BIOREMEDIATION OF PETROLEUM CONTAMINATED SOIL AND WATER Robert

BIOREMEDIATION OF PETROLEUM CONTAMINATED SOIL AND WATER Robert Rawson, President of Bioremediation International iws@sonic. net Alexis Gropper, lexgrop@gmail. com 1

Bob Rawson • President: International Wastewater Solutions Corporation, and Bio. Remediation International. • Director of Bio-Remediation Research for IOS-Corporation in Collaboration with CINVESTEV, and PRG Environmental Engineering, Mexico. • Inventor of US Patent No. 7, 658, 851 “AN APPARATUS AND METHOD FOR GROWING BACTERIA”, for use in wastewater treatment, biological pest management, bioremediation of soil and wastes. • Inventor of US Patent No. 8114659 “A DEVICE AND METHOD FOR THE CATALYTIC TREATMENT OF A MEDIA”. • Vice President: Northern California River Watch, Environmental Expert Witness for Clean Water Act, ESA, RCRA litigation. • Environmental Technology Instructor at Santa Rosa Junior College 27 years • Grade V Wastewater Treatment Operator 36 years Experience. 2

I wish to thank the Geographic Institute of Universidad San Francisco de Quito alongside El Frente, UDAPT, and everybody involved with organizing and participating in this program, “Reconstrucción Socio-Ecológica en el Norte de la Amazonía Ecuatoriana”. s 3

Review • Advantages of Bioremediation • International Success of IOS-500 Bacteria • Growing Methods and Applications of IOS-500 Bacteria • Non-pathogenic certifications of IOS-500 Bacteria • Multi-Kingdom Approach to Site Specific Treatments • Potential Proposals

Advantages of Bioremediation to Address Multiple Problems Efficiently Remediation efforts can serve multiple benefits: • Compensate the “Affected People” for the enormous health consequences that have been inflicted on them through: Healing environment, Providing Jobs, Economic Stimulation Optimizing the Labor and Capital needed to remediate the contaminated environment in favor of the affected communities, instead of strategies of highest economic gain distributed to few people.

Production Possibilities Curve to clean up the Petroleum contaminated The Y Axis of this Graph represents the money spent on Equipment, Chemicals and Energy required to clean up a given quantity of contaminated soil. OUTSIDE CAPITAL Greater Benefits Expanding out from Curves Q 0 to QL show greater Benefits like the Amount of Soil Bio-Remediated for a specific combination of Money as Capital and Labor. The X axis represents money paid to workers in the form of wages and benefits to clean up a given unit of contaminated soil. WAGES and LOCAL EMPLOYMENT

• BOBS SCHEME 7

Disadvantages of Conventional Remediation Methods • high chemical, mechanical and transportation costs • offsite and/or onsite pollution • not true remediation (burning, chemical, burying, relocating) 8

International Success of IOS-500 Bacteria on Petrol-Contaminated Soils • We will demonstrate several systems for growing and implementing non-toxic bacterial methods that are adequate for petroleum spills on land in various aquatic environments. 9

Santa Alejandra Swamp 11

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GROWING METHODS: Composting as Bacterial Farming • Utilizing bacteria in a composting process to treat waste products such as petroleum contaminated soil. 15

BP Cleanup Site in Egypt. 16

DILUTION! Bacteria and Carbon are the magic ingredients. It is necessary to dilute the concentration of contaminants below a level of 15% and provide Carbon mass to support bacterial survival and augmentation (Cow and Chicken manure are excellent supplements) 17

The Fire Triangle Analogy Critical components for the survival of aerobic Bacteria (like us we need food and oxygen to survive) 18

Why Bioremediation is an Art Composting microorganisms require the correct mix of carbon, nitrogen, oxygen and water to operate. The recipe: • • • Blending the correct ratios of raw materials during windrow formation is one of the key steps to successful composting. According to literature, the optimum recipe will have a carbon-to-nitrogen (C: N) ratio of between 20: 1 and 30: 1. This means there will be between 20– 30 units of carbon, by mass, for every unit of nitrogen present. The optimum moisture content of the recipe is 40 -60%. Feel is the best measure for determining proper moisture, because clay and sand hold different amounts of water and feel different in the hands. The materials must be blended or piled to allow for oxygen to infiltrate the pile — materials should not be compacted in a compost pile. Observations of poor practices by local contractors practicing in Lago Agrio 19

1) Dilution with Carbon sources and application of IOS-500 Bacteria 20

Example of Windrow layout and turning. 2) Aeration 21

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3) Taking the Temperature of the Pile 23

