Prepared by Gavin King, BluePlanet Consulting, Paarl, Western Cape, South Africa.

Page 2 : Introduction
Page 3 : Method
Page 5 : Results
Page 6 : Conclusions
Page 7 : Bibliography
Page 8 : Appendices

Introduction
Use of the vehicle workshop area at the Frito-Lay Simba Isando Plant has been discontinued due to outsourcing of the distribution chain. An area in front of the wash bay bounded by the workshops and boundary walls has been heavily contaminated by hydrocarbon pollutants. These pollutants were a mixed ranging of Petroleum and Diesel Organics (PRO and DRO). The total area contaminated covered approximately 300 square metres. The contamination occurred in three main areas, these being DRO in approximately 30 square metres under the removed diesel tank, a mix of DRO and PRO in approximately 120 square metres in front of the wash bay and an area of approximately 90 square metres of tarmacadam covered soil. The wash bay area was contaminated with a mixture of petroleum, diesel, oils, alkenes and kerosene. During initial sampling the average depth of contamination was found to be 150mm below the surface, and therefore a target depth for remediation was set at 250mm below the surface. In order to re-use this ground the Company needed to bioremediate the soil to a value below a Total Petroleum Hydrocarbon (TPH) value of 2000 mg/kg. This value was chosen based on the fact that the site was industrial and would not readily be used for agriculture or human occupation in the foreseeable future. Another factor affecting this target was that the Department of Water Affairs and Forestry (DWAF) had in the past recommended the use of a similar target for hydrocarbons at another industrial site (Snyman 1996).

The area was enclosed by concrete walls on three sides, with a brick workshop on the remaining side, creating a situation where In-Situ Bioremediation was the preferred method due to the following benefits:

No need from expensive removal of the contaminated soil for off-site clearing.
No need for a suitable dumping site for the concomitant soil expenses.
No expensive civil work to remove and replace the walls required.
No need for extensive replacement of soil and general site reconstruction.

Method
Due to Local EPA environmental audits, Company was looking speedy resolution to the contaminated soil problem. On an experimental basis, bioremediate was selected as the treatment protocol with a target of 12 weeks. While this is a short time span, but was considered appropriate for evaluation. In order to facilitate and create favorable conditions for the successful bioremediation of hydrocarbons in the soil, a number of factors needed to be considered:

The above conditions were satisfied using the following method:

Prior to the delivery of hardware and product, initial soil samples were taken as a baseline for the bioremediation and to ascertain the C:N:P ratio. An independent laboratory was used to analyze the soil samples. Two sampling areas were used determined an average value of the contaminated soil and a control sample of uncontaminated soil. The soil samples were taken by extracting a plug of soil which was then placed in a sample jar, inverted and kept at constant temperature in a polystyrene container until delivered to the laboratory.

The soil was then tilled to a depth of 250mm.

The initial values for the C:N:P ratio were given as 100:4:1 rounding to the nearest whole number. This value was deemed to be close enough to the required values of 100:5:1, therefore this ratio was not corrected with the addition of fertilizers.

An irrigation system comprising 25mm irrigation pipe was laid over the area. This consisted of a main pipe trunk line running along the west wall for 10 metres. This trunk main was fed via a 1kW water pump fed by a 60 litre drum. The drum was replenished from the water main using a regulator valve to maintain a constant volume of water in the drum. Five branch pipes were laid 2 metres apart, running at right angles from the trunk main eastwards across the contaminated area. Each 30 metre branch pipe was connected to the trunk via a tee piece and regulating valve. 360 degree irrigation spinners were fixed at 2 metre intervals along each branch pipe. This layout created an effective grid system of 2 metre squares fed from each irrigation spinner. The required flow rate of water was then manually set.

Once the required flow rate was confirmed by monitoring the soil moisture content for two days, ½ kg of AquaClean-HYDRO (See appendix for details) was spread evenly over the soil. This dosage of Aquaclean-HYDRO was repeated every 2 days from the inoculation date.

