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Introduction


Remtech has developed a proprietary slow-release enhanced bioremediation accelerator and biosurfactant product, HC-2000 (HC2), that cleans, disinfects, desorbs, reduces odors and degrades fuels, oils, transformer oils, lubricants, chlorinated and non-chlorinated solvents. HC-2000 works on soil, ballast, gravel, groundwater, surface water, wastewater, sludges, septic tanks, power substations, cleaning surfaces (clothing, concrete, asphalt, metal), and other matrices. Biosurfactants increase mass transfer of biochemical reaction accelerators to rapidly degrade and clean petroleum based contaminates. Slow release ingredients continue to deliver active ingredients over periods exceeding 1 year.


HC-2000 is a Green Sustainable Technology product.  HC2 restores the environment by accelerating natural systems (heterotrophic bacteria) to degrade petroleum and solvent based contaminants.  HC2 is non-toxic and removes contaminants with minimal economic disruptions to business and generally cost less than other remedial technologies. When treatment is complete, native conditions are restored.


HC-2000 has been used successfully on over 400 cleanup site remediations during the past 20 years.  HC-2000 is designed to work in freshwater applications on refined fuels and solvents not addressed by EPA’s NCP List of saltwater approved crude oil remediation and cleaning products.  HC-2000 is ten times less toxic to aquatic organisms than most of the NCP products.  Remtech's Bioremediation Design Optimization Primer with HC-2000 is presented here. Remtech's Biopile Bioremediation Design Optimization Primer with HC-2000 is presented here.


HC-2000 is particularly cost-effective where: service or business interruptions need to be minimized; access is limited; areas are environmentally sensitive; or sites are geotechnically, hydrologically or structurally sensitive or unstable, and where contamination exists around grounding grids at power plants or substations.


HC-2000 Enhanced Bioremediation Product Multi-Media Applications


Soil Treatment - treats surface soil passively (via topical or gravity feed systems for soil depths up to 3 to 5 ft) or actively (in combination with biovent/injection & biosparge/multiphase extraction systems for depths exceeding 5 ft).


Ballast, Gravel & Soil Treatment (Railroad Ballast, Power Plants, Substations & Tank Farms) - Gravel, soil and/or ballast can be treated with HC-2000 at railroad mainline and siding tracks, power plants, substations, and in above ground storage tank secondary containment areas. HC-2000 is particularly effective in railroad ballast (that functions like a trickling filter with elevated baseline heterotrophic bacteria plate counts). In place treatment minimizes rail or power service interruptions and disturbance of grounding and cathodic protection systems.


Groundwater Treatment - treats groundwater insitu passively (infiltration galleries, biofences) or actively (reactor trenches, sparge wells, and total fluids extraction wells).


Surface Water Treatment - removes sheens by lowering surface tension and allowing petroleum hydrocarbons to come in contact with particulate born bacteria for degradation. Removes light sheens and odors almost immediately and desorbs fuel from banks, leaves, limbs, and other organic matter. Degrades solution phase contaminates in aerobic, facultative, and anaerobic environments.


Pavement & Surface Cleaning - cleans and removes oils and solvents from asphalt, concrete, metal surfaces, parts, and other surfaces. Accelerates degradation rates of rinsates.


Sediment Treatment - desorbs flammable/combustible fuels and solvents from sediment in sewers, retention ponds, stream/river beds, and storage tanks. Flushing systems with HC-2000 accelerates solvent removal/desorption, degradation, and lowers flammable vapor concentrations. Insitu treatment methods minimizes erosion.


Solids & Sludge Breakup - breaks up sludge in digesters, sludge pits, septic tanks, and solids buildup in ASTs and USTs.


Forrest Canopy, Vegetation, River Banks, & Wildlife - cleans fuels and solvents from leaves, foliage, sod, etc., to minimize interference with photosynthesis and minimizes loss of foliage. Rehabilitates flora & fauna from oil exposures. Accelerates degradation of wash residues.


Wastewater Treatment - accelerates biological degradation in oil/water separators, truck and car washes, parts washing, septic tank systems, and conventional biological wastewater treatment systems.


Vapor, Odor Control & Disinfection - HC-2000 kills pathogenic and odor causing bacteria, reduces vapor pressure of volatile compounds, and activates bacterial odor degrading microorganisms.


