Bioremediation Services

Bioremediation Services

Altogen Labs has developed and manufactured several types of bioremediation products (soil-, site-, and environment-specific) and provide bioremediation services in USA and Canada. Our scientists developed fast, effective and safe bioremediation technology. We can provide quotes for price and timeline based on your bioremediation service project details (site location, type of spill, size). Please contact us at info@altogenlabs.com or call Altogen Labs technical support: 512-850-4740

Altogen Labs provides large-scale bioremediation services for soil contaminated with: crude oil, pesticides, herbicides, perchlorate, vinyl chloride (VC), carbon tetrachloride, chloroform, PAHs, petroleum, acetone, benzene, methyl ethyl ketone, toluene, xylenes, perchloroethylene (PCE), trichloroethylene (TCE), dichloroethylene (DCE), chlorofluorocarbon (CFC), aliphatic chlorinated hydrocarbons, other industrial and chemical manufacturing wastes and spills.

Our bioremediation services include:

– Sample collection and analysis
– Spill evaluation and characterization of environmental parameters
– Design of optimized bioremediation program
– Manufacture of site-specific bioremediation product (large-scale)
– Field services to apply bioremediation product
– Analysis (sample biomarker) and evaluations
– Report results

Altogen Labs developed effective, site-specific, and safe bioremediation technology based on accelerating bioremediation process by re-introducing large number of natural bacteria to the contamination site. Technology is optimized for variables in temperature, environmental conditions, and type of contaminant providing highly effective tool for bioremediation of large amounts of polluted soil and liquids.

Bioremediation services process:

Education and Training
Our professionally trained staff will assist in the training and understanding of the methodology associated with bioremediation techniques. Guidance is provided to ensure the viability and activity of the microorganism applied to the site. Troubleshooting and problem solving for successful bioremediation is also available.

Application of Products
Application of the product is determined upon site specificity and will be thoroughly examined. Our staff provides guidance or will assist in the application of the product upon request of the client.

Biostimulation Services
Biostimulation services are provided to optimize the effectiveness of the product by supplying the microorganisms with the necessary nutrients. Our trained staff will determine the need for biostimulation services in combination with our products based on site specifications.

 

Bioremediation Agent Effectiveness Test

Bioremediation technologies (bacteria, yeast, fungi) are based on accelerating the degradation of hydrocarbons by re-introducing large numbers of naturally occurring bacteria to a crude oil contamination site. Microbial agents are optimized for variables such as temperature, environmental conditions and type of contaminant, thus, providing a highly effective tool for environmental bioremediation of large amounts of polluted soil and liquids.

Altogen Labs offers comprehensive environmental services in evaluation of a developed bioremediation agent.  In order to be utilized in an oil cleanup, all products must be listed on the National Contingency Protocol (NCP) as required by the Comprehensive Environmental Response, Compensation, and Liability Act of 1980.  Upon the client’s data submission to the U.S. EPA, the submitter will receive a decision within 60 days if the product will be added to the NCP Product Schedule.

The Bioremediation Agent Effectiveness Test protocol determines a microbial agent’s ability to degrade oil.  Hydrocarbons (alkanes and aromatics) are measured by GC/MS and final data is presented as a percentage of remaining hydrocarbons in the test sample relative to the control sample. An accompanying required test is analysis to determine microbial agent viability during the effectiveness testing protocol.  The Most Probable Number (MPN) method is used as a microbial enumeration determination, or indication of microbial response, of viable microbial agents within the samples at all time points, which is an indicator of change in biomass relative to the control sample.

Altogen Labs will perform the EPA mandated Bioremediation Agent Effectiveness Test according to Subchapter J, Appendix C to Part 300.  As described in Appendix C to part 300, the resultant test results (GC/MS analysis and microbial viability) and supporting data will be submitted by the client to the NCP Product Schedule Manager.

