Bioremediation of PCB-Contaminated Soil:

An Arctic Case Study

Todd Adamsson

A thesis submitted to the Department of Chemicai Engineering

in conformity with the requirernents for

the degree of Master of Science (Engineering)

Queen's University

Kingston, Ontario, Canada

May, 1998

copyright C3 Todd losef Adamsson, 1998

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This research was conducted to assess the feasibility of remediating polychlorinated biphenyl

contaminated soil in northem Labrador using bactena. Studies were conducted on severd scales

of soil ranging fiom 20 grarns to 3 tonnes, and on both aerobic and anaerobic metabolic

processes.

The anaerobic process requires an environment with a redox potential of approximately -400mV.

It was found that the redox potential of containenzed saturated soil codd be significantly

reduced by stimulating indigenous oxygen scavenging organisms through the addition of

nutrients and a labile carbon substrate. Redox potentials were reduced to - 175mV in plastic pails

without proper seds or gas traps. An anaerobic culture was successfully introduced into this

system.

The aerobic degradation of Aroclor 1260 could be stirnulated in fieshly excavated soils solely by

ensuring oxygen availability and maintaining moisture at 40-60% of the water holding capacity

of the soil. Biopiles containhg Aroclor 1260 contaminated soil at a concentration of 200ppm

exhibited reductions of 20% during a six month incubation at 20°C.

Degradation could be M e r enhanced by amending the soil with nitrogen and phosphorous at a

ratio of 5: 1. At ratios lower than this, fertilization either had no effect or a negative impact.

Three groups of microorganisms are required to completely mineralize PCB into water, carbon

dioxide, and chloride ion. Al1 three groups are indigenous to the contaminated site in Labrador.

Although undesirable microbial succession occurred in stored soils, the less effective PCB

degraders which survived could be stimulated by the addition of finely milled sphagnum moss.

Addition of peat to fieshly excavated soils resulted in disrupting healthy bacterial populations

due to a reduction in pH.

Addition of metabolic inducers slightiy stimulated the degradation of PCB to chlorobenzoic acid

and chlorinated aiiphatic acid. However, the inducers proved to be toxic to the other species of

bacteria capable of rnineralizing these intermediates.

Acknowledgments

The author would like to acknowledge the collaboiative effort responsible for the research

presented in this thesis, and extend gratitude to some individuals which made the project not only

possible, but enjoyable.

Dr. Juliana Ramsay, as my thesis advisor, was dways willing to entertain new ideas, and provide

guidance and insight.

Dr. Ken Reimer of the Environmentai Sciences Group introduced this project to Queen's, and

made it possible to conduct field studies at the contaminated site in Labrador.

Allison Rutter, Stephen D m , Paula Whitney, Veronique Goffaux, and Cindy Cowan of the

Analytical Services Units at RMC and Queen's for providing PCB analysis.

Dr. Lyle Whyte and Luc Bourbonniere of the Biotechnology Research lnstitute aided in

preparing the I4c study and monitored it for six rnonths.

Jason Stow of the Environmental Sciences Group was the team leader of the site assessment and

clean-up in 1997, and provided support in terms of both logistics and personnel.

Members of the 1996 and 1997 Saglek site assessment team assisted in the labour intensive tasks

related to manuai excavation and sieving of soil. Special thanks are due to Zouzou K u y k who

filled the long days on Antenna Hill with her limitless enthusiasm.

- -

Table of Contents

1 . Introduction

2 . Literature review

2.1 Polychlorinated biphenyl

2.2 General biological requirements

2.3 Aerobic metabolism

2.4 Anaerobic metabolism

2.5 Site Assessrnent

2.6 Bioremediation implementation factors

2.7 Bioremediation techniques

2 .7 . 1 Phytoremediation

2 . 7 . 2 Landfarmhg

2.7.3 Windrows

2 . 7 . 4 Soii sluny reactor

2 . 7 - 5 Two-step remediation

3. Methods: Aerobic sîudies

3 . 1 2-CB mineralization microcosm study

3 .2 Aroclor 1260 degradation microcosm study

3 . 3 4kg biopile study

3 - 4 2.4kg biopile study

3 - 5 Moisme-nutrierît-peat optimization study

3 .6 Aqueous phase inducer assessrnent

3 - 7 Bacterial identification using API@ test strips

3 8 Soi1 bacteria enmeration

3 . 9 PCB d y s i s

4. Methods: Anaerobic studies

4 .1 Anaerobic inoculation test

4 .2 Anaerobic biopile study

4.3 Small scale deoxygenation study

4.4 Large scale deoxygenation study

5. Resdts: Aerobic studies

5.1 Introduction

5.2 Controls

5.3 Nutrient studies

5.4 Bullcing agent s u e s

5.5 Soil phase inducer studies

5.6 Aqueous phase inducer study

5.7 Preferential degradation of specific congeners

5.8 Mineralization of PCB

5.9 Soil texture

5.1 0 Microbial populations

5.1 1 Soil pH

5.1 2 Inoculation

5.13 Moisture-nutrient-peat optimization

6. Resdts: haerobic studies

6.1 Introduction

6.2 Redox potential manipulation

6.3 PCB degradation

6.4 Inoculation

7. Conclusions

References

Vita

List of Tables

Table 2.1 : Aroclor products and their respective homologs.

Table 3.1 : Experimental design of moisture-nutrient-peat optimization shidy.

Table 3.2 Inducers studied in aqueous phase inducer assessment.

Table 4.1 : Experimental design of small scde deoxygenation study.

Table 5.1 : Aroclor 1260 degradation observed in 2.4kg biopile study.

Table 5.2: Relative populations of soi1 microorganisms after 1.5 months and 5 months of incubation at 5 ' ~ (Aroclor 1260 rnicrocosm study).

Table 5.3: Soil pH of Aroclor 1260 rnicrocosms after 5 months of incubation at 5OC.

Table 5.4: Soil pH of 1997 biopiles after 18 days, 3 1 days, and 180 days of incubation at 20°C.

Table 6.1 : Redox potentials of anaembic biopiles after 5 months of incubation.

Table 6.2: Summary of amendments added to each treatrnent in the 1 kg soi1 deoxygenation study and the time to oxygen depletion.

List of Figures

Figure 2.1 : Some polychlorinated biphenyl molecules.

Figure 2.2: The biphenyVPCB upper metabolic pathway.

Figure 5.1 : 2-CB mineralization observed in 20g microcosms incubated at 5C (nutrient s tudy ).

Figure 5.2: Aroclor 1260 degradation observed in 5Og microcosms incubated at SC (nutrient study).

Figure 5.3 : koc lo r 1260 degradation observed in 4kg biopiles incubated for six months at 20C (nutrient study).

Figure 5.4: 2-CB mineralization observed in 20g microcosms incubated at 5C (bullcing agent study).

Figure 5.5 : Aroclor 1260 degradation O bserved in 50g microcosms incubated at SC (bulking agent study).

Figure 5.6: Aroclor 1260 degradation observed in 4kg biopiles incubated for six months at 20C (buiking agent study).

Figure 5.7: Aroclor 1260 degradation observed in 2.4kg biopiles incubated for eleven months at 20C (bulking agent study).

Figure 5.8: 2-CB mineralization observed in 20g rnicrocosms incubated at 5C (inducer stud y).

Figure 5.9: Aroclor 1260 degradation observed in 50g microcosms incubated at SC (inducer study).

Figure 5.10: Aroclor 1260 degradation observed in 4kg biopiies incubated for six months at 20C (inducer study).

Figure 5.1 1 : Linear inhibition of Aroclor 122 1 degradation due to biphenyl, arbutin, and naringin addition in aqueous culture.

Figure 5.12: Non-linear inhibition effects of vanillic acid, cinnamic acid and coumarin on the degradation of Aroclor 1221 in aqueous culture.

vii

Figure 5.13: Aroclor 122 1 removal kinetics for the controls and hwo inducer amended 5 1 (3 -25 mmoUL) liquid cultures.