Tank Cleaning - Removes petroleum hydrocarbons and solvents from above ground and underground storage tanks



Technology Selection Factors


Environmentally Sensitive Areas - food quality natural components in HC-2000 accelerate bioremediation in environmentally fragile areas such as wetlands, marshes, beaches, national parks, dunes, nature preserves, and forests without adverse environmental impacts on flora or fauna. HC-2000 synergistically accelerates bioremediation in the rhyosphere (root zone), and organically enriched and microbial diverse environments.


Structurally Sensitive/Restrictive Areas - treats soils and groundwater under buildings, around footings & building foundations, utilities, old fragile sewer systems, and grounding & cathodic protection systems.


Geotechnically Unstable or Erosion Sensitive Areas - treats drainage ditches, stream beds, river banks, and highway right-of-ways while minimizing erosion and adverse environmental impacts.


Security Sensitive Facilities - in access restricted areas, applications can be made quickly with minimal disruptions at airports, military and penal installations.



How Does HC-2000 Work?


Native heterotrophs are ubiquitous in the environment and are found in soil, water, groundwater, and waste in aerobic, facultative, methogenic, and anaerobic zones. Indigenous (insitu) bacteria are the predominant waste degraders in soil, groundwater, surface water, railroad ballast, gravel, and saltwater.


Numerous publications conclude that very little scientific data demonstrates that enhanced degradation (microbial addition or bioaugmentation) works on full-scale cleanups. According to peer reviewed literature, bioaugmentation appears to have little benefit for the treatment of spilled petroleum hydrocarbons in the environment. Microbial addition has not been shown to work better than nutrient addition alone in many field trials (10). Biostimulation has been proven to be a promising tool to accelerate and enhance the natural attenuation process.


Native soil populations capable of degrading petroleum hydrocarbons have been reported to range from 10 to 20% of all soil heterotrophic bacteria. Soil particles containing heterotrophs are found in lower concentrations in groundwater and surface water (2 x 106 cells/ml) than in soil (2 x 107  cells/gm) (11), albeit provide the metabolic and synthetic activity to digest petroleum hydrocarbons. Approximately 67% of all bacteria in groundwater sediment are attached to the silt and clay fraction, though this sediment fraction may account for a small fraction of the total sediment by weight.

Bulk Diesel Removed prior to
HC-2000 Treatment
HC-2000 Removes Sheens, Treats Surfacewater & Fuel Contaminated Sediment

HC-2000 Removes Sheens, Treats Surfacewater & Fuel Contaminated Sediment

Motor Oil Contaminated Ballast

Motor Oil Contaminated Ballast

Ballast Staining & Oil Concentrations Reduced by Over 70% after 4 months of HC-2000 Treatment

Ballast Staining & Oil Concentration Reduced by Over 70% after 4 months of HC-2000 Bioremediation

HC-2000 Degrades Diesel Fuel & Removes Stains & Odors in Railroad Ballast
HC2000 Gasoline Contamination
Wetland Remediation

HC-2000 90% Diesel Fuel Degradation over 4 Months & Removes Odors in Railroad Ballast

HC-2000 New York Solvent Remediation Under Building

85 to 99% Solvent Removal in 5.5 Months with HC-2000

HC2 Riverbank Treatment at
JP-8 Contaminated Golf Course
Prevents Erosion Claims
HC-2000/BioSparge Injection
Well at Gasoline GUST Trust
Fund Reimbursed Site
HC-2000 Desorbs & Degrades 
Lube Oil in Railroad Ballast

HC-2000 Desorbs & Degrades

Lube Oil in Railroad Ballast

98.7%  Gasoline Removal in

Wetland in 18 Weeks with HC-2000

HC2 Removes Diesel Fuel
in 10-6 cm/sec Conductivity
Aquifer under Building

HC-2000 Removes Diesel Fuel

in 10-6 cm/sec Conductivity

Aquifer under Building

Airport JP4 HC2000
Remediation in Restricted Area

99% JP4 Removal in 4 Months at Airport with HC-2000

Bulk Diesel Removed prior to

HC-2000 Treatment

94% JP-8 HC-2000 Degradation over 4 Months in Riverbank

Aerobic Mineralization of Petroleum Hydrocarbons

SIMPLIFIED

MICROBIAL AEROBIC WEATHERING/MINERALIZATION

OF PETROLEUM HYDROCARBONS

Complete mineralization in nature results from a consortium of microorganisms working together synergistically. Cometabolic activity (enzymes generated by an organism growing at the expense of one substrate) can also transform a different substrate that is not associated with that organism's energy production, carbon assimilation, or any other growth process - degradation of a compound only in the presence of other organic material that serves as the primary energy source (11). Multiple bacteria populations found in nature provide the mechanisms to break down petroleum compounds to CO2 and water that is not possible with a single bacterial strain.

