compounds released by plant cells during the induction process, including cyanide. The induction process also induces and stabilizes several enzymes found in  Rhodococcus  or  Norcardia , including but not limited to nitrile degrading enzymes and/or monooxygenase or a combination thereof within the bacteria. The present invention is described in full detail herein after; references are made to embodiments included in this application. 
         [0015]    Elements to monitor the efficiency of the biocatalyst can be attached to the apparatus to monitor carbon dioxide levels or pH levels in the media. Conversion of hydrocarbons to volatile compounds in a biocatalyst may comprise of additional features to permit continued circulation of air flow within the closed system. An individual skilled in the art could envision modifications for the apparatus to improve monitoring and controlling of the atmospheric conditions for the biocatalyst. 
         [0016]    One embodiment of the closure is illustrated in  FIG. 1A  (front view) and  FIG. 1B  (detailed operational view). In particular embodiments of the invention the biocatalyst are cultivated in a closed flask container, providing a closed system. For example, as shown in  FIG. 1A , the apparatus contains a tri-phasic system comprised of a solid, liquid, and gaseous phase. The solid phase ( 9 ) is comprised of 12 g of Bacto agar suspended into 300 ml of dH 2 O, autoclaved and cooled in a 2 L Erlenmeyer flask. However the solid phase can consist of any of a gelatinous structure, bead-like structure, and/or a matrix like structure that can support microbial cells and facilitate filamentous growth. The liquid phase ( 23 ) contains 300 ml of the induction media that consist of a heavy metal, ammonium chloride, and phosphate compounds. The gaseous headspace ( 24 ) was filled with 10-15% of hydrocarbon gas for 3-5 days at 30° C. and 120 rpm. The gaseous compounds can include ethylene and/or propylene. 
       Operation 
     First Embodiment FIG.  1 B 
       [0017]    A detailed operational view of the apparatus used for the cultivation of biocatalyst, shown in  FIG. 1B . The apparatus is 2 L Erlenmeyer flask ( 8 ), apparatus could consist of any closed system container that could support a tri-phasic system. The rubber stopper ( 12 ) prevented loss of gaseous media components. The agar base ( 9 ) provides a solid surface for biofilm formation; the solid phase is an essential component to enhancing bacteria ability to modify plant development. The liquid phase ( 23 ) contains induction media necessary to induce bacteria ability to modify plant development. The gaseous headspace ( 24 ) contains hydrocarbon gas, an essential inducer to enhance bacteria ability to modify plant development. 
       Headspace Collection—FIG.  1 B 
       [0018]    Gaseous components are added and removed from the headspace using a two way valve ( 1 ). The open/close flow and direction of the valve is controlled by knob ( 2 ). The entry port ( 6 ) is used to attach syringes to inject gas into the system, media or gas enters the connecting tubing through the exit port ( 3 ). The 3/16 inch rubber tubing ( 4 ), is used throughout the system, the tubing is flexible and autoclavable. Cells pass quickly through the rubber tubing with little resistance or backflow. The rubber tube is connected to ⅛ silicon tube ( 5 ), silicon is rigid and necessary for constructing entry and exit point through the holes ( 21 ) in the rubber stopper. Gas enters the headspace ( 7 ). 
       Sample Collection—FIG.  1 B 
       [0019]    Gaseous and liquid components are added and removed through a two way valve ( 18 ). The open/close flow and direction of the valve is controlled by knob ( 19 ). The entry port ( 17 ) is used to attach syringes to inject liquid media into the system, enters the connecting tubing through the exit port ( 20 ). The 3/16 inch rubber tubing ( 15 ) is used throughout the system. The rubber passes into a 50 ml falcon tube ( 13 ) and is loosely connected to ⅛ silicon tube ( 14 ). The falcon tube ( 13 ) acts as a reservoir for over flow during cultivation, exposed openings are sealed with silicon based epoxy. The ⅛ silicon tube ( 14 ) are inserted into the stopper hole ( 22 ). A portion of the silicon tubing enters the flask ( 11 ) and connects to 3/16 inch rubber tubing ( 10 ). The rubber tubing is perforated on the ends and slightly coiled into the medium to allow for direct bubbling of gaseous components into the medium. 
         [0020]    The present invention is generated in a closed system apparatus, comprising of a tri-phasic media condition. The tri-phasic condition consist of a solid porous base, aqueous phase, and a gaseous phase composed of a hydrocarbon and air mixture. The mechanism used for induction of the biocatalyst is not intended to be limiting by a particular enzyme, but may increase activity or expression of one or more enzymes, comprising of dehydratase, nitrile degrading enzyme, and/or monooxygenase, or a mixture thereof. The induction of one or more of these enzymes may play a role conversion of a hydrocarbon to a volatile compound by the biocatalyst. This present invention encompasses biocatalysts that produce, or are induced to produce, or are genetically modified to produce dehydratase, nitrile degrading, and/or monooxygenase enzyme, at a quantity or at an enzymatic activity level sufficient for the conversion of short chained hydrocarbons to volatile compounds that deter ethylene production in climacteric plants or fruit. These enzymes have been studied in depth in literature based publications, possessing recognized enzymatic activities. The abundance of reference material related to the enzymes assures that such enzymes are well known to individuals skilled in the art, and the enzymes discussed in this invention can be easily produced, engineered, or purified from the biocatalyst. 
         [0021]    The following embodiments are offered as examples, and are felt to be non-limiting and are meant to illustrate the invention but are not meant to be limiting in any way. 
       Examples 
       [0022]      Rhodococcus  cells were suspended in the induction media and exposed to the hydrocarbons for 3-5 days at 30° C. and 120 rpm. Biocatalyst exposed to the induction media were able to modify plant development process, and delaying ripening in climacteric plants and fruit. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Induction Media 
               
