Project Documentation

U.S. PATENT EXCERPTS

Abstract
Nematodes
PCE (Perchloroethylene)
TCE (Trichloroethylene)
Hydrocarbon Degraders

The following document contains excerpts from U.S. Patent No. 5,266,096. 
The bacterial mixture referred to as "Mystic Microbe" is the basic "mother" culture that has
 evolved a variety of strains, all of which are termed AgriCultures, under ACI Bioremediation.

This document includes the ATCC lab analysis results regarding the 
cultures' ability to degrade hydrocarbons and reduce substances 
such as PCE (Perchloroethylene)  and TCE (Trichloroethylene).

United States Patent
Patent Number: 5,266,096
Date of Patent: Nov. 30, 1993

Abstract Excerpts

"A microbial hydrocarbon degrader, soil amendment and growth-promoting composition comprising a mixture of bacteria,.......... which is capable of decomposing cellulose, fixing nitrogen, fighting plant pests and disease, and liberating phosphorous into the soil upon application and mixture to soil. The heterotrophic microbial composition further is capable of degrading hydrocarbons in contaminant events."

Excerpts from Background and Summary of the Invention

"The three basic types of bacteria, present in the [mixotrophic mixture], are very important in liberating minerals and nutrients already in the soil. Application of the [mixatrophic mixture] assists in achieving and maintaining balanced soils conditions to supply plants with nutrients and fend off disease and insects. Additionally, the [mixatrophic mixture] may be employed in a soil remediation mode as a precursor to planting or utilization of the soil for an ecosystem base. 

It is a principle object of the present invention to provide a microbial composition which acts as a multifunctional soil amendment to decompose plant matter into humus, act as a nitrogen fixer and produce biologically active substances which combat diseases or insect infestations.

It is another object of the present invention to provide a microbial composition which is capable of acting both as a soil amendment and as a hydrocarbon degrading composition for use with oil spills, oil contamination, or other hydrocarbon contamination events.

These properties are accomplished by the present invention..."

Nematodes
     "Scripto Bacteria [one of the types of bacteria in AgriCultures] fight diseases and use antibiotics which combat fungal diseases, nematodes, root rot and infestation."

Example: Photo showing  nematode effects on potatoes 

LEFT:  healthy tuber (potato) 

RIGHT: bumpy tuber (potato) caused by root-knot nematodes

PCE and TCE Reductions:

•      Perchloroethylene was provided as Mallinckrodt No. 1933 trichloroethylene
•       Sample preparation: Three 2 liter samples were prepared as follows:

      A.  From a previous trial it was determined that 450 ml of the [mixotrophic mixture] added to 1,550 ml of distilled water would produce an endogenous respiration rate of approximately 20 ml of oxygen per hour. 450 ml of the [mixotrophic mixture], 100 ml of Bushnell-Haas medium and 1,450 ml of distilled water were added to a first sample chamber.

      B.  Also from a previous trial it was determined that 90 ml of Solmar CH-118 pre-soaked supernatant in 1,910 ml of distilled water would produce an endogenous respiration rate of approximately 20 ml of oxygen per hour. 90 ml of the CH-118 supernatant, 100 ml of the Bushnell-Haas medium, and 1,810 ml of distilled water were added to a second sample chamber.

      C.  Samples in the Tech-line respirometer were aerated vigorously. Aeration may have caused the PCE to volatilize into the headspace gas causing a decrease in PCE concentration which is not attributable to metabolism. To compensate for this, a third sample chamber was a volatilization control. This third sample chamber was filled with 100 ml of Bushnell-Haas medium and 1,900 ml of distilled water.

      D.  After all three samples had reached endogenous respiration levels, the samples were moved from the sample chambers and placed in beakers. 4 ml of perchloroethylene were added to each sample. The samples were mixed for 20 minutes after which the excess perchloroethylene was allowed to settle of the bottom of each beaker. Each sample was siphoned from the top and placed back into the respirometers.

      E.  Three samples were continuously monitored for respirometric activity for 14 days. Respiration rates were determined on days 0, 3, 7, and 14 by withdrawing two, 40 ml aliquots from each sample for gas chromatography analysis in accordance with Environmental Protection Agency Procedure 8021. On day 10, 0.5 grams of synthetic sewage medium was added to each sample as a food source.

Table 1, below, details the test data reflecting respiration rates as milliliters of oxygen generated per hour over the fourteen-day test period, data for each of the [mixotrophic mixture], SOLMAR CH-118 and distilled water control.

      FIG. 1, which is a graph of respiration rate tests of the [mixotrophic mixture], illustrates a fairly constant rate over days one trough six, with decreased respiration days 7-9, and a sharp increase on day 10, reflecting the stimulated respiration due to addition of the synthetic sewage. Days 11 to 14 showed decreased respiration.
      FIG. 2, which is a graph of respiration rate tests of Solmar CH-118, illustrates a rapid decrease of respiration during days 0-3, with a steady rate of during days 4-9. Feeding on day 10 produced an increase in respiration, although the increase failed to reach a level greater than the original respiration rate on day 0. After day 10, the respiration rate fell rapidly back to the level prior to feeding.
      FIG. 3 is the control data of the distilled water sample. As expected, the control distilled water sample reflected no respiration during days 0-5. However, on day 6 there was a slight increase in respiration, with a 55 subsequent increase during feeding on day 10. The increased respiration is believed due to contamination.
      The data indicate that even though the [mixotrophic mixture] and CH-118 started at the same respiration rate, the [microbial] heterotroph maintained a much higher average respiration rate then the CH-118. To stimulate respiration synthetic sewage was added after day 9 and the respiration of all three samples increased. An increase in respiration was, however, also observed in the distilled water control. The increase in respiration rate in the distilled water is believed due to contamination on day 7 when the sample aliquots were withdrawn.