Many organic contaminants will serve as a substrate. However, for some hazardous chemicals, another substrate is necessary. The compound targeted for degradation may be at concentrations below that necessary for good biological response for bioremediation. The addition of a primary substrate or carbon source (contained in HC-2000) supplies additional energy to stimulate biological growth. The target contaminant compound to be degraded becomes the secondary substrate.

Heterotrophs have been demonstrated to be effective degraders of a variety of hazardous and non-hazardous wastes including petroleum hydrocarbons, chlorinated solvents, and other pollutants. A myriad of degradation data was generated in the 1980s during research on land farming of hazardous wastes (1). Ross McKinney in his book Microbiology for Sanitary Engineers states ... the best source of micro-organisms is soil. The soil can furnish all the microorganisms ever needed in waste disposal. My advice to all sanitary bacteriologists who seek a special culture is to look under their feet; the supply is inexhaustible (2). HC-2000 targets native heterotrophs that control the bioremediation process.

Heterotrophic degradation is limited by enzymes, co-factors, nutrients, electron donors, biosurfactants, and the proper environmental conditions that accelerate biochemical reactions. Natural attenuation can take years and generally reaches asymptotic concentrations due to insufficient nutrients or inadequate environmental conditions. HC-2000 supplies the limiting factors and reduces degradation times to months or longer periods for more resilient compounds.

HC-2000 is a non-toxic aerobically fermented natural organic product that is readily assimilated and eliminates long lag reaction/acclimation periods of inorganic nutrients. HC-2000 can be applied under various environmental conditions (aerobic, facultative, methanogenic, anaerobic).

What’s In HC-2000?

HC-2000 is an organic food quality indigenous soil bacteria biostimulation agent that accelerates and improves natural attenuation of crude oil and refined petroleum and solvent products when compared to EPA's synthetic inorganic fertilizer blend and non-spiked controls. HC-2000 contains aerobically fermented organic nutrients including Kjeldahl nitrogen, proteins, enzymes, vitamins, and biosurfactants. Pulp present in HC-2000 acts as a slow release mechanism that provides extended release of active ingredients over time that in many cases exceeds one year.

Organic Versus Inorganic Nutrients

Natural organic origin fertilizers in urea, uric acid, and HC-2000 are considerably less toxic than most synthetic oleophic fertilizers. Organic fertilizers have low solubility in water (contribute to slow release); organic nutrients in HC-2000 bind to petroleum hydrocarbons and are therefore available to bacteria which grow at the hydrocarbon-water interface.  Pulp in HC-2000 retards leaching of nutrients, enzymes, and biosurfactants making active ingredients available over extended periods of time.

The addition of organic carbon sources such as surfactants and organic nutrients will increase the bacterial respiration of petroleum degrading bacteria and enhance n-alkane biodegradation (12).

Role of Biosurfactants

Biosurfactants (a key ingredient of HC-2000) have been used widely to break down and/or desorb contaminant matrix size to allow greater contact with native bacterial degraders. Native bacteria are already distributed in environmental media and rapidly acclimate to spilled material. Biosurfactants are used to increase mass transfer rates, and desorb/mobilize contaminates for greater contact with soil microbes.

Biosurfactants assist in petroleum hydrocarbon availability either by chemical or biological means. Microbial attachment takes place mainly at the oil-water interface and increased mixing of petroleum hydrocarbons results in dramatic increases of the surface area for microbial colonization. Biosurfactants are amphiphilic compounds of microbial origin with considerable environmental advantages over chemical surfactants in terms of biodegradability and effectiveness at extreme temperatures or pH and have a much lower toxicity (12). Biosurfactants facilitate the bioremediation of hydrocarbons having limited water solubility by increasing bacterial cell surface hydrophobicity, via fatty acid moieties, which promotes their adsorption to hydrocarbons.

Biostimulation with organic nutrients and biosurfactants enables naturally occurring microbes to adapt better and faster to the contaminated environment resulting in shorter lag phase and faster petroleum hydrocarbon degradation. Biosurfactants also play a major role in cleaning surfaces.

