CAP 18 ME Anaerobic Bioremediation Product TECHNICAL BRIEF

Copyright 2010 rev. 1/14 form RX 1634

The information contained herein is accurate to the best of our knowledge. However, data, safety standards and government regulations are subject to change; and the conditions of handling, use or misuse of the product are beyond our control. Carus Corporation makes no warranty, either expressed or implied, including any warranties of merchantability and fitness for a particular purpose. Carus also disclaims all liability for reliance on the completeness or confirming accuracy of any information included herein. Users should satisfy themselves that they are aware of all current data relevant to their particular use(s).

Carus and Design is a registered service mark of Carus Corporation. CAP 18® and CAP 18 ME® are registered trademarks of Carus Corporation. The product CAP 18 ME listed here is covered by U.S Patent No. 8,608,968 (issued December 17, 2013). Responsible Care® is a registered service mark of the American Chemistry Council.

C A P 1 8 M E ® A n a e r o b i c B i o r e m e d i a t i o n P r o d u c t : A N e w S u b s t r a t e f o r S t i m u l a t i n g A n a e r o b i c B i o d e g r a d a t i o n T E C H N I C A L B R I E F

C A P 1 8 M E ® A n a e r o b i c B i o r e m e d i a t i o n P r o d u c t

INTRODUCTION CAP 18® anaerobic bioremediation product, an unemulsified oil is utilized to stimulate anaerobic biodegredation. Some of the key properties of CAP

18 include: low cost, easy application, effectiveness, and a long lifetime. The very long lifetime of CAP 18 in the subsurface (which has been several

years in all current case studies) is driven by the low solubility of the triacylglycerols that comprise CAP 18. The triacylglycerols slowly hydrolyze to

release free fatty acids, which are metabolized to produce hydrogen for reductive dechlorination and other processes (Figure 1).

However, the low solubility characteristic can also result in slow onset of strongly reducing conditions over a period of several months in the field,

but which are then maintained for years (Figure 2).

Many sites undergo a phased remedy design and application process beginning with a laboratory microcosm test, followed by a field pilot test, before

proceeding with a full-scale remedy. A general expectation is that the microcosm test and the field pilot test should be completed within a few months

for each phase, in order to make timely decisions on site remedies. Due to the lower solubility of CAP 18 relative to other substrates, results of

side-by-side lab and field tests conducted over a period of a few months may not result in adequate and appropriate data to evaluate CAP 18 as more

or less effective than other more soluble substrates. This is because CAP 18 may not generate as strongly reducing conditions in as quickly a period

of time. The benefits of the very long lifetime of the CAP 18, in terms of much lower lifecycle costs due to fewer re-injections, cannot be easily

evaluated in the relatively short monitoring periods of laboratory microcosms or field pilot tests.

Fatty Acid GlycerolHydrolysis

Fatty Acid

Fatty Acid

Fatty Acid

Fatty Acid

Fatty Acid

Microbial

Utilization β-oxidation,Fermentation,

Other Processes

Triacylglycerol

FIGURE 1. Schematic of triacylglycerol utilization to produce hydrogen .

-300

-250

-200

-150

-100

-50

0

ORP

(mV)

0

1

2

3

4

5

6

Fe(II) a

nd Me

thane

(mg/L)

Fe(II)Methane

Injection Event

0

20

40

60

80

100

120

11/5/2003 5/23/2004 12/9/2004 6/27/2005 1/13/2006 8/1/2006

TOC

(mg/L

)

FIGURE 2. Example pilot test results using CAP 18, exhibiting controlled,long-term substrate release resulting in progressively more anaerobic conditions, and persistence for nearly three years (to the most recentsampling event).