Figure 5.14: Preferential depletion of specific congenen of Aroclor 122 1. 52

Figure 5.15: Preferential depletion of specific congeners of Aroclor 1260. 53

Figure 5.16: Aroclor 1260 degradation observed in 2.4kg biopiles incubated for eleven months at 20C (inoculum study). 6 1

Figure 5.17: Results of optimization study. 62

Figure 6.1 : Aroclor 1260 losses observed in anaerobic biopiles incubated for six 68 rnonths at 20C.

viii

1. Introduction

Soils at former military installations across northem Canada are contaminated with

polychlorinated biphenyls (PCBs). As PCBs are a prionty pollutant under the Canadian

Environmental Protection Act (CEPA), al1 soil contamimted with the compound at a

concentration greater than 50ppm m u t be excavated and treated. Considering the

remoteness of the sites and the quantities of soil to be treated, excavation and off-site

remediation would introduce an enormous, and possibly unjustifiable cost;

irnplementation of an effective on-site remediation technique would be the preferred

strategy. Requirements for clean-up include: prevention of fûrther migration of

contaminants, minimization of technical complexity to reduce heavy equipment

requirements, and selection of either a rapid process or a stable, self regulating process to

reduce on-site labour demands. Bioremediation is one option that satisfies the site

requirements.

Bioremediation is a process which stimulates 2 target biotic community capable of

reducing organic contaminant concen~ations by transfonning them into non-toxic

inorganic compounds such as water and carbon dioxide. Petroleum contaminated soils

and water are commonly treated using microorganisms, and applications are rapidly

expanding to include sites contaminated with more recalcitrant compounds such as PCBs

(Harkness et al., 1993). Bacteria capable of metabolizing PCBs are ubiquitous in the

environment, and the aerobic degradation pathway has been extensively studied and

documented. Although bacteria have been the focus of most of the technical reports to

date, some fungi are capabte of detoxifiing the contaminant, and vascular plants may also

play a role by enhancing bacteriai degradation rates.

Bioremediation can be implemented in a wide variety of ways ranging from simple

fertilization to inoculating an agitated soi1 slurry reactor. As the degree of aggressiveness

is increased, degradation rates, extent of removal, and process reliability improve at the

expense of energy and maintenance requirements. Selection of the technique best suited

to a particular site is based on bdancing site limitations and clean-up requirements

against process effectiveness and eficiency.

The research presented in this thesis was undertaken to assess the feasibility of applying

bioremediation at a long range radar site (LAB-2) at Saglek Met, Labrador. Experiments

were conducted at severai scales ranging fiom strictiy controlled microcosms containing

20g of soi1 to biopiles containing 4kg of soil. Strategies for stimulating indigenous

bactena included addition of nutrients, bulking agents, and metabolic kducers.

2. Literature review

2.1 Polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are a class of compounds that span a wide range of

physical properties depending on the degree of chlorination. A PCB molecde is

comprised of a biphenyl skeleton to which one or more chlorine atoms are substituted at a

hydrogen position. Figure 2.1 illustrates the structure of biphenyl as well as some PCBs.

biphenyl a rnonochlorinated a trichlorinated a pentachlorinated biphenyl biphenyl biphenyI

Figure 2.1 : Some polychlorinated biphenyl moIecules.

Although there are 209 theoretically possible variations, or congeners, only about 100

actually exist. Stenc hindrance prevents the formation of the others. PCBs with the same

number of substituted chlorine atoms, but diffenng o d y in the position of the chlorines

are referred to as isomes, and the entire family of such isomers (i.e. mono-CB, di-CB,

tri-CB, etc.) are called homologs.

Despite two decades of investigation and widespread public concem about these

compounds, their toxicity is still under scrutiny. In some studies, tumors have developed

in the livers of rats which were fed Aroclor 1260 for two yean, suggesting that PCBs

may be carcinogenic (Kimbrough et al., 1995). Clinical reviews of individuals exposed

to the chernical in industrial settings, however, concluded that the only adverse health

effects attributable to high occupational PCB exposures are dermai (James et al., 1994).

The degree of toxicity of the different congeners varies, and ïncreases with the degree of

chlorination and proportion of meta- and para-substituted constituents (Tanabe, 1989).

This group of compounds was produced by direct chlorination of biphenyl in the presence

of a femc chioride andfor iodine catalyst which resulted in a mixture of ail congeners

(Huntzinger et al., 1983). The industrial product was M e r refined by hctional

distillation to produce mixtures of dozens of congeners that roughly correspond to the

different homologs. The main producer of industrial PCBs in North Amenca was

Monsanto. The mixtures were sold under the trade name Aroclor. The Aroclor produ

were designated by four digits: the first two digits, 12, refer to twelve carbon atoms per

molecule, and the last two digits refer to the percentage of chlorine in the mixture. Table

2.1 lists several of the Aroclor products.

Table 2.1 : Aroclor products and their respective homologs. The mixtures ody roughly

correspond to the homologs, and overlap of congeners exists between closely related

mixtures.

Aroclor 1221 1?32 1242 1248 1254 1260

Homolog mono- di- tri- tetra- penta- hexa-

oily b

honey-Iike l iquid grease

About 6 . 5 ~ 105 rnetric tonnes of PCBs were manufactured before the late 1970s, when

woridwide production was ail but terminated. It is estimated that over a quarter of this

has been released into the environment (Abramowicz et al., 1995). PCBs are

hydrophobie, sorb strongly to soil matrices, and biodegrade very slowly in the

environment. Due to these characteristics, the likely fates of PCBs include persistence at

the contaminated site, bioaccumdation, and accumulation in sediments where site runoff

water collecîs.

2.2 General biological requirements

To properly apply bioremediation, it is first necessary to have a basic understanding of

microbial requirements. With this knowledge, an implementation strategy c m be devised

which rnaximizes ce11 growth rates to quickly obtain a healthy, dense population. Once a

stable microbiai cornmunity is established, the process requires little intervention.

Altîough supplying nutrients and favorable environmental conditions are essentiai for

bioremediation, understanding the desired metabolic pathway gives insight into the

requirements of the population which will degrade the specific contaminant and often

suggests methods by which the degrading microbes can be selectively enriched. When

the nutrient requirements of the species and the specific requirements of the metaboiic

pathway have been ascertained practical amendments can be selected and the proper

quantities determined. The so iVamendmen t mixture can then be incubated under

environmental conditions (pH, kmperature, moisture) conducive to the growth of the

target species.

To establish a healthy microbial community, basic requirements must be met. These

include: nutrients which act as raw material for the production of biomass, an electron

transfer pathway to provide the necessary energy for biomass production, and an

environment in which the cells can survive and reproduce.

The main nutritional requirements are carbon, nitrogen and phosphorous although other

compounds such as sulfur, potassium and iron are also needed in relatively srnail

quantities. In nature, these compounds are derived fiom decaying organic matter and

minerals present in soil. Vascular plants can also provide sugars and amino acids in the

rhizosphere. These nutrients are incorporated into the cells as proteins, fats and

carbohydrates. In this way the parent ce11 grows until it divides to produce two daughter

cells. In addition to acting as the building blocks of the cells, the nutrients can ais0 be

used to produce enzymes, some of which act as catalysts in the reactions which break

down PCBs into more biodegradable compounds, and eventually to carbon dioxide, water

and chloride ions.

The production of biomass and enzymes fiom nutrients requires energy. Al1 non-

photosynthesizing biota obtain energy by enzymatically catalyzing exothermic redox

reactions in which an electron is transferred firom one compound to another. The energy

released by the process is captured by the cells in the bonds of special energy storage

molecules such as ATP and NADH. The storage molecules cm then be transported

within the ce11 to a site where energy is required the bond is then enzymatically broken,

and the liberated energy can be used for metabolism. Microbial processes are commonly

divided into two categories based on the terminal electron acceptor: aerobic processes

which use oxygen as the electron acceptor, and anaerobic processes which use oxidized

compounds such as femc iron or sulfate. Electron acceptors and donors should be

supplied to the biomass at a sufficient rate to ensure microbial activity is limited only by

the metabolic rate of the species. Both aerobic (Ahrned and Focht., 1973) and anaerobic

(Quensen et al, 1988) bacterial cultures capable of PCB degradation have been

discovered,

Once nutrients have been provided and an electron transfer route established, the

microorganisms require an environment in which they can survive and perform their

metabolic activities. Adequate incubation conditions span a broad range of humidity,

temperature and pH depending on the species of bacteria. For aerobic

chemoheterotrophs, maximum eficiency can be expected at a pH between 6 and 8 and

where the soi1 is well drained. The optimal temperature is species dependent, and could

be as low as SOC if the microbes are psychrophiles, or around 3 0 ' ~ if the microbes are

mesophiles (Brock, 1997).

2.3 Aerobic metabotism

The aerobic degradation of PCBs was first documented by Ahrned and Focht in 1973

(Ahmed and Focht, 1973), and research since then has illuminated many of the details of

the process (Funikawa et al., 1977) as well as methods by which it can be stimulated and

improved (Dercova et al., 1995; Fava and Grassi, 1996; Barriault and Sylvestre, 1993).

