Cost Effective In-Situ Soil Bioremediation

Petrox organisms yield cost-effective in-situ soil bioremediation to remove residual petroleum contamination.  Instead of ripping up the service station to excavate the soil or installing a slow vapor extraction remediation system, an environmental consultant in east Texas used Petrox microbes to remove high concentrations of benzene and xylene from soil at a gas station.  Petrox bioremediation saved the property owner time and money.

Benzene and xylene contaminated shallow soil at concentrations of 230 mg/kg and 9,200 mg/kg respectively.  Although benzene drove the environmental risk up, xylene, which has a high soil absorption factor, can be more difficult to remediate.  Vapor extraction would have had problems extracting xylene from the sandy, clay soil.

Petroleum metabolism, including benzene and xylene, requires oxygen.  A calcium peroxide compound was added to supplement the natural oxygen for Petrox metabolism.

The treatment area was 500 square feet with an assumed treatment depth to 11 feet.  The consultant injected 220 gallons of hydrated Petrox solution and 25 pounds of calcium peroxide.

Post-treatment sampling showed the contaminant concentrations decreased by 97% in less than 90 days.  The benzene concentration dropped from 230 to 4.0 mg/kg.  The xylene concentration decreased from 9,200 to 309 mg/kg.

The Petrox and calcium peroxide materials cost was less than $6,000.  On top of the cost savings, bioremediation did not interrupt business operations.


Naphthalene Bioremediation With Petrox Microbes

Naphthalene is often a recalcitrant petroleum compound that keeps sites from clean closure.  This resistance to remediation is due to naphthalene’s relative high adsorption and low solubility rates.  Naphthalene has an aqueous solubility of 3.1E1 mg/l compared to benzene at  1.75E3 mg/l .  Naphthalene has an affinity of adsorption expressed as the octanol/water partition coefficient  (log Kow) of 3.26, while benzene has a log Kow of 2.13.  For further comparison, acetone has a high water solubility at 1E6 mg/l and low log Koc of -0.24.  In a study of relative degradation rates, the USEPA found a first order kinetic constant of 14.0 L/hg for naphthalene and 0.234 for benzene in activated sludge.  Despite these challenges,  naphthalene bioremediation with Petrox microbes has been successful at many spill sites.

Microbes initiate metabolism of naphthalene by inserting two oxygen atoms into the naphthalene structure.  This initial step is catalyzed by naphthalene dioxygenase.  Tests of sites inoculated with Petrox microbes show naphthalene dioxygenase at 1E8 to 1E10.

Petrox microbes thrive on naphthalene to the extent that naphthalene is occasionally used in the QA/QC  process to verify Petrox viability and effectiveness.  In this test, the Petrox organisms are placed on an agar devoid of a carbon source.  The naphthalene is applied to the top plate as the sole carbon source.  The Petrox viability is then demonstrated by colony growth on the top plate as shown on the following picture.

Click here and here to view cases studies of naphthalene bioremediation case studies.

In Situ TPH Bioremediation with Petrox

With Petrox microbes, in situ TPH bioremediation reduced the concentration of petroleum in soil at a former gas station from 9,500 mg/kg to 52 mg/kg. The impacted soil was at more than 20 feet deep.  Injection of calcium peroxide provided the oxygen needed for metabolism of the petroleum by Petrox microbes.  The post treatment sampling was 90 days after the bioaugmentation, so that even though the average remediation rate was 105 mg/kg/day, the peak remediation rate must have been much higher when the Petrox population was higher.

This is another example of fast, reliable bioremediation by Petrox organisms.

In-situ Bioremediation of BTEX, TPH and PAHs in Groundwater


In situ bioremediation reduced ground water contamination by more than 99% in less than a year at a gas station in Florida.

Convenience Store in Florida

After three underground storage tanks were removed from the location of a convenience store, investigation showed that petroleum contamination was present in small area near the former dispenser island.  The vadose-zone contaminated soil was excavation and disposed off-site.  Ground water monitoring showed that the ground water impact was limited to a small area near the source of contamination. The decision was made to close the site by applying Petrox® bioremediation to the ground water.

Ground Water Contamination

The ground water contamination was in a shallow water table aquifer that had a medium permeability.  The contamination was a suite of BTEX, PAHs, methylnaphthalene isomers and TRPH.