Fate of Degradation Products in the Environment

HC-2000 desorbs and degrades contaminants. Biostimulation bioremediation projects require adequate reaction zones and retention periods to complete degradation. Offsite migration can be minimized by removing as much of the mobile free product phase or grossly contaminated matrix as as possible at the beginning of the project and monitoring downgradient receptors to determine if additional downgradient HC2 injection points and/or containment/leachate recovery systems are required.  Controlled addition of HC2 can also enhance removal of bulk fuels or solvents by washing and desorbing for recovery by conventional mechanical recovery systems.

On soil or ballast applications, the desorption properties of HC-2000 accelerates the removal of mobile phase constituents and brings residual contaminates in contact with an increased number of soil bound degraders.

On water applications, the mobile phase is removed first by conventional methods. Non-recoverable sheens are removed with HC-2000 by temporarily lowering surface tension (not emulsifying) and bringing contaminants in contact with particulate born degraders. Oil skimmers, oil/water separators, and other conventional free product recovery devices are not adversely impacted by HC-2000 since HC2 does not emulsify contaminants.

Examples of leachate containment systems that may be used to provide adequate treatment periods include: filtration/adsorption dams, containment booms, interceptor trenches, cut-off walls, perimeter monitoring wells, downgradient air curtains or sparging, or groundwater depression/vacuum extraction systems.

Overcoming Mass Transfer Challenges


Insitu treatment is limited by mass transfer. Treatment technologies must come in contact or mix with contaminants and the environment to be effective. Native heterotrophic bacteria are already dispersed and rapidly acclimate to contaminates around them.


The ability to deliver treatment agents uniformly to heterogeneous media (such as soils and groundwater) is required to achieve complete site treatment. HC-2000 supplies wetting agents and biosurfactants that improve mass transfer. Bound or adsorbed contaminants in soil pores (frequently account for up to 60% of the total contamination) restricts bioavailability. Biosurfactants in HC-2000 desorbs or washes contaminates moving them towards microbial degraders. This same desorption mechanism is a major factor that facilitates cleaning surfaces.


HC-2000 enhances both passive and aggressive delivery methods. Passive insitu technologies rely on advection (bulk groundwater flow) and dispersion to disseminate reactants. Aggressive pulsed (air and/or reagent) injection enhances mixing and mass transport and generally produces more rapid and uniform results.


HC-2000 Delivery Systems


For bioremediation or cleaning, a HC-2000 and water mixture may be applied by a diaphragm, roller, or centrifugal pump with a coarse fire nozzle. Trace quantities of pulp may clog finer nozzles and piston pumps (especially pressure washers). HC-2000 may be injected past pressure washer pumps and applied with chemical feed nozzles followed up with a high pressure water chase (cold or hot - <120 oF) to drive HC2 into the media. Best results are achieved when surface solid media (soils, gravel, ballast, etc.) are mixed (tilled) or agitated with the initial application and several times during the treatment period. Agitation of hard surfaces (manual or power brooms, pressure or hot water washers), followed by a reaction period (approximately 5 to 20 minutes) and water rinse enhances surface cleaning effectiveness.


For subsurface soil applications, HC-2000 may be applied through infiltration galleries, horizontal or vertical biovent systems or reaction trenches/fences. HC-2000 may be applied as foam to enhance movement through and contact with soil. Soil moisture should be maintained at 70% of field holding capacity (or 20% moisture).


For subsurface groundwater applications, HC-2000 may be applied through horizontal or vertical biosparge systems, reaction trenches/fences, or in conjunction with total fluids vacuum extraction systems. HC-2000 may be injected as a liquid or foam to increase contact with pollutants.


For insitu soil and groundwater applications, reaction zones are generally defined by areas where 10% of the applied air pressure is observed at response points. Radius of influence or reaction zone tests should be performed to determine injection grid layouts and spacing. Application of HC-2000 during high water table conditions may avoid contamination desorption from the smear zone.


Suggested Monitoring & Dosage Rates


Optimal degradation conditions are present when total heterotrophic plate concentrations are elevated and maintained during the treatment period. Elevated plate counts indicate that sufficient nutrients, moisture, and environmental conditions are present. Secondary parameters that may be monitored include; respiration by-products, moisture, dissolved oxygen, pH, and redox potential. Monitoring the reduction of the contaminant(s) of concern determines when treatment is complete and/or when natural attenuation can complete the degradation process.