Aerobic degradation has also been observed in the environment (Flanagan and May,

1993). The main advantage of aerobic PCB degradation is that it c m lead to complete

mineraiization of the compound. A major drawback however, is that the degradation

rates are congener specific. The lowly chlorinated compounds can be degraded relatively

quickly, whereas the highly chlorinated congeners are more recalcitrant.

The upper metabolic pathway is illustrated in Figure 2.2. The rate limiting step is

generally considered to be the dioxygenation of the arornatic nucleus, which is catalyzed

by biphenyl dioxygenase, an enzyme system encoded by the bphA genes (Focht, 1995).

Biphenyl dioxygenase is not a constitutive enzyme; it is only produced by the ce11 in the

presence of an inducer. In laboratory studies, biphenyl has traditionally been used as the

inducer, but the ubiquitous nature of this gene combined with the fact that biphenyl is

rarely found in the environment suggests that the natural inducer of the bph gene is some

other compound. It has been hypothesized that the natural inducers of the pathway are

phenolic compounds which are produced by vascular plants (Domeily et al., 1994). The

cells produce the enzymes with these compounds as the intended carbon source, and the

PCBs are fortuitously cometabolized. Metabolism via the upper pathway results in the

transformation of PCBs to chlorobenzoates and five carbon aliphatic acids (Focht, 1995).

Bacteria which possess the genes required to perform the upper metabolic pathway do not

usually possess the required genes to complete the lower metabolic pathway: the

mineralization process (Hernandez et al., 1995). The intemediates can usually be

H NAD+ NADH + H' H H,O C W H

OHPDA

Figure 2.2: The biphenyl/PCB upper rnetabolic pathway.

degraded by other species of soi1 bactena (Hemandez et ai, 1999, and mixed cultures

capable of mineralking PCB often develop at long-time contaminated sites.

2.4 Anaerobic metabolisrn

The anaerobic dechlorination of PCBs was first suggested by observed shifts in congener

profiles in long-time contaminated sediments from the Hudson River. It has also been

observed at Silver Lake, the Sheboygan River, Waukegan Harbour, and the Hoosic River

(Bedard and Quensen, 1995). The process is much slower than the aerobic one, and

results in the depletion of highly chlorinated congeners with a cornmensurate increase in

the quantity of Iowly chlorinated congeners.

The cultures responsible for dechlonnating PCBs are il1 defined, but are believed to be

mixed populations of sulfate reducing bactena that utilize PCBs as an altemate electron

acceptor in the absence of sulfate. As an electron is hansferred from the carbon source to

the PCB, a chlonde ion is released, usually fiom a meta or para position. Several

dechlorination processes have been observed both in the environment and in laboratory

studies. The processes are designated M, Q, H', H, P, and N, and differ in congener

selectivity. It is currently believed that the difEerent processes are performed by different

consortia (Bedard and Quensen, 1995). The anaerobic process is most effective at

dechlorinating highly chlorinated congenen.

As the PCB is not consumed by the microorganisms, a carbon source must be provided to

satisfy the nutritional requirements of the population and allow for biornass production.

2.5 Site assessrnent

Site characteristics and soil properties play an integral role in the rernediation selection

process. Site-specific assessments are especially important for bioremediation as the

selection of suitable amenciments and determination of quantities required are dependent

on nutritional and environmental conditions of the existing contaminated soil.

The climate at these remote Arctic sites is harsh with long cold winters, reducing the time

in which bioremediation can be reliably applied. On-site, abandoned structures or

module greenhouses could provide a more controlled environment in which the

remediation season can be extended.

Although there is low biodiversity among the sparse vascular plant community, Pua

arctica (a grass species) and Asfiagalus alpinus (a legume) are both native to Saglek, and

c m potentially be used for phytoremediation application.

Characteristics of the soi1 itself also restrict which implementation strategies are

applicable to the site. The first major observation that cm be stated about the site on

Antema Hill at Saglek is that the soi1 is densely packed. This fact has a two-fold affect

on rnicrobial populations, and, to some extent, may explain the persistence of typically

biodegradable compounds, such as diesel, at the site. In addition to preventing oxygen

fiom reachuig subsurface soils, min is not able to penetrate the ground, but runs off as

surface water. Due to the arid climate, even if the soil was less packed one would expect

it to be relatively dry during most of the year. Arctic soils are also characteristically low

in nitrogen and organic matter.

Bioavailability of the contaminant is ofien a rate limiting factor at long-the

contaminated sites (Sugiura, 1992). Over decades of weathering the more soluble, lowly

chiorinated congeners may be preferentially biodegraded or washed fiom the site while

the remaining contaminants migrate into the mineral ma& of the soil particles. The

compounds sorbed to the exterior of the particles or dissolved in the mineral oil layer

surroundhg the particles may be degraded quickly, but the trapped molecules must first

migrate out of the mineral matrix, a very slow process, before the microorganisms have

access to them.

2.6 Bioremediation implernentation factors

Mauitainïng soil moisture is one of the main strategies to foster a microbial population.

If a solid-phase treatment is selected, action m u t be taken to eiûure that the soil is not

allowed to become dry at any point; otherwise the bacterial cornmunity will have to be

reestablished. Irrigation systems are usually used for land famiing and windrows, and

addition of water-absorbent organic matter such as milled peat moss would improve

water retention.

The low nutrient concen&itions characteristic of tundra soils may necessitate the addition

of nitrogen and phosphorous. Water soluble fertilizers are commercidly available in

slow to rapid release foms.

The soil pH at the sites is close to neutrai, which is the desired set point. As metabolites

are produced however, the soil can be expected to slowly become acidic. Monitoring soil

pH and making adjustments by adding a liming agent may be a maintenance requirement

of the system.

Most soil bacteria attach themselves to a solid support and grow as biofilms to control

their own microenvironment and facilitate propagation. Recalcitrant plant matter may

provide a support preferable to the minera1 soil particles. Adding an amendment such as

peat moss or wood chips may allow for denser microbial populations.

The aerobic pathway requires an inducer to stimulate biphenyl dioxygenase production.

Althcugh biphenyl has been demonstrated to be an effective inducer (Yagi et al, 1980;

Focht and Brumer, 1985; Harkness et al, 1992), uniform application is difficult due to its

low water solubility. If it can be shown that plant-produced phenolic compounds are as

effective as biphenyl, they may provide a more practical, and more acceptable alternative.

The mode by which the soil should be aerated is detennined by the remediation strategy

used. Periodic tilling of the soil is effective for land farrning applications, and aeration

can be improved through the use of bulking agents. Biopiles often make use of forced

aeration through a plumbing system. In a soil slurry reactor, air or oxygen is sparged into

the system at the reactor base.

Anaerobic dechlorination processes require the addition of an easiiy degradable carbon

substrate. As PCB is not assimilated into biomass, the added carbon serves as both an

electron donor and matenal for biomass production. Pyruvate (Moms et al., 1992),

glucose, acetate (Nies and Vogel, 1990) and soluble starch are potential amendments.

Anaerobic dechlorination only occurs at redox potentials less than about -400mV (Bedard

and Quensen, 1995). If these conditions cannot be achieved by incubating the soil with

an easily degradable carbon source until aerobic organisms scavenge the oxygen, a

reducing agent can be used to bring the system to the desired redox potential.

Penodic inoculation of the soils aid in maintaining dense microbial populations, and

ensuring that undesired microbial succession does not occur.

In addition to providing aeration in aerobic processes, d g would homogenize the soi1

matrix and al10 w for more uniform degradation.

2.7 Bioremediation techniques

The process of selecting a bioremediation option that will be irnplemented at a particular

site entails determining which techniques will be effective at the site, ranking the

alternatives according to energy and labour demands, and nnally choosing one option

based on cleanup requirements and time constraints. The first option which should be

examined is intrinsic remediation, or the "do nothing approach", in which contaminant

concentrations are tracked over time to determine the efficacy of the naturd ecosystem to

reduce pollutant concentrations. As the site has been contaminated for over two decades,

and concentrations are still above regulated levels, this is not a viable option at Saglek.

The next technique which should be explored is Ni-situ biostimulation. This technique

has the advantage that excavation is not required, but due to the high soi1 density, low

porosity and contaminant depth, this option is also not feasible. The following is a list of

remediation strategies ordered f b m Ieast to most energy intensive, or dtematively, fiom

ieast to most reliable. Large scale implementation of PCB bioremediation has not been

attempted using any of the following techniques. Although pilot studies would have to

be conducted to determine the actual rates of degradation and the extent of pollutant

removal which could be achieved, it is reasonable to assume that process behavior would

Vary depending on the remediation scheme used.