ESTERIFICATION PROCESS

Different formulations of CAP 18® anaerobic bioremediation product, comprising different source oil blends and additives, were tested to determine

if the initial onset of strongly reducing conditions at sites treated with CAP 18 could be accelerated. Formation of emulsions with various compounds

was not desirable, due to the tendency of emulsions to be more rapidly consumed and for the emulsion to break apart in the subsurface after

placement. Most other additives are immiscible with CAP 18. However, fatty acid methyl esters are miscible with CAP 18. Previous workers have

found that methyl esters readily support biodegradation. The glycerol backbone is already cleaved and thus the very slow hydrolysis step (Figure 1) is

avoided, resulting in more rapid availability of the fatty acids for microbial utilization. The fatty acid methyl esters used to produce CAP 18 ME®

anaerobic bioremediation product are prepared from the original CAP 18 formulation and thus have the same beneficial, long-chain (C18) unsaturated

fatty acids. Thus addition of methyl esters to the CAP 18 was evaluated with laboratory and field testing to determine if the blended product resulted

in a more rapid onset of reductive dechlorination.

The fatty acid esters are formed by reacting CAP 18 with sodium methoxide (prepared from sodium hydroxide and methanol). This cleaves the

triacylglycerol molecule and the free fatty acids react with methyl alcohol to produce a fatty acid methyl ester. The methyl esters are then separated

from the glycerin and purified. The methyl esters are blended with CAP 18 and then transferred to drums or totes for storage and shipment. The

liquids are miscible; there is no separation of the products over time. The resulting blend is stable indefinitely as long as the product is kept under

normal temperature conditions and out of direct sunlight. The blended product is CAP 18 ME.

Laboratory microcosm tests were conducted to evaluate relative treatment efficiency of CAP 18 and CAP 18 ME. Laboratory tests were performed

by an independent laboratory. The microcosms were prepared by loading 250 mL glass bottles with 200 mL of anaerobic mineral salt medium, and 1

mL of a water saturated with trichloroethylene (TCE) to reach a target TCE concentration of approximately 5 mg/L. The bottles were capped with

leak-tight closures to allow repetitive sampling of the bottle with minimal volatile organic compound (VOC) loss and to allow amendment addition, if

needed. All microcosms were constructed in duplicate. The microcosms were sampled and incubated in a covered, anaerobic chamber. The bottles

were placed horizontally to minimize VOC losses through the closure. Microcosms were incubated for a period of up to 99 days at room temperature

(22ºC).

CAP 18 and CAP 18 ME were tested concurrently. Two sets of duplicate microcosms were prepared for each formulation. One set of microcosms

for each formulation was bioaugmented and the other set of microcosms was not bioaugmented (as a control). Microcosms were bioaugmented on

Day 0 to a target Dehalococcoides concentration of 106 cells per liter. Then on Day 1, 100 µL of CAP 18 or CAP 18 ME was added to the respective

microcosms, corresponding to a dosage of 0.05% CAP 18 or CAP 18 ME on a volume basis.

The results from two tests, highlighting the onset of reductive dechlorination based upon the initial degradation of TCE, are presented in Figure 3. In

both tests, TCE degradation began more quickly with CAP 18 ME than with CAP 18. In Test 1, TCE was degraded approximately 50% faster with CAP

18 ME than with CAP 18, while in Test 2 the difference was smaller. The average degradation rates (k) for the two tests were 0.34 day-1

(corresponding to a half-life of 2.1 days) for CAP 18, and 0.70 day-1 (corresponding to a half-life of 1.1 days) for CAP 18 ME.

MICROCOSM TESTING






After the first 10-20 days, the differences between the degradation rates for CAP 18® and CAP 18 ME® anaerobic bioremediation products are less

clear. Results for formation and degradation of cis-1,2-dichloroethylene (DCE) are presented in Figure 4 (only Test 2 was continued long enough for

complete cis-1,2-DCE degradation). The evolution of cis-1,2-DCE concentrations in the microcosms was very similar, except that the final degradation

occurred more rapidly with CAP 18 ME than with CAP 18.