      Table 2 below details the data based on the gas chromatography performed on aliquots of the three samples taken on days 0, 3, 7, and 14. The data is presented for both perchloroethylene and trichloroethylene, the volatilization product of perchloroethylene.

The data in Table 2 reflects a large volatilization loss of perchloroethylene as trichloroethylene. FIGS. 4-6 graphically present both the perchloroethylene and trichloroethylene concentrations as percent perchloroethylene and percent trichloroethylene of the total concentration for each of the [microbial]  heterotroph
       (FIG. 4), SOLMAR CH-118 (FIG. 5) and the distilled water control (FIG. 6). In each instance, the perchloroethylene concentration decrease was accompanied by an increase in trichloroethylene.
       Perchloroethylene concentration reduction was further calculated by averaging the concentration of perchloroethylene from the PID and Hall detectors. Table 3 lists the data for days 0, 3, 7 and 14.

       It will be appreciated from the data in Table 3 that the majority of perchloroethylene reduction occurred in the first three days. The rate constant of perchloroethylene reduction of each sample was determined by a curve fit to the first order non-linear equation:

•        in which: x is an independent variable; in this case, time in days;
•        y is a dependent variable; in this case, percent reduction of perchloroethylene;
•        C-infinity sign is the value of y when x equals 0;
•        C-infinity sign is the value of y as x equals infinity;
•        and k is the rate constant; this number represents the rate of reaction; in this case metabolism and volatilization.

       Using the above equation the rate constant for the [microbial]  heterotroph was 0.918; for SOLMAR CH-118, 0.727; and for the distilled water control 1.602. A linear correlation between the rate constants versus concentration of perchloroethylene for each sample indicates that there is minimal correlation between the rate constants and the initial perchloroethylene concentrations. The lack of statistically significant correlation indicates that variation in rate constants cannot be accounted for by variations in initial perchloroethylene constants.
       It is assumed, therefore, that a combination of metabolism and volatilization accounts for the perchloroethylene reduction in the [microbial]  heterotroph and the SOLMAR CH-118 samples, and that volatilization alone accounts for the reduction in the distilled water control.
       From the distilled water control, it was noted that the majority of volatilization occurred within the first three days. Accordingly, to enhance observation of perchloroethylene reduction attributable to metabolism, the first three days of data were eliminated. Reduction of perchloroethylene was recalculated from the chromatography data from days 3, 7, and 14, and calculated in parts per billion and as a percent of initial concentration. The results are detailed in Table 4, below.

       A first order nonlinear curve fit was applied to each reduction figure in accordance with Equation 1, above. Rate constants were calculated for the [microbial] heterotroph as 0.606; for SOLMAR CH-118 as 0.079; and for the distilled water control as 0.551. The rate constants are dramatically different, and percentage 50 reductions observed in the distilled water control were less than the reductions in the two other samples.
       Subtracting the distilled water control data from each of the [mixotrophic mixture] and SOLMAR CH-118 data for perchloroethylene reduction, yielded the data detailed in Table 5.

       A first order non-linear curve fit was applied using Equation 1, above, and the rate constants calculated for .[mixotrophic mixture] as 0.430 and for SOLMAR CH-118 as -0.339. The curve fits are illustrated in FIG. 7. The negative rate constant for the SOLMAR CH-118 indicates that the curve has a positive concavity, while the [mixotrophic mixture] curve has a negative concavity, relative to the y-axis. From day 3 to day 12 the inventive heterotroph demonstrated a much larger reduction of perchloroethylene than the commercially available SOLMAR CH-118. In fact, the SOLMAR CH-118 did not begin significant perchloroethylene reduction until day 6. The lag period to day 12 may be indicative of a lack of acclimation of SOLMAR CH-118 to perchloroethylene.
       Thus, the inventive heterotrophic mixture  rapidly metabolizes perchloroethylene in the above tests. It is apparent from the test data that the inventive microbial heterotroph more rapidly metabolizes perchloroethylene than a commercially available microbial mixture SOLMAR CH-118, which is sold and used as a soil amendment.
       While the tests were run only on perchloroethylene, the test data is believed representative of a general activity of hydrocarbon metabolism. The [microbial]  heterotroph is, therefore, capable of degrading hydrocarbons. To test the capacity of the heterotrophic mixture to metabolize complex hydrocarbons, titers of gasoline in water, diesel fuel in water, waste oil in water and bunker fuel in water were made at serial dilutions of 10-1 to 10-6. The inventive heterotroph was diluted with the hydrocarbon titers, plate spread on BiTek Agar and cultured at 25 degrees C. for a period of 480 hours.
       Background heterotrophic bacteria was 1.4 x 108 colony forming units per ml of dilute sample. The plates were then examined for the presence of colony growths. The colonies were counted and colony forming units were calculated per milliliter of dilute sample.
       The results are set forth in Table 6 below, where C.U. indicates colony forming units, n.a. means not applicable, and TNTC means too numerous to count:

       Those skilled in the art will appreciate from the foregoing data, that the inventive heterotrophic bacteria exhibits the ability to metabolize, and hence, degrade hydrocarbons.