Customer responsibilities:

  • Company contact information
  • Primary distributors or sales outlets of proposed bioremediation agent
  • Special handling, storage, additives and other necessary information
  • Application procedures
  • Physical properties

Altogen Labs responsibilities:

  • Chemical Analysis
  • Gravimetric Analysis
  • Microbiological Analysis

Bioremediation Agent Effectiveness Test Protocol

Briefly, the sample preparation procedure includes an initial incubation of seawater plus test agent. Microbiological analysis (viability) of microbial agents are tested on Day 0, 7 and 28 in triplicates, and organics are extracted into dichloromethane (DCM) with a subsequent solvent exchange into hexane for chemical and gravimetric analysis via GC/MS. Below is a summary of the procedure as reported in Subpart J, Appendix C to Part 300 for bioremediation agents.

 

Bioremediation Agent Effectiveness Test Protocol

The Bioremediation Agent Effectiveness Test includes an initial incubation of seawater plus test agent, followed by microbiological analysis of microbial agent viability and a chemical analysis of the organic compounds extracted into a solvent for analysis via GC/MS. Below is a summary of the procedure as reported in Subpart J, Appendix C to Part 300 for bioremediation agents.

Experimental Setup

  1. Sterile Borosilicate glass Erlenmeyer flasks (250–mL) are labelled with the appropriate sample information: control/nutrient/product, sample day, and letter indicating replicate.
  2. 100 mL of seawater is added to each flask. For nutrient and product treatments that require the addition of nutrients, seawater containing the nutrient solution is prepared.
  3. The labelled flasks containing seawater and other nutrients (as necessary) are placed on the balance and the flask is tared. The appropriate amount of oil (0.5 g) is added drop by drop using a sterile, large bore Pasteur pipette. Care is taken to avoid splashing the oil or getting it on the sides of flasks. Precautions are taken when handling the flasks to minimize the likelihood of contamination by exogenous microbes. This includes using a new sterile pipette for each series of flasks.
  4. The weight of the oil is recorded in the laboratory notebook.
  5. The test product is prepared and added to the appropriate flasks according to the manufacturer’s or vendor’s instructions.
  6. Flasks are carried upright and carefully placed in the holders on the shaker table to minimize the amount of oil that might adhere to the side of the flasks. Flasks in which a significant amount of oil is splashed on the sides are discarded and reassembled.
  7. The prepared flasks are shaken at 200 rpm at 20°C until such time that they will be removed for sampling.
    1. Sampling: The control and treatments (nutrient and product flasks) are sampled three times over a 28-day period: day 0, day 7, and day 28. The entire flask is sacrificed for analysis; a 0.5 mL aliquot is removed from each flask for the Microbiological Analysis and the remainder of each flask is used for the Chemical Analysis. At the time of each sampling event, physical observations of each flask will be recorded.

Microbial Analysis

  1. 5 mL of water from each flask is removed and added it to a tube of 4.5 mL sterile phosphate buffer (1:10 dilution). Using sterile technique, mix and perform serial dilutions (0.5 mL of previous dilution to 4.5 ml of sterile phosphate buffer) to 10-9 dilution.
  2. Using sterile technique, 1.75 mL of BH broth is added to each well.
  3. 1 mL of fluid from each dilution tube is added to the appropriate well, starting with the most dilute sample.
  4. After transferring all dilutions, 20 μL of sterilized No. 2 fuel oil is added to the top of each well.
  5. The plates are incubated at 20°C.
  6. After 14 days of incubation, 100 μL of p-iodotetrazolium violet dye (50 mg/10 ml of D.I. water) is added to each well to determine growth.
  7. Plates are viewed against a white background to determine if color is present. Development of a purple or pink color upon standing for 45 minutes constitutes a positive test.
  8. The number of positive wells and the dilutions at which they occur are recorded.
  9. Data is entered into a computerized enumeration method using ‘‘MPN Calculator’’ software program (version 2.3 or higher) by Albert J. Klee, U.S. EPA Office of Research and Development, Risk Reduction Engineering Laboratory, Cincinnati, OH.