In order to dose the Aquaclean-ACF-32 (See appendix for details), 12 liters of AquaClean ACF-32 as an inoculation dose was poured into the 60 litre drum feeding the pump. The main water inflow to the drum created sufficient turbulence to mix the product with the water. This mixed product was then pumped into the irrigation system and evenly distributed via the 360 degree spinners. A quantity of spare spinners was kept available for replacement for any blocked spinners. These were replaced as and when required. An AquaClean ACF-32 dosage of 4 litres was then repeated every 2 days from the inoculation date.

This dosing regime ran for 44 days from 15 May 2010 to 28 July 2010. The total product utilized was therefore 11kg of AquaClean-HYDRO and 36 liters of AquaClean ACF-32.

The soil was then tilled to 250mm once every two weeks.

Results

The table below gives the results of the laboratory analysis.

Column 1 is the date the samples were taken.
Column 2 is the sample number.
Column 3 gives the TPH value for the sample taken in the contaminated area.
Column 4 gives the TPH value for the sample taken in the uncontaminated area.
Column 5 is the percentage reduction in TPH relative to Contaminated Sample 1.

The TPH values are shown graphically below.

It can be seen from Series 1 that the contamination was reduced from a initial TPH value of 17630 mg/kg to 16012 mg/kg (Sample 2) during the initial 3 weeks after inoculation. This reduction indicates that the bacterial consortium from the AquaClean ACF-32 had begun to take hold and establish itself in the soil. The growth of the consortium reaches a peak in the second month where the bacterial colony has grown to a level where the colony/nutrient source optimizes the oxidation of the hydrocarbons in the soil. Sample 3, at 1684 mg/kg, indicates a drop of over 90 percent and attainment of the goal This oxidation process continues at this level for a period of 4 weeks until the nutrient content supplied by the hydrocarbons has been depleted to the point where the bacterial colony begins to die off in relation to the depleted nutrient source.

During the final 3 weeks of the remediation the bacterial colony has once again reached equilibrium with the nutrient source, however at a much lower level. This is the expected outcome of the natural bell curve growth of organisms in the presence of a finite nutrient source.

Series 2 shows a small upward trend in the TPH value reaching approximately the same level of TPH value given by series 2. This upward trend is due to the action of the water/product mix creating a osmotic effect in the soil where some of the hydrocarbon contamination is spread throughout the treatment area.

It is shown in the results that the target TPH value of 2000 mg/kg was reached and exceeded in 10 weeks with the final value being 1681 mg/kg. This is seen to be a reduction of 90.465%.

Conclusions
Despite the short time frame given for the bioremediation trial of the hydrocarbons in the workshop area at Frito-Lay Simba-Isando, the AquaClean Methodology chosen for the in-situ reduction of hydrocarbon contamination in the soil has proven the efficacy of method. The required result of reducing the contamination to an acceptable level below 2000 mg/kg TPH was reached in 10 weeks. The method proved to be extremely cost efficient when compared to any other alternative, and very effective in meeting the remediation goals.

In short a successful bioremediation was achieved using the method as proposed by BluePlanet LLC and Ecological Laboratories. AquaClean ACF-32 and AquaClean-HYDRO has proven their ability to degrade the DRO and PRO Hydrocarbons.

This report provides an overview of the contamination issues found and remediated in the contaminated workshop area of Frito-Lay Simba-Isando. Every effort has been made to present the data based on the actual findings, and the conclusions are based on the total experience gained from the trial. Where research materials and white papers have been used in the preparation of this report and proposal, they are listed in the attached bibliography.

Credits

The author would like to credit the following for the assistance given during the entire process.

John Morrell – President of BluePlanet LLC for treatment plan and application.

Mark Kurpka – Technical Vice President of Ecological Laboratories for method design and analysis.

Lesiba Kgoogo – Frito-Lay Simba for on-site dosing of the product and the tilling of the soil.

Bibliography

The following white papers were used in the process and compiling of this report.