BP Cleanup Site in Egypt. 24

Windrow Composting Rows 25

WHAT ARE THE IOS-500 BACTERIA? ? 26

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IOS-500 TM contains • Pseudomonas species Breaks down chemical ring structures like phenol and nitrogenous compounds. Capable of plasmid transfer. (sharing information with other bacteria and borrowing from them) • Bacillus Species- Breaks down fats, oils, greases, and cellulose. 29

Aromatic Ring Compounds Benzene Naphthalene Anthracene Pyridine Quinoline Isoquinoline Pyrazine Quinoxaline Acridine Pyrimidine Quinazoline

Pseudomonas in the aerobic root zone of soil 31

Bacillus Subtilus Enzymes for bio-remediating lipids. 2. 4. Metabolism of lipids BG 10013 yyb. R; unknown; similar to ester hydrolase [SP: P 37486] BG 10305 bkd. B, bfm. B 2, bfm. BB, bkd; branched-chain alpha-keto acid dehydrogenase E 2 subunit (lipoamide acyltransferase) [EC: 2. 3. 1. -] [SP: P 37942] BG 10306 bkd. AB, bfm. B 1 b, bfm. BAB, bkd; branched-chain alpha-keto acid dehydrogenase E 1 subunit (2 oxoisovalerate dehydrogenase beta subunit) [EC: 1. 2. 4. 4] [SP: P 37941] BG 10307 bkd. AA, bfm. B 1 a, bfm. BAA, bkd; branched-chain alpha-keto acid dehydrogenase E 1 subunit (2 -oxoisovalerate dehydrogenase alpha subunit) [EC: 1. 2. 4. 4] [SP: P 37940] BG 10632 ywf. H, ipa-86 r; unknown; similar to 3 -oxoacyl-carrier protein reductase [SP: P 39644] BG 10646 glp. Q, ybe. D; glycerophosphoryl diester phosphodiesterase [EC: 3. 1. 4. 46] [SP: P 37965] BG 10679 lip, lip. A; extracellular lipase [EC: 3. 1. 1. 3] [SP: P 37957] BG 11012 pss. A, pss; phosphatidylserine synthase [EC: 2. 7. 8. 8] [SP: P 39823] BG 11013 psd; phosphatidylserine decarboxylase [EC: 4. 1. 1. 65] [SP: P 39822] BG 11040 dgk. A, yqx. F; diacylglycerol kinase [EC: 2. 7. 1. 107] [SP: P 19638] BG 11048 yox. D; unknown; similar to 3 -oxoacyl-carrier protein reductase [SP: P 14802] BG 11153 sco. A, yxj. D; probable succinyl Co. A: 3 -oxoacid Co. A-transferase (subunit A) [EC: 2. 8. 3. 5] [SP: P 42315] BG 11154 sco. B, yxj. E; probable succinyl Co. A: 3 -oxoacid Co. A-transferase (subunit B) [EC: 2. 8. 3. 5] [SP: P 42316] BG 11225 ycs. D; unknown; similar to hydroxymyristoyl-(acyl carrier protein) dehydratase [SP: P 42961] BG 11239 acd. A; acyl-Co. A dehydrogenase [EC: 1. 3. 99. -] [SP: P 45867] BG 11305 ywi. E; unknown; similar to cardiolipin synthetase [SP: P 45860] BG 11310 ywj. E; unknown; similar to cardiolipin synthetase [SP: P 45865] BG 11319 mmg. A, yqi. L; acetyl-Co. A acetyltransferase [EC: 2. 3. 1. 9] [SP: P 45855] BG 11320 mmg. B, yqi. M; 3 -hydroxybutyryl-Co. A dehydrogenase [EC: 1. 157] [SP: P 45856] BG 11321 mmg. C, yqi. N; acyl-Co. A dehydrogenase [EC: 1. 3. 99. -] [SP: P 45857] BG 11373 pgs. A, ymf. N; phosphatidylglycerophosphate synthase [EC: 2. 7. 8. 5] [SP: P 46322] BG 11383 acc. B, fab. E, yqh. W; acetyl-Co. A carboxylase (biotin carboxyl carrier subunit) [EC: 6. 4. 1. 2] [SP: P 49786] BG 11384 acc. C, yqh. X; acetyl-Co. A carboxylase (biotin carboxylase subunit) [EC: 6. 4. 1. 2] [SP: P 49787] BG 11417 ykh. A; unknown; similar to acyl-Co. A hydrolase [SP: P 49851] BG 11535 fab. G, ylp. F; beta-ketoacyl-acyl carrier protein reductase [EC: 1. 100] [SP: P 51831] BG 11536 acp. A, acp. P; acyl carrier protein [SP: P 80643] BG 11611 ugt. P, ypf. P; UDP-glucose diacylglycerol glucosyltransferase [SP: P 54166] BG 11701 yqh. M; unknown; similar to lipoate protein ligase [SP: P 54511] BG 11714 yqi. D; unknown; similar to geranyltransferase [SP: P 54383] BG 11719 yqi. K; unknown; similar to glycerophosphodiesterase [SP: P 54527] BG 11722 ptb, bkd, yqi. S; probable phosphate butyryltransferase [EC: 2. 3. 1. 19] [SP: P 54530] BG 11723 bcd, bkd, yqi. T; leucine dehydrogenase [SP: P 54531] BG 11724 buk, bkd, yqi. U; probable branched-chain fatty-acid kinase (butyrate kinase) [SP: P 54532] BG 11725 lpd. V, bkd, yqi. V; probable branched-chain alpha-keto acid dehydrogenase E 3 subunit (dihydrolipoamide dehydrogenase) [SP: P 54533] BG 11746 yqj. Q; unknown; similar to ketoacyl reductase [SP: P 54554] BG 11836 fab. D, ylp. E; malonyl Co. A-acyl carrier protein transacylase [EC: 2. 