2.7.1 Phytoremediation

Phytoremediation is an innovative technology which uses vascular plants to aid in the

stabilization and proliferation of bacterial cultures in the rhizosphere. It has been

demonstrated to significantly improve degradation rates of recalcitrant compounds such

as PAHs (Rooney et al., 1995) and m T (Watanabe, 1997), but application to PCB

contamuiated soils is not documented in prominent joumals.

In general, the macrophytes aid rnicrobiai populations by improving water retention,

aeration and nutrient concentrations. As the roots grow and protrude downwards, the

sunoundhg soi1 is loosened. This allows for the transport of both water and air into the

sub-surface. In addition to this, some species of vascular plants are able to actively

translocate oxygen fiom the atmosphere, through the stem, into the root zone. Most

plants exude amino acids and other metabolites through their roots which can act as

nutrients for microbial populations. Legurnes, such as Astrugalus alpinus (Burt, 199 1)are

especially promising as potential arctic phytoremediation species due to the symbiotic

relationship they form with bacteria capable of fixing atmospheric nitrogen and

converting it into an assimilable nutrient. The plants would also produce phenolic

compounds which may act as inducers of PCB degradation (O'Connel1 et al., 1996).

Application would be limited to sites where the depth of contamination is less than about

30cm, as arctic plants typically have shallow root systems.

The plants would be introduced to the location and culhwd until their community

becomes stable. At this point, the system would be self regulating, and, if the engineered

ecosystem survives the winten, d l that remains is to occasionally monitor PCB

concentrations unûl they meet site cleanup requirements.

If phytoremediation were used at Saglek, a barrier would have to be constmcted to ensure

that migratory caribou do not graze at the site and introduce PCBs into the food chain.

2.7.2 Land f m i n g

Land farming is another low maintenance remediation strategy. It is a proven, cost

effective technology for many recaicitrant compounds, and several land farrning

companies are operational in Canada.

Implementation involves excavation of the soil and containment within a bermed

structure. The treatment ce11 is lined with an impermeable membrane to prevent the

transport of contarninants, metabolites and nutrients out of the system in run-off water.

Nutrients and other amenciments are added to the soil, and the system can be inocdated.

Occasionai watering and tilling are required to maintain sufficient moisture and aeration.

Nutxitional composition and soil pH should be rnonitored and adjusted as required.

Although the technique can be applied outdoors, shelter will Lengthen the remediation

season as well as provide irnproved process control.

2.7.3 Windrows

A windrow is an altemate soil phase remediation strategy. Amenciments and nutrients are

added to the soil as they would be in a land f m i n g application, but instead of spreading

the soil to a depth of up to two feet, the soil is piled into windrows which can be as hi&

as ten feet. This method has the advantages that less area is required and the system is

betier insuiated against the environment. Oxygen addition can be irnproved by

occasionally tuming the windrows with a fiont-end Ioader or via a forced aeration system,

which increases technical complexity and energy demands.

2.7.4 Soil slurry reactor

Soil slurry reactors are more reliable and more easily controlled than subsaturated soil

remediation strategies, but energy requirements, technical complexity and costs are

greatly increased. The soil is mixed with water in a ratio of approximately 1 :3, and the

slurry can be agitated either in a rotating d m or through the use of impellen. If aerobic

degradation is desired, oxygen can be spaïged into the base of the system. Automated

systems are comnonly used to achieve strict environmental control so process efficiency

can be maxirnized.

2.7.5 Two-step remediation

To avoïd d e degradation limitations imposed by the highly c h i o ~ a t e d congenen, a N o -

step process can be applied. Anaerobic biotransformation or a chemical dechlorination

process can fïrst be used to reduce the degree of chlorination. The treated soil,

contarninated with lowly chlorinated PCBs and biphenyl, can then be reliabiy treated

using a low maintenance aerobic procedure such as land farrning.

3. Methods: Aerobic studies

3.1 2-C B muieralization microcosm study

This study was conducted at the Biotechnology Research Institute in Montreal, and

consisted of eight distinct 20g microcosms each prepared in ûiplicate. Amendments

which were tested include nutrients, peat, and potential induces. Soi1 was spiked with 14 C labeled 2-chlorobiphenyl, and production of CO^ was monitored for six months.

The soil was obtained fiorn Antema Hill at Saglek and stored at 4 ' ~ for eight months

until use. The soil was passed through a #10 sieve to remove particles larger than 0.25cm

to reduce sampling error. M e r sieving, the PCB concentration was 85ppm Aroclor 1260.

Abiotic controls were prepared by adding l%(w/w) AgN03 to the soil followed by

autoclaving for 20 minutes at 120'~. After being sieved, only water was added to the soil

used for the biotic controls.

1.04kg of 4%(w/w) moist soil, the equivalent of 1.00kg dry soil, was amended wirh 5ml

of a nutrient solution containing 2 . 8 6 ~ NH4N03, 0.88g KH2P04 and 0.1 3g MgSO,. The

solution was added in lm1 portions, and mixed with the soi1 on a tray. The soil was then

placed in a g l a s cylindrical vesse1 and roll mixed for two hours to ensure homogeneity.

This soil was used in the nutrient, peat and inducer studies.

Microcosms were prepared in 1251111 s e m vials. Each via1 received 20g of the

appropriate soil, and the moisture content was adjusted to 70% of the water holding

capacity of the soil. Amendments were then added and mixed into the soil. A small test

tube containing an aqueous potasium hydroxide solution, as a CO2 solvent, was placed in

each vid before closing with Teflon-lined caps and sealing with aluminum crirnp seals.

Ortho-chlorobiphenyl, spiked with a trace quantity of radiolabeled 2-CB, was added to

the soil through the septum using a syringe to a concentration of IOppm, correspondkg to

100,000dpm (disintegrations per minute). The vids were incubated at SOC for six

months. Al1 microcosms were prepared in triplicate. Production of 14c02 was

determined by removing a small amount of the KOH solution through the septum using a

syringe, and quantifihg the dpm of the solution using a scintillation counter.

a) Nutrient shidy

The nutrient study consisted of an abiotic control, a biotic control, and a nutrient

amended microcosm. The object of the study was to detemine if indigenous PCB-

degrading microflora could be stimuiated solely by nutrient addition. The final nutrient

concentration was 0.5g available nitrogen (as ammonium ion), 0.2g phosphorous (as

phosphate ion), and 0.035g sulfur (as sulfate ion) per I kg dry, sieved soil.

b) Peat study

The object of the peat study was to determine if the presence of a bulking agent further

stimulated the indigenous bacteria over nutrient amendment alone. The study consisted

of an abiotic control, a biotic control, a nutrient amended microcosm, and a peat and

nutrient amended microcosm. The milied peat moss was obtained fiom a garden centre

in Kingston, and was further milled using a blender to disperse aggregates. The peat and

nutrient amended microcosms were prepared by placing nutrient amended soils into the

triplkate vids in addition to 0.4g of the fmeiy milled peat moss to achieve a peat

concentration of 2%(w/w). Vial contents were homogenized by roll mixing pnor to

addition of 14c-2c~.

c) Inducer study

The inducer study consisted of seven microcosms: an abiotic control, a biotic control, a

nutrient amended rnicrocosm, and four nutrient amended microcosms which were

supplemected with inducers: biphenyl at 1 OOppm and 1000ppm and cimamic acid and

quercetin at 1000pprn. Inducers were added fiom stock solutions of the compounds in

acetone, and the acetone was allowed to evaporate before the vials were roll mixed and

sealed.

3.2 Aroclor 1260 degradation rnicrocosm study

This study consisted of six distinct 50g microcosrns each prepared in triplicate. It was

run in parallei with the mineralization study conducted at BRI. Radioactive compounds

permanently contaminate gas chromatograph coliuiuis. This study allowed for

determination Aroclor 1260 transformation.

The same soil used in the mineralization study was used in this study. Soi1 was prepared

as stated in Section 3.1 with the exception of the abiotic controls. A chernical

sterilization agent was not used, but soils were autoclaved for 20 minutes at 120°C on two

successive days.