Dosage rates are site and contaminate(s) specific. Dosage rules of thumb range from three (3) to ten (10) cubic yards of contaminated media (soil, groundwater, railroad ballast, etc.) per gallon of concentrate over a five-week treatment period. Concentrate dilution ratios of one part of HC-2000 to sixteen parts of water is recommended for soil, ballast or gravel applications.


Groundwater dosage rates vary from 1:16 to 1:30 (HC-2000 to water mixture). Always add HC-2000 concentrate to water to avoid excessive foaming. For example, to treat 10 cubic yards of contaminated media (assuming one gallon of concentrate treats 10 cy), weekly dosages would be 0.2 gallons in 3.2 gallons of water. Assuming a 16-week treatment period, then 16 x 0.2 gallons or 8 gallons of concentrate would be required.  For surface cleaning applications a 1:5 to 1:10 ratio may be used. For pressure and/or hot pressure washers chemical feed ratios of 1:2 to 1:5 are recommended.


Transformer Oil Removed from Containment Structure at 
Power Plant

Transformer Oil Removed from Concrete Containment Structure at

Power Plant

HC-2000 Treatment Algorithm for Ballast, Gravel, Soil at Rail, AST, and Power Facilities

Bioremediation Agent and Biosurfactant, Green Sustainable Product White Paper HC-2000

Purchase from Remtech Store

HC2000 Treatment Steps

A.  Phase 1:  Locate, Contain, and Monitor Mobile phase

B.  Phase 2:  Remove/Desorb Mobile Phase

Activation & Stability of HC-2000

HC-2000 has a shelf life that exceeds five (5) years (as long as it is stored in its concentrated state and at temperatures below 120oF).  HC-2000 is activated when it is mixed with water according to the contaminated matrix and applicable dilution ratios. Diluted mixtures of HC-2000 have a shelf life of a couple of weeks and should be utilized as soon as possible after mixing with water.

Regulatory Approvals

Regulatory authorities frequently favor (and generally quickly approve) the acceleration of natural degradation processes with a Green Sustainable Technology as apposed to addition of foreign microbes or toxic materials into the environment. Stimulating natural biochemical processes reduces the possibility of toxic by-product formation and allows multiple native species (operating under a variety of environmental conditions) to reduce contaminates to minimum levels. When treatment is complete, native conditions are restored.

HC-2000 has been approved by Georgia, Florida, and other states for the treatment of soil and groundwater on a case -by-case basis. No specific approval is required for soil applications in Georgia. Georgia frequently only requires three (applications of HC-2000 to soil without performance sampling to complete a cleanup.

HC-2000 Benefits

HC-2000 is a Green Sustainable Technology.  HC2 restores the environment by accelerating natural systems (heterotrophic bacteria) to degrade petroleum and solvent based contaminants.  HC2 is non-toxic and removes toxic contaminants with minimal economic disruptions to business and generally cost less than other remedial technologies. When treatment is complete, native conditions are restored.

Other remedial technologies may leave residual contaminants that require additional treatment. These technologies include; pump and treat, soil venting and air sparging, and total fluids extraction. Why not use a technology that can finish the job?

Site remediation costs with HC-2000 typically range from $15 to $225/cy of contaminated media. Costs are site specific and are affected by the type and amount of contamination, local geology, volume of contaminated media, and contaminant location. HC-2000 performs best in formations where adequate communication and mass transfer are present or can be established.

HC-2000 is non-toxic, non-allergenic, and contains food quality ingredients. Accelerating the natural degradation process (Biostimulation) with HC-2000 is generally received favorably by regulatory authorities and the general public.  Native heterotrophs are already acclimated and distributed in the environment. All that is required is to deliver HC-2000 to the degraders and provide adequate environmental conditions. When treatment is complete, native conditions are restored.

HC-2000 goes right to work by energizing native heterotrophs. Chemical oxidization with permanganate, peroxide, and ozone frequently oxidize materials other than target contaminants, i.e., a significant mass of reagents are wasted. Chemical oxidization and bioaugmentation may form toxic by-products that are not normally associated with cometabolic native biochemical reactions.

Oxygen and hydrogen release compounds singularly introduced generally rely on passive slow release mechanisms and depend on advection and dispersion to transport the reagent to the contaminant. Limiting nutrient deficiencies, enzymes, and co-factors are not addressed. Adsorbed and soil pore bound contaminates are only addressed by sufficient concentration gradients to draw reactants to contaminants. HC-2000 provides contaminant desorption with biosurfactants that is generally more effective than concentration gradients. Aggressive pulsed reagent injection (used with HC-2000) generally provides better mass transfer and mixing.