3. 1. 39] [SP: P 71019] BG 11843 pls. X, ylp. D; involved in fatty acid/phospholipid synthesis [SP: P 71018] BG 11946 lcf. A; long chain acyl-Co. A synthetase BG 11951 lip. B, yfi. P; extracellular esterase [EC: 3. 1. 1. 1] BG 12023 ycl. B; unknown; similar to phenylacrylic acid decarboxylase BG 12080 ydb. M; unknown; similar to butyryl-Co. A dehydrogenase BG 12089 acp. S, ydc. B; probable holo-acyl carrier protein synthase [SP: P 96618] BG 12143 yde. P; unknown; similar to cinnamoyl ester hydrolase BG 12221 fab. L, yfh. R, yga. A; enoyl-acyl carrier protein reductase [EC: 1. 3. 1. 9] BG 12241 yis. P, yuc. D; unknown; similar to phytoene synthase BG 12331 ysi. B; unknown; similar to 3 -hydroxbutyryl-Co. A dehydratase BG 12456 ywh. B; unknown; similar to 4 -oxalocrotonate tautomerase BG 12483 ywn. E; unknown; similar to cardiolipin synthase BG 12496 ywp. B; unknown; similar to hydroxymyristoyl-(acyl carrier protein) dehydratase BG 12557 acc. A; acetyl-Co. A carboxylase (alpha subunit) BG 12575 cds. A; phosphatidate cytidylyltransferase [EC: 2. 7. 7. 41] BG 12679 sqh. C; squalene-hopene cyclase [EC: 5. 4. 99. -] BG 12729 cyp. C, ybd. T; fatty acid beta-hydroxylating cytochrome P 450 BG 12809 ydz. F; unknown; similar to cinnamoyl ester hydrolase BG 12914 yfj. R; unknown; similar to 3 -hydroxyisobutyrate dehydrogenase BG 12994 yha. R; unknown; similar to enoyl Co. A hydratase BG 13021 yhd. O; unknown; similar to 1 acylglycerol-3 -phosphate O- acyltransferase BG 13029 yhd. W; unknown; similar to glycerophosphodiesterase BG 13048 fab. HB, yhf. B; beta-ketoacyl-acyl carrier protein synthase III [EC: 2. 3. 1. 41] BG 13055 yhf. J; unknown; similar to lipoate-protein ligase BG 13057 yhf. L; unknown; similar to long-chain fatty-acid-Co. A ligase BG 13063 yhf. S; unknown; similar to acetyl-Co. A C-acetyltransferase BG 13064 yhf. T; unknown; similar to long-chain fatty-acid-Co. A ligase BG 13127 fab. HA, yja. X; beta-ketoacyl-acyl carrier protein synthase III [EC: 2. 3. 1. 41] [SP: O 34746] BG 13128 fab. F, yja. Y; beta-ketoacyl-acyl carrier protein synthase II BG 13152 fab. I, yjb. W; enoyl-acyl carrier protein reductase [EC: 1. 3. 1. 9] [SP: P 54616] BG 13173 yjd. A, yid. A; unknown; similar to 3 -oxoacyl-carrier protein reductase BG 13328 ykw. C; unknown; similar to 3 -hydroxyisobutyrate dehydrogenase BG 13408 upp. S, ylu. A; probable undecaprenyl pyrophosphate synthetase [EC: 2. 5. 1. 31] [SP: O 31751] BG 13429 ymf. I; unknown; similar to 3 -oxoacyl-carrier protein reductase BG 13457 yng. F; unknown; similar to 3 -hydroxbutyryl-Co. A dehydratase BG 13458 yng. G; unknown; similar to hydroxymethylglutaryl-Co. A lyase BG 13460 yng. I; unknown; similar to long-chain acyl-Co. A synthetase BG 13461 yng. J; unknown; similar to butyryl-Co. A dehydrogenase BG 13518 des, yoc. E; membrane phospholipid desaturase BG 13523 yoc. J; unknown; similar to acyl-carrier protein phosphodiesterase BG 13544 yod. R; unknown; similar to butyrate-acetoacetate Co. A-transferase BG 13545 yod. S; unknown; similar to 3 -oxoadipate Co. A-transferase BG 13870 ytk. K; unknown; similar to 3 -oxoacyl-carrier protein reductase BG 13900 ytp. A; unknown; similar to lysophospholipase BG 13926 acc. D, ytt. I; acetyl-Co. A carboxylase (beta subunit) BG 14022 yus. J; unknown; similar to butyryl-Co. A dehydrogenase BG 14023 yus. K; unknown; similar to acetyl-Co. A C-acyltransferase BG 14024 yus. L; unknown; similar to 3 -hydroxyacyl-Co. A dehydrogenase BG 14029 yus. Q; unknown; similar to acyloate catabolism BG 14030 yus. R; unknown; similar to 3 -oxoacyl-carrier protein reductase BG 14031 yus. S; unknown; similar to 3 -oxoacyl-carrier protein reductase BG 14063 yva. G; unknown; similar to 3 -oxoacyl-carrier protein reductase BG 14144 yvr. D; unknown; similar to ketoacyl-carrier protein reductase 32