A pilot study was completed to determine feasibility of Petrox bioremediation combined with a peroxide oxygen supplement.  The pilot study reduced contaminant concentrations by up to 92%.  A full-scale application completed the site remediation.


The pilot study and full-scale applications of Petrox and an oxygen supplement reduced BTEX compounds by >99%, naphthalene by 99%, TRPH by 96% and naphthalene isomers by 88% to achieve the remediation goals and site closure.  The microbial cost for the pilot study and full-scale treatment was less than $2,000.

To learn more, click here to download the case study.

Bioremediation With Horizontal Injection for BTEX and Naphthalene Remediation

In situ remediation is a contact sport, requiring contact between the microbes and contamination.  Clay-rich soils are particularly challenging as the low permeability limits effective distribution of inoculants.  While numerous closely-spaced injection points can improve the distribution of inoculants, this approach is often not possible at active properties and can be costly.  Horizontal drilling was used on the subject site to overcome the combined challenges of low permeability and limited site access to inject Petrox® microbes for bioaugmentation.  The combined technologies reduced the total BTEX concentrations in ground water from 2,771 µg/L to 645 µg/L in less than one year.


The site is a former gas station located in the Florida panhandle.  The site soils are mixed silt and clayey silts that are typical of the coastal setting.  The depth to the water table varied seasonally from 13 to 18 below ground surface.

The underground storage tanks and contaminated soil had been removed prior to the ground water treatment.  Residual ground water contamination exceeded Florida Ground Water Cleanup Target Levels (GCTLs) for benzene, toluene, ethylbenzene, xylene, naphthalene and methylnaphthalene isomers.

The proposed treatment area was approximately 2,500 square feet.  The treatment depth was from 10 to 25 feet below grade.  The treatment depth included the capillary fringe to treat residual contamination above the seasonal low water table.

Horizontal Injection

 Access to the treatment area was limited by the current property use, the proximity of roads on two sides and a building on the third.  Horizontal drilling was selected by the site consultant, Advanced Environmental Technologies ( to deliver Petrox® to the contamination.

The horizontal drill rig was set back from the treatment zone on the opposite side of the building.  The horizontal injection wells were set in four horizontal sets of eight wells. The horizontal layers were at 10, 15, 20 and 25 feet deep.  The wells in each layer were five feet apart.  A total of 32 injection wells were closely spaces for excellent coverage through the treatment zone.

Petrox® was injected into the ground water in two treatment events – November 6, 2008 and June 24, 2009.  Petrox® was delivered in each injection well as the drill stem was withdrawn through the treatment zone.  The injection was monitored for accuracy so that 0.2 gallons of Petrox® was injected per foot of injection zone.  A total of 320 gallons of Petrox® slurry was injected.

Following the Petrox® injection, air was injected periodically through vertical sparging wells to increase the oxygen availability for the microbial metabolism.


Ground water samples were collected from a monitoring well inside the treatment area to track the progress of the bioremediation.  Approximately 60 days after the first Petrox® treatment, analysis of ground water samples showed 84% reduction in the benzene concentration and 35% reduction in the total BTEX compounds concentrations.  There was an apparent increase in the xylene concentration due to ground water mixing and induced increase in solubility due to the bioaugmentation injections.

A second sampling event approximately 30 days after the second injection showed additional reduction in the contaminant concentrations.  After the second injection, the total BTEX concentrations were 23% of the original concentration with xylene decreasing from 1,200 to 95 µg/L.

In addition to the BTEX compounds, naphthalene and methylnaphthalene isomer concentrations decreased through both treatments.

Sampling Date Benzene Toluene Ethylbenzene Xylene Naphthalene
Pre-treatment 11/11/05 3,000 42 1,100 1,100 230
10/23/08 1,700 18 460 454 280
11/6/08 1,600 41 370 760 310


1/22/09 420 43 140 1,200 43
7/30/09 490 2.6 57 95 55


Horizontal drilling and injection made it possible to remediate ground water at this site of petroleum contamination with limited access and low natural permeability.  Without disturbing the property use, the horizontal injection of Petrox® provided effective distribution of the microbes for bioremediation.  The injection may have also made the contamination more available for bioremediation by increasing the contaminant solubility as shown by temporary increases in concentration.

This case study demonstrates that in situ bioaugmentation may be a feasible solution for sites with limited permeability and access restrictions.  For more information contact CL Solutions at