Chemical Analysis

  1. After 0, 7, and 28 days of rotary shaking and incubating at 20 °C, the reaction vessels are sacrificed. Prior to the chemical analysis, a 0.5 mL sample of the aqueous phase is removed for the microbiological analysis (see Microbial Analysis above).
  2. A surrogate recovery standard is prepared in the following manner: 1,000 mg of d10-phenanthrene and 1,000 mg of 5α-androstane are measured into a 500 mL volumetric flask and DCM is added to the mark to produce a 2,000 ng/μL stock solution.
  3. A 100 μL aliquot of the surrogate solution (from step 2) is added to each test flask. The final concentration of surrogates in each flask is approximately 4 ng/μl of solvent in the final extract. The aliphatics and marker data should be corrected for percent recovery of the 5α-androstane surrogate and the aromatics for the d10-phenanthrene surrogate.
  4. The contents of the flask are placed into a 250 mL separatory funnel.
  5. A total volume of 50 mL DCM is measured for use in the extraction process. Three 10 mL fractions of DCM is used to rinse the flask into the funnel and the remaining aliquot of DCM is transferred to the funnel.
  6. The separatory funnel is stoppered and mixed vigorously by shaking (approximately 50 times) while ventilating properly.
  7. Each funnel is set aside to allow the DCM and water layers to partition. This may take 5–10 minutes for some products, or up to 3 hours if the product has caused the formation of an emulsion.
  8. The first 10 mL of the DCM (bottom) layer is drained, capped, uniquely labelled, and used for gravimetric analysis (see below). The remaining 40 mL of the DCM layer is drained and dried by passage through an anhydrous sodium sulfate column.
  9. The DCM from step 8 is concentrated using a Kuderna-Danish (KD) concentrator by attaching a Snyder column to an evaporation flask and graduated concentrator tube. The setup is aligned vertically with concentrator tube partially immersed in a water bath. The water bath is set to the appropriate temperature to maintain proper distillation.
  10. The de-watered extract is collected into the KD concentrator.
  11. DCM is evaporated to approximately 10 mL, then approximately 50 mL of the exchange solvent (hexane) is added and the volume is concentrated to 10 mL.
  12. The flask is rinsed into the concentrator tube with 50 mL hexane and concentrated to 10 mL. This is repeated one more time with 50 mL of hexane.
  13. The concentrator tube with the recovered 10 mL of sample volume is removed from the apparatus. The heavier residual material may be present as a precipitate (bottom layer).
  14. The precipitate fraction is centrifuged to aid the separation of the hexane.
  15. The hexane-soluble fraction (top layer)—approximately 1.0 ml—is placed into a GC/MS vial for analysis.
  16. The samples are analyzed by GC/MS using the conditions determined by the U.S. EPA; U.S. EPA Method 8270.
  17. The remaining seawater is drained into a storage sample vial/container, sealed and stored frozen. This water layer is kept in case additional extractions are necessary.

Gravimetric Analysis

  1. The 10 mL of DCM extract (from Sample Procedure step 8 in Chemical Analysis above) is placed in a small vial and concentrated to dryness by nitrogen blowdown techniques using a steady stream of nitrogen (pre-purified gas). If the oil is severely biodegraded, a larger volume of DCM (>10 mL) may be necessary for the gravimetric analysis.
  2. The residue is weighed 3 times for the gravimetric weight of the oil and recorded.
  3. Statistically (p <0.05), the weight of the product treatment versus the weight of the control from each respective time period is compared. If a significant decrease in weight is observed in the treatment flask (indicating degradation of the crude oil), then proceed with the GC/MS portion of the Chemical Analysis.

Determination of the presence of the following within the applied Microbial Agent:

  • Salmonella, Fecal Coliform, Shigella, Staphylococcus Coagulase positive, and Beta Hemolytic Streptococci

see also:
Bioremediation Products | Oil-degrading Bacteria | Bioremediation Services