Growing and Application Methods of the IOS-500 33

IOS-500 microbes are grown with a White Knight inoculator contained inside a Nursery Tank such as pictured. A microbial inoculation and aeration device is depicted in this picture.

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Multi-Kingdom Applications to Site Specific Treatments • Bacteria (bio-remediation) • Mushrooms (myco-remediation) • Plants (phyto-remediation) Ecosystem pioneers and their roles in decomposition and distribution of the nutrient cycle 38

Possibilities with Mycoremediation Chemical structure of lignin in wood Chemical structures of petroleum

Documenting species thriving directly in oil spills

Taking it from Laboratory (ex-situ) to In-situ • In the laboratory • Controlled Experiments(Ex-Situ) • At a contaminated site (In-Situ)

Myco-Reactor Array

This conference has provided a unique collaboration between local, international and multi-disciplinary experts >> a conference of complimentary strategies We’ve seen so many examples of projects terminating prematurely due to various reasons, perhaps due to temporary international help leaving, or funds drying out… How do we guarantee the continuation of these social and environmental projects? Short term, Long term planning. 43

Potential proposals • Present here today we have universities, indigenous, regional organization leaders, municipalities-- each with their individual strengths. • A Communication platform to elaborate on the social and environmental problems to work to find the adequate solutions • Ideally, an institution where we can collectively train local leaders, organizations, students, professors in these multi-disciplinary themes and investigations • Ideally, a location for on-site remediation demonstrations and continued training Criteria: security, electricity, containment, capacity, credibility, etc. (potential sites like Municipality of Cascales where criteria are met) • Such pilot projects can provide important opportunity to organize our collective strategies in preparation for the larger scale remediation that will be possible when the Texaco Case is successfully concluded. 44

A Proposed Example of Stacking Multiple Functions that hypothetically could provide jobs for Affected Communities, and improve the economy. • Grow Enormous numbers of Bacteria using waste products in the manner that Alvaro Borja is in process of demonstrating at the City of Cascales and then using these bacteria to treat the landfill leachate to reduce the strength of the pollution being discharged to rivers. • These same bacteria can then be used in many other applications including: Bio-Remediation of Petroleum contaminated soil, and in agriculture to treat fungal and bacterial plant pathogens including Black Pod which is attacking the Cacao Crops, and other agricultural commodities. 45

• WE APPRECIATE YOUR PRESENCE, YOUR ATTENTION, AND YOUR FUTURE COLLABORATION OF IDEAS AND PROJECTS. Robert Rawson, President of Bioremediation International iws@sonic. net Alexis Gropper, lexgrop@gmail. com 46

Controlling temperature is important 47

Windrow pile construction. • 4 feet (1. 25 meters) or more high, by as much as 12 feet (3. 5 meters) across in layers of matrix such as manure or green chop and contaminated soil. I like a 9 foot high pile. • Consider Mass Effect in determining the size. More mass more heat and more moisture retention. Cold and warm climate are considerations. 48