Microcosms were prepared by placing 50g of the appropriate soil in flat-walled, 200d

glass vials with screw caps. Amendments were added, tap water was used to b ~ g the

moisture content to 70% of the water holding capacity of the soil, and the via1 contents

were homogenized by roll mixing. The vials were incubated on their sides to maximize

the soiVair interface; soil depth was approximately 0.5cm. Incubation was at S O C for six

months, and al1 microcosms were prepared in triplicate.

a) Nutrient study

The nutrient study consisted of an abiotic control, a biotic control, and a nutrient

amended microcosm. The object of the study was to determine if indigenms microflora

could be stimulated solely by nutrient addition. The final nutrient concentration was 0.5g

available nitrogen (as ammonium ion), 0.2g phosphorous (as phosphate ion), and 0.035g

sulfûr (as sulfate ion) per Ikg dry, sieved soil.

b) Peat study

The object of this study was to determine if the use of a bulking agent would M e r

stimulate indigenous bacteria over nutrient amendment done. The study consisted of an

abiotic control, a biotic control, a nutrient amended microcosm, a nutrient and peat

amended microcosm, and an autoclaved peat amended microcosm. The nutrient and peat

amended microcosm was prepared by adding 1 g of finely milled peat moss to nutrient

amended soil to achieve a bulking agent concentration of 2%(w/w). The sterile peat

amended microcosm was prepared by autoclaving a 2%(w/w) mixture of peat in soil for

20 minutes at 120'~ on two successive days.

c) Inducer study

An inducer study was conducted to determine if biphenyl addition to fertilized soils could

stimulate indigenous bacteria to a greater degree than fertilization done. Biphenyl was

added to nutrient amended soils in an acetone stock solution, and the acetone was ailowed

to evaporate before the vials were seaied. Biphenyl was added to achieve a final

concentration of 1000ppm.

3.3 4kg biopile study

This biopile shidy was conducted in the technical service module (TSM) at Saglek. It

consisted of ten distinct 4kg biopiles each prepared in triplicate. The amendments tested

in this experiment include nutrients, four bulking agents, and three potential induces.

The soil was obtained from Antenna Hill at Saglek. It was sieved though a 5/16" sieve to

reduce sampling error, and roll mked in a 205L barre1 to achieve homogenization. The

soil concentration of ArocIor 1260 after being sieved was 195ppm.

The biopiles were prepared in 45cmx30crnx 15cm plastic basins. Each biopile consisted

of 4kg of soil with the appropriate amendments. The three replicates of each biopile were

stacked on one another, and incubated at 2 6 " ~ for six months. The moinire content of

the soil was adjusted to approximately 50% of the water holding capacity of the soil when

required.

The abiotic controls were sterilized with a 4.0% sodium hypochlorite bleach solution,

whereas the biotic controls received only water. At the commencement of the experiment

2OOrnl of the appropriate liquid was added to the soils. M e r 1 I days, an additional 50ml

was added, and two more measures of 2001111 each were added on days 25 and 3 1. The

biopiles were sealed with shrink wrap in September to reduce moisture loss until

February 1998 when they were again sampled.

a) Nutrient study

Soils used in the nutrient amended biopiles were supplemented with 200 ml of medium

when the study was begun. M e r 1 1 days, 50ml was added, and two more measures of

200ml each were added on days 25 and 3 1. This corresponds to a total available nitrogen

(as ammonium ion) concen~ation of 0.09g/kg, and a total phosphorous concentration (as

phosphate ion) of 0.4gkg.

The medium was prepared by diluting 77.5ml of PA concentrate and lOml of PAS salts

concentrate in 1L of tap water. The PA concentrate consisted of 57g/l K2HP04, 22gA

KH,P04, and 28gA N H Q The PAS salts concentrate consisted of 19.5gA MgSO,,

5.0g/l MnSO,.H,O, 1 .Og/l FeS0,.7H20, and 0.3gA CaCI2.2H2O.

b) Bulking agent study

Four bulking agents were tested for their ability to stimulate aerobic PCB degraders:

rnilled peat rnoss, PearliteQ, arctic bluegrass (Poa Arctica) flowers and Aminoplast@.

The AminoplastB biopile had no replicates. n i e milled peat moss and Pear l i td were

purchased in Kingston at a garden supply store, the Aminoplast@ was obtained as a test

sample, and the arctic bluegrass was collected on-site at Saglek. Peat, pearlite, and

bluegrass were added at a concentration of l%(w/w), and Aminoplast was added at a

concentration of 0.2%(w/w). In addition to the bullcing agents, these soils were arnended

with the nutrient solution on the sarne schedule as the nutrient amended biopiles.

c) Inducer study

The inducer study consisted of four distinct biopiles: a nutrient and peat arnended biopile,

and three nutrient and peat amended biopiles which were nipplernented with either

biphenyl, quercetin, or coumarin. Approxirnately 1 g of the suspected inducer was mixed

into the soils at the commencement of the experiment, and three more additions of l g

each were made on days 1 1,25, and 3 1. The soils were amended with the nutrient

solution on the same schedule as the nutrient arnended biopiles. Peat concentration was

1 %(w/w).

3.4 2.4kg biopile study

This biopile study was conducted in the TSM at Saglek. It consisted of 15 distinct 2.4kg

soil biopiles each prepared in duplicate. The factors which were examined included

fertilizer concentration, bulking agents, and inoculum preparation.

The aerobic culture used in these studies was enriched fiom soil collected fiom the

contaminated site at Saglek Inlet. Cells were grown aerobically at 5OC in 1251111

Erlenmeyer shake flasks. The Erlenmeyer flasks were filled with 50 ml of the medium

described in Section 3.3, and biphenyl in acetone was added to the flasks to achieve a

final concentration of 500 mg/l after evaporation of the solvent.

The soi1 was obtained nom Antema Hi11 at Sagiek Inlet. It was manually excavated,

sieved through a #10 sieve to rernove Iarger particles, and rnixed on a tarpaulin to

mi aimite heterogeneity.

Wooden fiames were constructed in Kingston and transported to the site. The &unes

were approximately 30cmx20cmx4cmy the bottoms were made of fiberglass screen to

allow air to circula~e ihrough the soil, and they were elevated by about 4cm. 2.4kg of

treated soi1 was placed in each frame, and the h e s were then placed in

45cmx30cmx 15cm plastic basins. At the commencement of the experiment, water was

added to the plastic containers to a depth of 4cm.

a) Nutrient study

This study examined the effects of three different fenilizer concentrations and two N:P

ratios. Soils were measured into 2.4kg quantities, and each was amended with 50g of

finely milled sphagnum moss, nitrogen (So-GreenTY ammonium nitrate 33-0-0) and

phosphorous (So-Greenn" superphosphate 0-20-2) before inoculation. At a N:P ratio of

2.5: 1, the total available nitrogen (as ammonium ion) concentrations tested were 0.02g,

0.05g, and 0.07gl kg soil corresponding to phosphorous (as phosphate ion) concentrations

of 0.008gy 0.02gy and 0.028g/ kg soil. Using the intermediate nitrogen concentration as

the basis, a biopile was prepared with a N:P ratio of 1 : 1.

b ) Bulking agent study

Two potential amenciments which could act as bulking agents were tested in this study:

fmely milled sphagnurn moss and red pine bark chips. Soi1 was arnended with nutrients

at a concentration of O.OSg/kg total available nitrogen (as ammonium ion), and O.O2g/kg

total phosphorous (as phosphate ion). When peat was w d as the bulking agent, 50 g was

added to each 2.4 kg biopile, corresponding to a concentration of 2%(w/w); when the

bark chips were used, 100 g was added to each 2.4 kg biopile, corresponding to a

concentration of 4%(w/w).

To test the effectiveness of chitin as a CO-buking agent, biopiles were prepared in which

13g ground crab shells were added to 2.4 kg of soil containing peat moss at a

concentration of 2%(w/w).

c) Inocdum study

Three different inoculation techniques were assessed in this study. They are designated

prepared peat, concentrated live suspension, and fieeze-dried culture.

The prepared peat inocula were made by adding a centrifbged culture to coarse sphagnum

moss in the Kingston laboratory. The immobilized cells were then placed in sterile petri

dishes, and kept cool (5OC) for two weeks until inoculation on-site. The inocula were

added to the biopiles by dispesing the support particles evenly throughout the soil. The

concentrated live suspension was prepared by centrifuging lOOml of a dense liquid

culture. The culture was kept cool (5OC) for one week until resuspension on-site. The

fieeze-dried culture was prepared in Kingston one week before resuspension on-site.

Both these inocula were resuspended on site in a mineral salts medium (described in

section 3.3) with biphenyl crystals as the sole carbon source. After one week of

incubation at room temperature (20'~) on an orbital mixer, the cultures were supported

on coarse peat moss. The inocula were added to the biopiles by tearing the sphagnum

into small (-2 g) fragments and dispersing them evenly throughout the soil.