H.I.Atagana – Bioremediation of creosote-contaminated soil in South Africa by landfarming - School of Earth Sciences, Mangosuthu Technikon, Jacobs, Durban, South Africa - October 2003

M.J. Ayotamuno , R.N. Okparanma , E.K. Nweneka , S.O.T. Ogaji , S.D. Probert - Bioremediation of a sludge containing hydrocarbons - Agricultural and Environmental Engineering Department, Rivers State University of Science and Technology, School of Engineering, Cranfield University - May 2007

Reuben N. Okparanma, Josiah M. Ayotamuno and Peremelade P. Araka - Bioremediation of hydrocarbon contaminated-oil field drill-cuttings with bacterial isolates - Department of Agricultural and Environmental Engineering, Rivers State University of Science and Technology. - May, 2009

Okoh, A. I. and Trejo-Hernandez, M. R. - Remediation of petroleum hydrocarbon polluted systems: Exploiting the bioremediation strategies - Department of Biochemistry and Microbiology, University of Fort Hare. - December, 2006

Krishan Ramluckan - The Evaluation of Liquid Hydrocarbon Contamination of Soil around Petrochemical Tank Farms at a Durban Refinery – 2004

AQUACLEAN ACF-32 is a highly active liquid culture consortium designed specifically for use in all industrial and municipal wastewater systems. AQUACLEAN ACF-32 contains a blend of 12 select microorganisms with a specification of 387/450 million microorganisms/ml. AQUACLEAN ACF-32 cultures accelerate the biological oxidation of slow to degrade organic matter in aerobic, facultative anaerobic and anaerobic wastewater systems, ponds, lagoons and wastewater retention tanks. AQUACLEAN ACF-32 enhances the biological oxidation of slow to degrade organic compounds significantly improving overall system performance and stability.

AQUACLEAN ACF-32 select cultures promote increased biological degradation capabilities in all types and designs of biological wastewater systems. They increase the degradation rate of slow to degrade, difficult to degrade and many non-biodegradable compounds that have shown to negatively impact wastewater system performance. They resist high concentrations of inhibitory constituents; improve system performance, reduce system upsets and biomass kills common to industrial and municipal wastewater systems.

AQUACLEAN ACF-32 increases overall microbial oxidation rates, significantly increasing organic degradation performance. This novel microbial consortium provides reductions in final effluent BOD, COD, SS, and improves settling in final clarifiers, while reducing waste sludge volume in difficult to degrade compounds such as, fatty acids, various chemical compounds, hydrocarbons and fibrous matter.

Effects

AQUACLEAN ACF-32 contains purple sulfur and non-purple sulfur microorganisms capable of controlling biological gaseous reactions such as H2S and other associated biological gaseous reactions, as well as associated corrosion. Specifications:

Warning: Avoid excessive contact with skin, wash hands after use; may irritate eyes, flush with water for 15 minutes. Do not take internally.

AQUACLEAN ACF-32 contains naturally occurring Non-Toxic soil microorganisms, environmentally safe for wastewater systems; will not harm humans, animals or aquatic life.

AQUACLEAN/HYDRO is a highly active dry culture consortium design specifically for use in petrochemical, refining, power generation, coal gasification, textile, steel cocking and land farming facilities to enhance overall biological oxidation capabilities.

AQUACLEAN/HYDRO contains a blend of select microorganisms that are designed to accelerate the biological oxidation of slow to degrade, difficult and non-biodegradable compounds and to enhance the biological oxidation of hydrocarbon compounds, such as petroleum products.

AQUACLEAN/HYDRO - Contains microorganisms...Specifications:

Storage/Handling - Do Not Freeze, store in cool dry place

Warning: Avoid excessive contact with skin, wash hands after use; may irritate eyes, flush with water for 15 minutes. Do not take internally.

AQUACLEAN/HYDRO contains naturally occurring Non-Toxic soil microorganisms, environmentally safe for wastewater systems; will not harm humans, animals or aquatic life. See MSDS for product data. AQUACLEAN is designed for use in biological wastewater systems by environmental wastewater service professionals.

SECTION 1

SECTION II - HAZARDOUS INGREDIENTS

SECTION III - PHYSICAL INFORMATION

SECTION IV - RESULTS OF ANALYSIS