Test 2

0.0

0.2

0.4

0.6

0.8

1.0

0 10 20 30 40 50 60 70Days

Norm

al Co

ncen

tratio

n (C/C

o)

CAP18CAP18-ME

Test 1

0.0

0.2

0.4

0.6

0.8

1.0

0 3 6 9 12 15Days

Norm

al Co

ncen

tratio

n (C/C

o)

CAP18CAP18-ME

Test 2

0.0

0.2

0.4

0.6

0.8

1.0

0 3 6 9 12 15Days

Norm

al Co

ncen

tratio

n (C/C

o)

CAP18CAP18-ME

FIGURE 3. Laboratory microcosm test results for TCE degradation.

FIGURE 4. Laboratory microcosm test results for cis-1, 2-DCE degradation.






Results for vinyl chloride and ethene are presented in Figure 5. Initially the CAP 18 ME® anaerobic bioremediation product microcosms exhibit much

faster vinyl chloride degradation rate than the CAP 18® anaerobic bioremediation product microcosms, which is coupled with an earlier achievement

of peak ethene concentrations.

Based upon the promising laboratory microcosm tests results, CAP 18 ME was injected at six sites for pilot testing purposes. The first injections were

conducted in July 2006. Initial results are available for one site, at which CAP 18 ME was applied to augment an earlier CAP 18 injection. The site

(Figure 6) is located in northern Indiana. The treatment zone is from the water table at 4.6 m below grade to 9.1 m below grade. VOC concentrations

are generally low, with maximum onsite concentrations of 0.1 mg/L perchloroethylene (PCE) and 0.099 mg/L cis-DCE. Concentrations within the

treatment area (monitoring well (MW)-5S and MW-7) are lower. The initial design objective for the CAP 18 injection was to construct a treatment

barrier to address the groundwater plume before it migrates offsite. Approximately 3,773 kg of CAP 18 were injected in 14 points arranged in two

21 m long barriers on each side of a public street in November 2004, and monitoring wells MW-5S and MW-7 were utilized as monitoring locations.

MW-5S initially exhibited decreased TCE concentration and detectable but very low concentrations of cis-DCE (maximum of 0.018 mg/L), while

MW-7 exhibited a variable TCE concentration but the first detections of cis-DCE approximately 11 months after injection. Since that time, TCE

concentrations stabilized in MW-5S and cis-DCE dropped to non-detectable, and in MW-7 the TCE has remained variable while cis-DCE remained

elevated (Table 1). Potential explanations were that the CAP 18 was not hydrolyzing sufficiently rapidly to generate strongly reducing conditions, or

that the injection point density was insufficient. Both factors could be overcome by CAP 18 ME.

Test 2

0.0

0.2

0.4

0.6

0.8

1.0

0 20 40 60 80 100 120 140Days

Nor

mal

Con

cent

ratio

n (C

/Co)

CAP18CAP18-ME

Test 2

0.0

0.2

0.4

0.6

0.8

1.0

0 20 40 60 80 100 120 140Days

Nor

mal

Con

cent

ratio

n (C

/Co) CAP18

CAP18-ME

Vinyl Chloride

Ethene

Ethene

FIGURE 5. Laboratory microcosm test results for vinyl chloride and ethene.

FIELD PILOT TESTING






In September 2006, 193 kg of CAP 18 ME® anaerobic bioremediation product was injected via five injection points located upgradient of MW-5S

(Figure 6). The objective was to evaluate if the injection point density in the barriers was insufficient to address the plume, and if the CAP 18 ME

resulted in more rapid degradation of the VOCs. Two quarterly sampling events have been conducted since the September 2006 injection event. At

MW-5S, PCE has decreased to 0.031 mg/L from 0.070 mg/L, and cis-DCE has increased to 0.0083 mg/L from <0.001 mg/L (Table 2). TCE remains

undetectable.

FIGURE 6. Site 2 layout map showing injection points for CAP 18 (green triangles) and CAP 18 ME (red triangles).






