(Shale Formation)

The Bio-Enhance ISBR is an extension of the highly successful Bio-Trap® passive microbial sampler. Deployed in groundwater monitoring wells, Bio-Traps have been used for many years all over the world to provide converging lines of evidence to evaluate bioremediation as a treatment mechanism and direct site management activities.

The Bio-Sep® beads (below) within the Bio-Trap sampler have been shown to be rapidly colonized with high densities of indigenous microorganisms, which are released into groundwater when the carrying capacity of the beads is reached. Thus, the beads can act as a continuous source of degraders for release in the aquifer environment.  These properties combine to make Bio-Sep an ideal medium for an in situ biotreatment system (Bio-Enhance ISBR) where indigenous bacteria with biodegradation capabilities are concentrated for treatment and transported away from the wellbore area and into the aquifer matrix.


A fuel oil release occurred beneath a residential structure. Soil excavation was not feasible, as structural supports could not be installed due to the shallow fractured bedrock and unique construction of the house.

An aerobic in-situ bioreactor (ISBR) system was deployed in a fractured shale aquifer impacted by the release. The ISBR was installed in an existing two-inch monitoring well (BR1), with four additional 1-inch monitoring wells (MW1–MW4) installed down-gradient. Groundwater flow was greatly influenced by a sump and associated pump located 10 ft away where approximately 6,000 gal of groundwater was removed during the course of the incubation of the ISBR. A site map is shown below.

To monitor monthly progress, Bio-Trap samplers were incubated in the ISBR well and the down-gradient monitoring wells.  Data from this field study demonstrated that the operation of the ISBR resulted in an increased concentration of microbial degraders as well as increased expression of functional genes associated with aerobic hydrocarbon degradation.

Bio-Traps were analyzed for both DNA, and RNA. Detecting DNA genes involved in biodegradation indicates that microorganisms are present. However, detecting the target functional gene in messenger RNA (mRNA) indicates that the functional gene is actually being transcribed and translated for enzyme production. In other words, while DNA analysis represents the microbial community’s genetic potential for biodegradation, mRNA analysis indicates the actual expression of the functional gene and represents biodegradative activity.

Site map. The ISBR was installed in BR1. Downgradient wells MW1, MW2, MW3 and MW4 were monitored to determine the ISBR's radius of influence.

Site map. The ISBR was installed in BR1. Downgradient wells MW1, MW2, MW3 and MW4 were monitored to determine the ISBR's radius of influence.

Messenger RNA (mRNA) was extracted from all Bio-Trap samplers to be analyzed for total eubacteria (EBAC) and functional genes associated with aerobic hydrocarbon degradation, phenol hydroxylase (PHE) and naphthalene dioxygenase (NAH).

Background contaminant concentrations were measured 123 days before the ISBR was fully operational, and additional groundwater samples were collected on three occasions during the operation of the ISBR, on Days 36, 78, and 251.


Changes in the microbial community in response to treatment were observed in both the ISBR well and the monitoring wells, confirming the effects of the ISBR system had spread beyond the immediate wellbore area.

Results of mRNA analysis of Bio-Traps are shown to the right. The vertical line indicates the start of operation of the complete ISBR system. Prior to this time the ISBR did not contain any Bio-Sep in the packed-bed section although the reactor did receive air sparging and/or air sparging plus nutrient addition.

In addition to the increase in microbial activity and expression of genes linked to aerobic hydrocarbon biodegradation, significant decrease in hydrocarbon concentrations were observed in BR1. Following initial stimulation, growth of hydrocarbon degraders became limited by the availability of hydrocarbon as a carbon and energy source. The only hydrocarbon available for degraders in the BR1 wellbore area was that carried in by groundwater or diffusion.


Installation and operation of the Bio-Enhance aerobic ISBR resulted in stimulation of aerobic hydrocarbon bio degradation as indicated by a concurrent decrease in concentrations of BTEX and naphthalenes in groundwater and an increase in expression of genes associated with aerobic hydrocarbon biodegradation. 

This degree of stimulation was not produced by air sparging and nutrient addition alone and required the presence of Bio-Sep beads in the ISBR to serve as a reservoir and source of hydrocarbon degraders released into the aquifer.

The stimulation aerobic hydrocarbon bio degradation was not confined to the well housing the ISBR but was evident in downgradient monitoring wells within 30 days of operation of the complete ISBR system.  Therefore, the effect of the ISBR is not confined to the installation well of the ISBR exerts a positive effect on biodegradation downgradient.