HC-2000 is easily assimilated by native bacteria without a lag time. Commercial fertilizers and synthetic surfactants may initially inhibit microbial degradation. Nutrient and carbon sources such as molasses, sugars, and vegetable oil need to be broken down further prior to assimilation by microbes. Organic nitrogen and proteins (contained in HC-2000) are a preferred source of nitrogen over nitrates, ammonia, and other compounds containing inorganic nitrogen.



References


  • Monitor leachates and soil degradation targets (visual observation of stormwater runoff - no sheens), sample ballast fines and downgradient soil and leachate for total petroleum hydrocarbons. Supplemental water may be required to maintain moisture levels at 70% of field holding capacity.
  • Apply HC-2000 per label instructions and adjust for site conditions.
  • Repeat until regulatory limits, risk based closure, or until natural attenuation can reach regulatory limits.
  • Remove/close leachate control systems.
  • Close HC2 injection systems.

  • Determine site access and available site service interruption time, ie, track time, business interruptions, environmental receptors, sensitive environments and structures, and cleanup and risk based targets
  • Locate contamination and mobile phase pathways. Look for short circuits, i.e., sewers, swales, drains, backfill, utilities, streams using test pits, monitoring wells, geoprobe points
  • If mobile phase present, install containment or free product migration monitoring system. Install leachate/migration control systems (straw bale filtration dams, collection trenches, pits, drainage tiles & sumps, or temporary detention pools). For track applications, instal leachate control near toe of ballast and/or in drainage swales. Apply HC-2000 followed by oxygen saturated water to increase penetration, moisture, and oxygen.
  • Protect structural integrity of track, grounding, and cathodic systems - Do not disturb ballast below ties or within a 45% slope of tie edges.
  • Monitor and contain free product movement.

C.  Phase 3:  Degradation Phase

  • Remove mobile phase or saturated soil/solids hot spots and HC2 injection method and grid.
  • Apply HC-2000 (followed by water chase to accelerate removal of mobile phase and assist with product desorption. Bound contaminates account for over 60% of contamination. Fuels may become mobile as they are desorbed and broken down into shorter hydrocarbon chains.
  • Ballast Treatment – Apply HC-2000 topically followed by a water chase or through injection probes. Fouled ballast - mixing or agitation enhances HC2 delivery and treatment. Pressurized water (3,000 to 5,000 psi) or a cribbing bucket loosens ballast next to ties.
  • Repeat HC-2000 applications until mobile phase dissipated.

  • Hazardous Waste Land Treatment, Environmental Protection Agency, Solid Waste & Hazardous Waste Research Division, Cincinnati, Ohio, April,1983.
  • McKinney, Ross, Microbiology for Sanitary Engineers, McGraw-Hill Book Company, Inc.,1962.
  • Suthersan, Suthan S., Natural and Enhanced Remediation Systems, Lewis Publishers, 2002.
  • Suthersan, Suthan S., Remediation Engineering, CRC net Base,1999.
  • Ryckman, Mark D., et. al., Enzyme helps Remediate at Lightning Speed, Soil and Groundwater Cleanup Magazine, February - March 1997.
  • Ryckman, Mark D., et.al., Cut Project Life Cycle Costs, Soil and Groundwater Cleanup Magazine, January - February 1996.
  • Enhanced Bioremediation with HC-2000, Remtech Engineers News Letter, Vol. 6, December, 2002.
  • HC-2000 Multi-Media Applications, Remtech Engineers News Letter, Vol. 9., November, 2004
  • Remtech Engineers, Confidential Client Project Report Files,1997- 2016.
  • Venosa, A., Literature Review on the Use of Commercial Bioremediation Agents for Cleanup of Oil-Contaminated Estuarine Environments, USEPA, National Risk Management Research Laboratory, Cincinnati, OH, July, 2004.

11. Cookson, John T., Bioremediation Engineering - Design & Application, McGraw-Hill, Inc., 1995.

12. Nikolopoullou, M., Kalogerakis, N., Enhanced Bioremediation of Crude Oil Utilizing Lipophilic Fertilizers Combined

with Biosurfactants and Molasses, Marine Pollution Bulletin 56 (2008) 1855-1861., Elsevier Ltd, 2008