&EPA
                                    United States
                                    Environmental Protection
                                    Agency
                 £PA/540/MR-93/527
                 November 1993
                                    SUPERFUND INNOVATIVE
                                    TECHNOLOGY EVALUATION
                                      Demonstration  Bulletin

                   Augmented In Situ Subsurface Bioremediation Process™

                                                   BIO-REM, inc.
Technology Description: BlO-REM's technology is an in-situ
subsurface bioremediation process for the treatment of hydrocar-
bon  contaminated soils.  BIO-REM uses a proprietary product,
"H-10", of microaerpphilic microorganisms and micronutrients, in
conjunction with minor components of surface tension depres-
sants and penetrants used to enhance introduction into the pore
spaces of the soil. The H-10 slurry does not contain hydrogen
peroxide  or any other oxygenating compounds. The BIO-REM
process was demonstrated on JP-4 (jet fuel hydrocarbons) con^
taminated soils at Williams Air Force Base, Arizona between May
1992 and June 1993.

BIO-REM claims that the microaerophilic bacteria require, minimal
oxygen to survive and  utilize hydrocarbons as electron acceptors.
Therefore, subsurface oxygen concentration is not a limiting fac-
tor since the microorganisms can exist and propagate under very
low oxygen tensions.  They state that no additional sources of
oxygen nor additional nutrient treatments are necessary during
the course of the remediation.

The H-10 slurry is prepared on-site and above-ground and placed
directly into borings and/or wells previously drilled into the con-
taminant plume.

According to BIO-REM, implementation of the technology con-
sists of the following steps:

   1.  Definition and characterization of the contaminant plume
      including the geology, hydrogeology, and gradient of the
      site, and the dimensions of the contaminant plume.


   2.  Determination of application methodology. In permeable
      soils, borings and wells drilled into the contaminant
      plume are sufficient. Soils containing fine sands and
      clays may also require lancing.


   3.  Initialization and propagation of H-10, and placement
      (inoculation) of the batch solution into the contaminant
      plume. The batch solution is placed into the contaminant
      plume by the application methods described in Step 2.
      The quantity of culture and batch solution placed into the
      contaminant plume is determined by a proprietary formula,
      and is based on the type and level of contamination.

Waste Applicability: According to BIO-REM, a wide range of
hydrocarbon contaminants can be treated by their bioremediation
process. Typical contaminants include gasoline, diesel, JP series
aviation fuels, and other wastes containing polyaromatic hydro-
carbons, pentachlorophenols, trichloroethene, and dichloroethene.
BIO-REM claims that over two hundred hydrocarbon compounds
have been identified as being amenable to their treatment pro-
cess. '!       .  .                             •

Demonstration Activities: The BIO-REM in situ process was
tested at the former Liquid Fuels Storage Area on Williams Air
Force'Base near Phoenix, Arizona. Soils at the 0.9 acre site are
contaminated with hydrocarbons derived from JP-4 jet fuel as a
result of leaking underground storage tanks and pipelines. The
tanks 
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 the progress of the remediation, BIO-REM utilized periodic vapor
 analysis for hydrocarbons from several open boreholes through-
 out the site.

 Tha progress and effectiveness of the bioremediation technology
 was evaluated by the SITE Program by comparing baseline (time
 0} soli contamination levels to contamination levels at one month,
 three  months,  and  six months  after inoculation.  During each
 sampling event,  twenty  borings,  within a highly contaminated
 portion of the site, were  used to evaluate the  process. These
 borings were divided into four depth intervals from four feet BLS
 to twenty-five feet BLS. All samples were analyzed for benzene,
 toluene, ethylbenzene, and xylenes  (Method SW846 5030/8021)*
 and Total Recoverable Petroleum Hydrocarbons (TRPH) (Method
 SW846  3550/EPA 418.1). In addition, selected samples were
 analyzed for Total Petroleum Hydrocarbons (TPH), as per the
 California Leaking Underground Fuel Tanks Manual Method for
 dlesei contaminated soils.

 Demonstration Results: Results from the Demonstration indi-
 cate that the BIO-REM process was unsuccessful in reducing
 target contaminants in the soil to the project clean-up levels.

 Baseline sampling indicated that a  majority of the soil samples
 were significantly higher than the cleanup levels of 130 ppb for
 benzene and 100 ppm  for TRPH.  Furthermore,  soil samples
 analyzed one and three  months after inoculation did not show
 s^ntfteant reductions in benzene or TRPH contamination (Table
 1). The lack of progress in the remediation prompted concerns
 regarding the effectiveness of the technology. It was jointly de-
 cided between the SITE Program and BIO-REM to collect sixteen
 samples (four boreholes)  at six months to determine the progress
 of the remediation at the predicted end of the  project. Results
                                from the six month sampling event also indicated  a lack of
                                significant reduction in contaminant concentrations.

                                Based on these results, BIO-REM submitted a request to the Air
                                Force to re-inoculate the site based on their assessment that
                                sub-surface lithological conditions inhibited the remedial process.
                                In  March of 1993 BIO-REM re-inoculated the site by injecting
                                approximately 35,000 gallons of H-10 slurry into 104 boreholes
                                deepened to a depth of 23 feet below land surface. The inocula-
                                tion to deeper  depths was implemented to overcome the sub-
                                surface lithological conditions identified by BIO-REM.  In June of
                                1993 a confirmatory sampling event initiated by the Air Force, in
                                conjunction  with  the SITE Program, indicated that  significant
                                contamination existed at the site, and that the re-inoculation was
                                unsuccessful in reducing the target contaminants to the project
                                specific clean-up levels. Based on these results, these site activi-
                                ties were concluded.

                                A final report will be available in the summer of 1994

                                For Further Information:

                                EPA Project Manager:
                                Kim Lisa Kre'rton
                                U.S. EPA Risk Reduction Engineering Laboratory
                                26 West Martin Luther King Drive
                                Cincinnati, OH 45269
                                513-569-7328

                                BIO-REM, Inc.:
                                David O. Mann
                                P.O. Box 116
                                Butler, IN 46721
                                800-428-4626
                                                                                    •U.S. Government Printing Office: 1903 — 650-067/80116
Tabla 1. Frequency Distribution of Benzene and TRPH for Demonstration Sampling Events
Number of Samples
                        Benzene (ppb)
 <50       50-130    130-1,000      1,000-10,000      10,000-100,000
Baseline
One Month
Throe Months
33
27
37
4
3
3
13
20
12
26
25
22
4
5
6
Number of Samples
        Total Recoverable Petroleum Hydrocarbons (ppm)
<100                    100-1,000                1,000-10,000
Baseline
One Month
Three Months
  27
  29
  22
33
34
30
20
17
28
    United States
    Environmental Protection Agency
    Center for Environmental Research Information
    Cincinnati, OH 45268

    Official Business
    Penalty for Private Use
    $300
                                                                 BULK RATE
                                                            POSTAGE & FEES PAID
                                                                     EPA
                                                               PERMIT No. G-35
    EPA/S40/MR-93/527

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