3.5 Moisture-nutrient-peat optimization microcosm study

This was a factorial design experiment to determine the optimum operating conditions of

moisture, nutrient concentration and peat moss concentration. The Aroclor 1260

contaminated soi1 was collected fiom Lab-2 and spiked with Aroclor 122 1.

The soil was obtained fkom the beach area at Saglek. It was stored at CFB Kingston in a

205L barre1 for 2 months at room temperature, and then stored at O°C for 2 months before

use. The soil was passed through a #IO sieve to remove particles larger than 0.25cm.

The Aroclor 1260 concentration after sieving was 230ppm. A 2.3kg portion of soil was

placed in a glass cylindncal vesse1 and roll mixed ovemight before addition of Aroclor

122 1. Seven vials of 50mg Aroclor 122 1 were dissolved in 501-111 of acetone, and added

to 2kg of soi1 2nd at a t h e , allowing for evaporation and roll mixing between additions.

Afier addition of the stock solution, air was passed over the soil for one h o u to ensure

complete evaporation of the acetone. The contaminated soil was then homogenized by

roll mixing for 24 hours.

The microcosms were prepared in 2 0 0 d Bat-walled glass vial with screw caps. To each

vial lOOg of soi1 was added. The soils were then amended with water, peat, nutrients and

biphenyl according to the experimental design. The vials were incubated on their sides to

maximize the soiYair interface; the depth of the soil was approxïmately lcm. Incubation

was at 5 ' ~ for one month.

The experiment consisted of 15 distinct microcosms of varyirig moishire content, nutrient

concentration, and peat concentration. Additional microcosms included an abiotic control

which was sterilized with 0.2%(w/w) sodium azide, a dupiicate of the median microcosm,

and a biphenyl amended (500ppm) microcosm.

The nitrogen source used was m 4 N 0 3 , the phosphorous source was KH,P04, and the

ammonium N:P ratio was held constant at 5: 1. The experimental design is summarized in

Table 3.1.

A moisture content range of 20% to 60% of the water holding capacity of the soil was

selected because it is within this range that aerobic soil bacteria are most prolific

(Cookson, 1995). An upper peat concentration of Z%(w/w) was selected to limit the

amount of foreign matter amended to the soil; also, above this concentration, the

acidification effects of peat would geatly hinder bacterial activity. The N:P ratio and

ammonium concentration were selected based on other PCB biodegradation -dies

reported in prominent joumals (Dercova, 1995; Barriault and Sylvestre, 1993).

Table 3.1 : Experirnental design of moisture-peat-nutrient optimization study.

Microcosm moisture content (%) peat concentration (%) ammonium N

concentration (gkg)

1

2

3

4

5

6

7

8

9 (median)

10

1 I

12

13

14

15

16 (abiotic)

17 (duplicate)

18 (biphenyl)

3.6 Aqueous phase inducer assessrnent

The objective of this study was to evaluate the potential of phenolic compounds to

stimulate the aerobic degradation of Aroclor 1221. Five phenolic compounds and

biphenyl were tested for their ability to induce PCB metabolism.

The inocula were grown at 5OC in a 1 Litre Erlenmeyer flask on a platform shaker. The

flask contained 400ml mineral sdts medium with biphenyl as the sole carbon source.

The experiments were incubated on a platform shaker at 5OC in 2 5 d glass scintillation

vials with aluminum lined caps. Each scintillation via1 was filled with 7 ml medium.

The individual phenolic compounds were dissolved in appropriate solvents (Table 3.2)

and added to the vials in one of three concentrations: 1.63 mmol/l, 3.25 mmoV1, or 4.88

rnmoV1. m e r evaporation of volatile solvents, 2 ml of a dense inoculum was added to

each vial. A 0.3 ml aliquot of Img/ml Aroclor 1221 in acetone was then added to each

vial to achieve a final concentration of 33 ppm afler evaporation of the solvent. The

abiotic controls were sterilized using perchloric acid.

The inducers which were assessed are biphenyl, arbutin, vanillic acid, cinnamic acid,

coumarin, and naringin. Their structures are shown in Table 3.2. Naringin has a rhamno-

glucose moiety and arbutin has a glucose moiety; to ensure that variations in observations

were due only to the phenolic compounds, sugars were added to the cultures to achieve a

1 : 1 : 1 rnolar ratio of rhamnose: glucose: inducer. Sugars were also added to the biotic and

abiotic controls. Six replicates were made for each experiment, and three were sampled

at each sampling time.

Table 3.2: The chernicai structures of the inducers examined and the solvents in which

they were solubilized before addition to the medium.

bipheny 1

cinnamic acid r- 1 vanillic acid

Structure Solvent

medium

(water + nutrients)

--

acetone

acetone

acetone

Three concentrations were tested for each potential inducer. The basis for the selection of

the concentrations was the biphenyl concentration at which the cells were grown for

inocula purposes. This concentration is 500 mg, or 3.25 mmol/l. Because the induction

process is a function of the number of inducer molecules in the vicuiity of the bacteria,

the molar units were selected as opposed to the m a s based concentration. The medium

concentration was therefore chosen as 3 -25 mmoV1, the low concentration was half this

value (1.63 mmoV1)), and the hi& concentration was selected to be 4.88 mmoV1.

The PCBs were extracted from the cultures using a surfactant followed by hexane

washing. The procedure followed was a modification of a method described by Bedard

(Bedard et al., 1986). The vials were opened and Triton-X 100 was immediately added to

a final concentration of 1 % (dv). This surfactant disrupts ce11 membranes and increases

the solubility of PCBs by several orders of magnitude. Sodium sulfate (final

concentration of 0.0 1 g/l) was added to each via1 to prevent the formation of a stable

emulsion. The solutions were then washed with three successive aiiquots of 3 r d of

hexane; the fust wash lasted 20 minutes, the second for one hour, and the last wash was

ailowed eight hours to approach equilibrium. The viais were shaken on a platform shaker

during the extractions.

3.7 Bacterial identification using APIO test strips

A bacterial species was isolated from the soils exhibithg the most effective PCB

removals in the Aroclor 1260 degradation microcosm study. The species was tentatively

identified using the API-Rapid NFT system. This system is designed to identiS,

clinically significant species: identification of environmental species may not be

accurate.

The system consists of a plastic strip containing 20 cupules of dehydrated substrates for

the demonstration of either enzymatic activity or the assimilation of carbon sources.

Each cupule is inoculated with a few drops' of a metabolically active culture, and

incubated for 24 hours at 30°C.

The result of each test is recorded as either positive or negative as evidenced by either a

color change or turbidity. A simple numerical algorithm is then followed to obtain a

seven digit number. The species is then identified using an automated telephone system

operated by API Laboratory Products Ltd.

3.8 Soil bacteria enurneration

Soil bacteria were enumerated in the Aroclor 1260 degradation microcosm shidy using

the spread plate technique. A 1 g sample of test soi1 was diluted in 9ml of the medium

described in Section 3.3. A I d aloquot of this dilute slurry was transfered to a test tube

containing 9ml of medium, and two more 10: 1 serial dilutions were made resulting in a

final solution containing 10' g soi1 / ml medium. A 0.5rnl aliquot of this dilute solution

was transferred, using a pipette, to petri plates prepared with lOml of aga-solidified

medium. A sterile g las rod was used to spread the culture evenly over the surface of the

agar medium before being sealed. The plates were then incubated at SOC.

3.9 PCB analyses

Al1 Aroclor analyses were performed by the Analytical Sciences Unit at the Royal

Military College. The standard method used quantifies PCB concentration based on six

prominent GCECD peaks which correspond to six congeners in the Aroclor mixture.

Approximately log of air ciried soil was spiked with an aliquot of decachlorinated

biphenyl, used as an internai standard. The soil was then extracted with dichlorornethane

on a soxlet apparatus for six hours at 4-6 cycles per hour. The extract was then

concentrated by roto-evaporation to approximately 1 mi, 5rnl of hexane was added, and

the sample was again evaporated to 1 ml. This was repeated two more times. The lm1 of

hexanePCB solution was then passed through a Florisil column to remove lipid

contaminants. The col- was rinsed with hexane, and the eluant was used to dilute the

sample to 10.0ml. This sample was anaiyzed by gas chromatography with an electron

capture detector (GCIECD).