Laboratory and field applications of CAP 18® anaerobic bioremediation product have demonstrated that the product is long-lasting, easy to apply, and

relatively low cost compared to other substrates. The long lifetime of the CAP 18 is due to the slow hydrolysis reaction that cleaves the triacylglycerol

compound and releases free fatty acids, which in turn are utilized for microbial metabolism. However, a trade-off of the slow hydrolysis and long

lifetime is a slow onset of strongly reducing conditions. Within the typical monitoring period of a field pilot test, CAP 18 was often perceived as less

effective than other substrates because results were not apparent within a few months; the much longer lifetime of the less soluble product was often

not evaluated or considered. Rapid achievement of reducing conditions was given more value than overall project costs based on the mass of hydrogen

released per pound of product injected, the cost of equipment and labor for injection, and the maintenance of mechanical recirculation systems. CAP

18 ME® anaerobic bioremediation product was developed to overcome the obstacle of achieving rapid reducing conditions, while simultaneously

preserving all of the cost and performance benefits of CAP 18.

CAP 18 ME is a blend of CAP 18 with fatty acid methyl esters derived from CAP 18. The fatty acid methyl esters provide a more readily available

supply of substrate to support microbial metabolism and to stimulate reductive dechlorination. Laboratory microcosm tests have confirmed that the

onset of TCE degradation occurs as much as 50% faster with CAP 18 ME than with CAP 18. Field pilot tests have just recently been initiated. Results

for one field pilot, application of CAP 18 ME substantially accelerated PCE degradation in a biobarrier application. Overall, CAP 18 ME appears to

accelerate the onset of biodegradation without sacrificing the long lifetime, cost advantages, or ease of application provided by CAP 18.

CONCLUSIONS

PCE TCE cis-DCE VC Ethene Ethane Methane TOC

12/14/2006 0.031 <0.001 0.0083 <0.001 <0.001 <0.00092 0.019 NS

9/29/2006 0.062 <0.001 0.0019 <0.001 <0.01 <0.01 0.026 NS

5/26/2006 0.07 <0.001 <0.001 <0.001 <0.01 <0.01 0.20 NS

5/4/2006 0.045 <0.001 <0.001 <0.001 <0.01 <0.01 0.20 NS

1/12/2006 0.042 <0.001 <0.001 <0.001 <0.01 <0.01 <0.01 23

10/7/2005 0.031 <0.001 <0.001 <0.001 <0.01 <0.01 <0.01 18

7/26/2005 0.089 <0.002 0.00487 <0.002 <0.00001 <0.00001 0.0014 25.3

4/8/2005 0.1 <0.001 0.018 <0.001 <0.01 <0.01 <0.01 29

4/16/2004 NS NS NS NS NS NS NS NS

12/14/2006 0.066 0.003 0.0065 <0.001 <0.01 <0.01 0.220 NS

9/29/2006 0.062 <0.001 0.0019 <0.001 <0.01 <0.01 0.026 NS

5/4/2006 0.0041 <0.001 0.099 <0.001 <0.01 <0.01 1.30 NS

1/12/2006 0.0026 <0.001 0.14 <0.001 NS NS NS NS

10/7/2005 0.07 <0.001 0.0016 <0.001 <0.01 <0.01 0.04 21

7/26/2005 0.038 <0.005 <0.005 <0.005 <0.00001 <0.00001 0.051 28

4/8/2005 0.05 <0.001 <0.001 <0.001 <0.01 <0.01 0.01 32

4/16/2004 0.033 <0.005 <0.005 <0.002 NS NS NS NS

MW-5S

MW-7

Location / Date

TABLE 1. Site 2 Groundwater Data (Concentrations in mg/L)

Note: Only data for MW-5S and MW-7 are provided because the other wells are not in the treatment zone. NS indicates not sampled.

Documents

CAP 18 ME Anaerobic Bioremediation Product TECHNICAL BRIEF