               
                   
               
             
             
               
                 M9 Stock Media (autoclaved) 
               
             
          
           
               
                   
                 Na 2 HPO 4 —7H 2 O 
                 64 
                 g/L 
               
               
                   
                 KH 2 PO 4   
                 15 
                 g/L 
               
               
                   
                 NaCl 
                 2.5 
                 g/L 
               
             
          
           
               
                   
                 NH 4 Cl 
                 5.0 g/L-6.0 g/L 
               
             
          
           
               
                   
                 dH 2 O 
                 1 
                 L 
               
             
          
           
               
                 Working Solution M9 Media (Sterile) 
               
             
          
           
               
                   
                 M9 media 
                 200 
                 ml 
               
               
                   
                 1M MgSO 4  (sterile) 
                 2 
                 ml 
               
               
                   
                 1M CaCl 2  (sterile) 
                 100 
                 μl 
               
             
          
           
               
                   
                 Propylene and/or 
                      (10-15%) 
               
               
                   
                 Ethylene (v/v) 
               
             
          
           
               
                   
                 Cobalt or Iron 
                 0.201 
                 g/L 
               
               
                   
                   
               
               
                   
                 Table 1: Media contained ammonium chloride and heavy metals induced bacteria to delay fruit ripening. 
               
             
          
         
       
     
         [0023]    Fruit were collected from experiments, a Brix measurement were determined stages to validate that fruit stored with biocatalyst ripening process was delayed compared to control samples. ° Brix (U) measures physiological changes, such as changes in soluble carbohydrate concentrations. Brix (U) increase and continue to increase throughout the ripening process. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Brix Measurements 
               
             
          
           
               
                   
                   
                 Cobalt 
                 Cobalt, Urea, 
               
               
                   
                 Control 
                 &amp; Urea 
                 &amp; Propylene 
               
               
                   
                   
               
             
          
           
               
                 Brix° Unit 
                 15 ± 0.50 
                 13 ± 1.5 
                 10 ± 1.2 
               
               
                 (% Sugar Content) 
               
               
                   
               
               
                 Table 2: Brix° (U) measurement determines % of soluble sugars from apple samples. Apples were incubated with live biocatalyst for 12 days at 21° C. Control initial measurement ~9° (U) Brix. 
               
             
          
         
       
     
         [0024]    Exposure to hydrocarbons induced nitrile degrading activity in  Rhodococcus  cells. Increased nitrile degrading activity suggests bacteria cells converted hydrocarbons into volatile compounds. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Nitrile Degrading Activity 
               
             
          
           
               
                   
                   
                   
                 Post 
                 Post 
               
               
                 Carbon 
                   
                   
                 Induction 
                 Fruit Exposure 
               
               
                 Source 
                 Inducers 
                 Substrate 
                 Δ(U) 
                 Δ(U) 
               
               
                   
               
               
                 Glucose 
                 N.A. 
                 Acrylonitrile (1 g/L)   
                     2 ± 4.3 
                     4 ± 0.7 
               
               
                   
                   
                 *Cyanide (0.05 g/L)   
                 N.D. 
                 2.0 ± 0.6 
               
               
                 Glucose 
                 Cobalt, 
                 Acrylonitrile (1 g/L)   
                 41 ± 11 
                  55 ± 1.0 
               
               
                   
                 Urea 
                 *Cyanide (0.05 g/L)   
                     1 ± 0.0 
                     3 ± 0.3 
               
               
                 Propylene 
                 Propylene 
                 Acrylonitrile (1 g/L)   
                  27 ± 2.0 
                     48 ± 10.5 
               
               
                   
                   
                 *Cyanide (0.05 g/L)   
                 1.0 ± 0.0 
                 3.0 ± 0.3 
               
               
                 Propylene 
                 Propylene, 
                 Acrylonitrile (1 g/L)   
                 75 ± 23 
                 96 ± 17 
               
               
                   
                 Cobalt, 
                 *Cyanide (0.05 g/L)   
                 5.30 ± 3.0  
                 4.0 ± 1.2 
               
               
                   
                 Urea 
               
               
                   
               
               
                 Table 3:  Rhodococcus rhodochrous  DAP 96253 cultured in tri-phasic conditions. Nitrile degrading assay measures (U) units of activity. Units 1 uM of acrylonitrile converted to 1 uM acrylamide in 1 min, pH 7.2 at 30° C. 
               
               
                 *Units 1 uM of KCN converted to 1 uM formic acid in 1 min, pH 7.2 at 30° C. 
               
               
                 N.D. = Not Detected; 
               
               
                 N.A. = No Addition 
               
             
          
         
       
     
       CONCLUSION, RAMIFICATIONS, AND SCOPE 
       [0025]    Accordingly the reader will see that, according to one embodiment of the invention, I have developed a low cost method to expose PGPB to hydrocarbons and heavy metal compounds to induce bacteria to modify the plant development process and delay fruit ripening in climacteric fruit and plants. While the above description contains many specifications, these should not be construed as limitations on the scope of any embodiment, but as exemplifications of the presently preferred embodiments thereof. Many other ramifications and variations are possible within the teachings of various embodiments. For example, the heavy metals used in this experiment can include iron or cobalt. Both metals can induce enzymatic activity to produce volatile compounds required to delay fruit ripening, but enzyme activity varies in the presence of iron verses the presence of cobalt. 
         [0026]    Thus the scope of the invention should be determined by the appended claims and their legal equivalents, and not by the given examples.