Each sample was analyzed using an HP 5890 series II Plus gas chromatograph equipped

with a %Ji electron capture detector, and S P B ~ ~ - I hsed silica capillary column (30m,

0.25 mm ID x 0.25 pm film thickness) and the HPChem station software. The

chromatographic conditions were as follows: 2 pL sample volume; splitiess injection;

1 OO'C for 2 minutes initial temperature; 1 O°C/min ramp to 1 50°C ,5'C/min to 300°C;

final tirne 5 minutes. The carrier gas used was heliurn at a flow rate of I d m i n .

Nitrogen was used as a make-up gas for the ECD. Al1 values were reported as ppm

(pg./g) on a dry weight basis an corrected for recovery using the intemal standard.

4. Methods: Anaerobic studies

4.1 Anaerobic inoculation test

A consortia of PCB-dechiorinating bacteria was enriched by Dr. W. Mohn of UBC, and

used to inoculate 8kg of sanirted soil. This experirnent was conducted in the TSM at

Saglek. Soils used in this study were obtained fiom Antenna Hill, and passed through a

5/16" sieve to remove larger particles.

This experirnent consisted of two vessels (1OL pails) each containing 8kg of 1SOppm

Aroclor 1260 contarninated soil. The soils are satwated and covered by an aqueous phase

to a depth of approximately 15cm. The soils were incubated for five months at room

temperature in the TSM at Saglek. The control soil did not receive any arnendments.

The test soil was mixed with log corn starch, log rice, 4.0g NH,CI, and 2.7g

Superphosphate (0-20-0) before the addition of water. The mixture was incubated for

two weeks, to reduce the redox potential, before inoculating with 8 125ml via!s

containing a PCB dechlorinahg bacteriai consortia.

4.2 Anaerobic biopile study

This study consisted of four distinct 3kg biopiles each prepared in triplicate and located

in the TSM at Saglek. The study examines addition of nutrients, carbon, and anaerobic

sediment.

The soil used in this shidy is the same soil which was used for the 4kg biopile study. The

soi1 onginated fiom the summit of Antenna Hill, and was sieved through a 5/16" sieve to

reduce sampling error. The soi1 was roll mixed in a 205L barre1 to achieve homogeneity.

Each of the 12 biopiles were prepared in 35cm x 20cm x 15cm basins with Lids. Each

basin received 3kg of soi1 to which the solid amendments were added as required. The

soils were then thoroughly mixed with a trowel before adding 8L of the aqueous phase

resulting in a liquid depth of approxirnately 8cm over the saturated soil. The biopiles

were incubated at room temperature (20°C) for five months.

Four distinct biopiles were prepared in triplicate.

The biotic control consisted of 3kg of soil in a basin to which 8L of tap water was

added,

The nutrient amended biopile was prepared by adding 8L of a nutrient solution

containing 2g/L NH4CI and 1gL KH2P0, over the 3kg of soil.

The nutrient and carbon amended biopile was prepared by adding the 8L of nutrient

solution over 3kg of soi1 Uito which 3g each of corn starch and soluble starch had

been rnixed,

4. The nutrient, carbon and sediment amended biopiles are sirnilar to the nutrient and

carbon arnended biopiles with the exception that 30g of anaerobic sediment was dso

mixed into the soil before addition of the nutrient solution. The sediment was

collected fiom a lake which receives the Antenna Hill runoff.

4.3 Small scale deoxygenation study

This experiment was conducted in the Technical Services Module (TSM) at Saglek.

Several ziplock bags were filled with lkg of soil with varying nutrient and carbon

concentrations; time to deoxygenation was rneasured. The soil was obtained fkom the

summit of Antenna Hill, and passed through a 5/16'' sieve to remove larger particles.

Each ziplock bag contained 1 kg of soil plus amendments, and was moistened with 50ml

of either water or nutrient solution. An oxygen indicator strip was placed in the bag

before being sealed. Incubation was at room temperature (20°C).

Seven distinct weatments were prepared in duplicate. Nutrient and carbon source

concentration were the parameten which were varied. The nutrient source was the media

described in Section 3.3, and the carbon source was corn starch. The experimentai design

is summarized in Table 4.1.

Table 4.1 : Experimental design of small scale deoxygenation study.

Treatment Amendments

control 50ml water

low nutrient 5Oml medium

no carbon

low carbon 1 g/kg cornstarc h

medium carbon Sgkg cornstarch

high carbon lOg/kg cornstarch

high nuaient 50d medium (x2 conc.)

low carbon 1 g/kg cornstarch

medium carbon 5 glkg CO mstarc h

4.4 Large scale deoxygenation study

This expenment was conducted to assess the feasibility of PCB dechlorination while soils

are being stored in 2m3 crates. Carbon and nutrient sources were mixed with the soil as

the crate was filled, and redox potentials were measured afler 1 month.

The soil was excavated fiom Antenna Hill using a back-hoe, and passed through a 2.5"

sieve into a hopper. The hopper was then emptied into tarpaulin and plastic lined

4 ' x 4 ' ~ 4 ' wooden crates using a forklift.

Two crates were prepared; one was not treated, whereas the other received arnendments.

Water was added to the surface of the soil several times over the span of two days to

achieve adequate moisture. The plastic and tarpauiin linings were folded to encase the

soil. The crates were stored on A n t e ~ a Hill, and were therefore exposed to daily

temperature fluctuations.

The control soil was not rnanipuiated afier removal of Stones larger than 2.5" by the

hopper grate. Amenciments were added to the soil between hopper loads. niree hoppers

were required to fil1 the crates, and one-third of the amendment mixture was added after

each load and mixed to a depth of about six inches using a garden hoe. The amendment

mixture consisted of 2kg corn starch, 3kg blood meal, 0.7kg superphosphate (0-20-O), and

2 kg limestone.

5 . Results: Aerobic studies

5 .1 Introduction

Five soil phase studies and one aqueous phase aerobic study are discussed in this chapter.

The soi1 studies include: 2-CB rnineralization microcosms, Aroclor 1260 degradation

microcosms, 4kg biopiles, 2.4kg biopiles, and nutrient-rnoisture-peat opthkation

microcosmst

In the 2-CB mineralization microcosm study , the production of l4co2 was monitored as

an indication of the lower metabolic pathway: the transformation of c h l o ~ a t e d benzoic

and aliphatic acids to carbon dioxide, water, and chloride ion. The microcosms consisted

of 20g of Antenna Hill soil in 125d serum vials, and were incubated for six mon& at

soc.

In the Aroclor 1260 degradation microcosm study, the disappearance of Aroclor 1260

was monitored as an indication of the upper metabolic pathway: the transformation of

PCB to chlorinated benzoic and aliphatic acids. This study was nui in parailel to the

mineralization study, and the sarne soil was used for both. The microcosms consisted of

50g of Antema Hill soil in 200ml glass vials, and were incubated for six rnonths at soc.

In the 4kg biopile study, the disappearance of Aroclor 1260 was rnonitored as an

indication of the upper metabolic pathway. The biopiles consisted of 4kg of Antenna Hill

soil in plastic basins, and were incubated for six months at 2 0 ' ~ .

In addition to sterilized abiotic controls and biotic controls which received only water

addition, the four studies descnbed above also included preparations to assess the effects

of nutrient, bulking agent, inoculum and inducer amendment.

In the 2.4kg biopile study, the disappearance of Aroclor 1260 was monitored as an

indication of the upper rnetabolic pathway. The biopiles consisted of 2.4kg of Antema

Hill soil in plastic basins, and were incubated for eieven months at 20°C.

In the moisture-nutrient-peat optimisation microcosm study, the disappearance of Aroclor

122 1 was monitored as an indication of the upper metabolic pathway. The microcosrns

consisted of 1 OOg of Saglek beach soil in 200ml glas vials, and were incubated for one

month at SOC. This was a factorial design experiment to detennine the optimum

operating conditions of rnoishire content, peat rnoss concentration and nutrient

concentration.

In the aqueous phase inducer assessment, the disappearance of Aroclor 122 1 was

monitored as an indication of the upper metabolic pathway. A pure culture, enriched

fiom Saglek soil, was used to inoculate 7ml aliquots of sugar and ArocIor 1221 amended

mineral salts medium in 25ml scintillation vials. The effectiveness of several suspected

inducers to improve degradation was assessed. The cultures were incubated for one

month at 5°C.

Reported confidence intervals were calculated as the standard deviation fkom the mean of

the replicates.

The results discussed in this section focus on two microcosm midies (2-CB

mineralization and Aroclor 1260 degradation) and the two biopile studies (4kg and

2.4kg). The abiotic controls were treated using various sterilization techniques which are

descnbed in Chapter 3. The biotic controls received water amendment only.

The degree of mineralization of 2-CB after 180 days of incubation was greatest in the

biotic control where 28.2 1.8% was converted to ' ' ~ 0 ~ (Figure 5.1). The rate of

mineralization in the biotic control was still increasing when the last data were collected.

This suggests that the potential mineralization end point would be much higher than

indicated here. Mineralization was not observed in the abiotic controls.

I Time (days) - - 1 j +abiotic control + biotic control 4 nutrient amended

Figure 5.1 : 2-CB mineralization observed in 20g microcosms incubated at 5C

(nutrient study).

In the study which was conducted paraIlel to the ' 4 ~ study, hoclor 1260 degradation was

not observed in either the biotic or abiotic controls (Figure 5.2).

15 n

E IO .b

t O .- Y

m 5 , - O 2 E O cl

-5

Time (days) f

j -+- abiotic control + biotic control +nutrient amended 1

Figure 5.2: Aroclor 1260 degradation observed in 50g microcosms incubated at

5C (nutrient study).

The biotic controls fiom the 4kg biopile study exhibited the greatest degree of Aroclor

1260 losses at 18.7%. No losses were observed in the abiotic controls (Figure 5.3).

I

-10.0 - - .- -

abiotic control

--- -

biotic control

I

Figure 5.3: Aroclor 1260 degradation observed in 4kg biopiles incubated for six

months at 20C (nutrient study).

PCB degradation was not observed in the biotic controls prepared for the 2.4kg biopile

study. Abiotic controls were not prepared.

As the soils used in the two microcosm studies were identical, the observed discrepancy

in the biotic controls was likely due to the increased chlorination of the Aroclor 1260

relative to the single congener of rnonochlorinated biphenyl.

The biotic controls fiom the 4kg biopiles exhibited significant degradation of Aroclor

1260 although the soil was similar to that used in the microcosm studies. Soi1 used in the

microcosm studies, however, was stored for eight months before use, whereas fkeshly

excavated soil was used for the biopile studies. During the storage period, the microflora

composition of the soil had probably changed significantly. Organisrns capable of

degrading the highly chlorinated congeners may have been present in the fiesh soil, but

not in the soil uçed for the microcosm studies. The higher incubation temperature may

also have been pady responsible for the increase in PCB losses.

As the 2.4kg biopiles were not covered to prevent moisture loss the soil became very dry.

The desiccation of these soils may have resulted in the cessation of metabolic activity at

an earIy stage in the incubation.

5.3 Nutrient studies

The results discussed in this section focus on two rnicrocosm studies (2-CB

mineralization and Arocior 1260 degradation) and the two biopile studies (4kg and

2.4kg). Nutrient addition has been shown to greatly improve bioremediation of

hydrocarbon-contaminated soils. As Arctic soils are characteristically low in nitrogen,

attempts were made to stimulate the indigenous organisms by amending the soil with

fertilizers.

Amending the soils with O.Sg/kg ammonium, O.Zg/kg phosphate, and 0.035gkg sulfate

resulted in lowering 2-CB mineralization relative to biotic controls (Figure 5.1). At the

conclusion of the experiment, only 12.4 I 3.6 % of the compound was mineralized. The

observed reduction in mineraiization rates caused by nutnent addition indicates that one

of the constituents may be toxic to these organisms. Psychrotrophic organisms are very

sensitive to changes in environmental conditions, and small variations may resdt in

drastic changes to species distribution. An altemate explmation is that the nutrients

stimdated another soi1 organism which had a negative impact on one or more of the three

target populations leading to the complete mineralization of 2-CB. This may have been

due to either direct competition for carbon and energy sources or due to the production of

a toxic metabolite.

In the paralie1 microcosm study, there was no significant reduction in Aroclor 1260

concentration in the nutrient amended rnicrocosms after 153 days of incubation (Figure

Addition of a minera1 salts medium (0.09gkg ammonium, 0.4gkg phosphorous plus

trace magnesium, manganese, iron and calcium) did not stimulate PCB degradation above

biotic control levels in the 4kg biopiles. The same average amount of degradation was

observed, but reproducibiiity was not as good (Figure 5.3).

Fertiiizer addition (0 .O5 @kg ammonium and O.OZg/kg phosphate) alone did not increase

PCB losses in the 2.4kg biopiles (Table 5.1). The more intricate nutrient study was

conducted on soils also amended with 2%(w/w) peat rnoss. At a N:P ratio of 2.5: 1, with a

nitrogen concentration of O.OSg/kg soil, a 14% loss was observed. When the phosphorous

concentration was increased, reducing the ratio to 1 : 1, the results exhibited a large standard

deviation. When the N:P ratio was kept constant at 2.5: 1 and the nitrogen concentration

\:-as raised to 0.07gkg soil, the highest level of degradation (20%) was obtained. At the

sarne N:P ratio with a lower nitmgen concentration of 0.02gkg soil, a 1 1% loss of PCB was

observed.

Table 5.1 : Aroclor 1260 degradation observed in 2.4kg biopile study. Biopiies were

uicubated at 2 0 ' ~ for 1 1 months.

Treatrnent PCB losses (%)

biotic control O

M=SOmg/kg; N:P=2.5: 1 4* 10

M=20mg/kg; N:P=2.5: 1 ; with peat 11 * 1

m]=SOmg/kg; N:P=2.5: 1 : with peat 14 (no replicate)

[N]=70mg/kg; N:P=2.5: 1 ; with peat 2015

M=SOmg/kg; N:P= 1 : 1 ; with peat 6* 13

The Aroclor 1260 microcosm study as well as the 2.4kg biopile suggest that nutrient

addition, at an N:P ratio of 2.5: 1, does not stimulate the target biota to a greater degree

than solely maintaining moimire and oxygen availability. Nitrogen concentrations in

these two studies spanned an order of magnitude from 0.05 to OSgkg. At a nitrogen

concentration of 0.9g/kg and an N: P ratio of 1 :4, no increase in PCB degradation was

observed in the 4kg biopiles. The I4c study indicated that nutrient addition can actuaily

be detrimental to the lower metabolic pathway. Other researchers working with

mesophillic bactena have found that nutrient addition can stimulate the aerobic

degradation of PCB (Barriadt and Sylvestre, 1993)

As s h o w by the 2.4kg biopile study, nutrient addition when combined with 2%(w/w)

peat amendment may improve PCB biodegradation. A N:P ratio of 2.5: 1 stimulatecl

degradation better a N:P ratio of 1 : 1, and, within the range tested, increasing nutrient

concentration resulted in improved degradation. These biopiles were not protected fiom

rnoisture loss, and the increased degradation was probably due to a combination of the

moisture retention property of the peat and the additional organic matter available as

substrate.

5.4 Buiking agent studies

The results discussed in this section focus on two rnicrocosm studies (2-CB

mineralization and Aroclor 1260 degradation) and the two biopile studies (2.4kg and

4kg). Bulking agents aid in aerating soils and improving moisture retention as well as

providing a matrix for microbial support (Bossert and Cornpeau, 1995). The aromatic

structures in peat moss rnay also improve PCB degradation by inducing the bph gene, and

chitin c m act as a nitrogen source as well as a liming agent.

The presence of 2%(wlw) peat moss in the 2-CE3 minerakation study reduced the

observed mineralization even lower than nutrient amendment alone. A total

mineralization of 7.1 2.5 % was achieved after 1 87 days (Figure 5.4). Addition of peat

moss lowers the pH of the soil matrix. This change may have been detrimental to the two

groups of organisms involved in the lower metabolic pathway. Again, direct cornpetition

may have been a factor contributing to the observations. In addition to indigenous soil

organisrns lacking the lower bph gene, introduced species could also be playing a

competitive role in these microcosrns. The peat which was added to the soil also had a

microbial community associated with it. It is unlikely that addition of peat-associated

organisms reduced the rate of the upper metabolic pathway. As peat contains nany

aromatic rings, the associated microorganisms are usually able to degrade these

structures. However, the introduced species rnay have interfered with the effectiveness of

the lower metabolic pathway which leads to CO2 production. Mineralization rates of the

peat amended soi1 were still increasing when the last data were collected. This suggests

that the potential mineralization end point would be greater than indicated here.

20 40 60 80 100 120 140 160 180 2

Time (days)

j + abiotic control + biotic control + peat amended

Figure 5.4: 2-CB mineralization observed in 20g microcosms incubated at 5C

(bulking agent study).

Ln the Aroclor 1260 microcosm study, the greatest disappearance was observed in the

autoclaved 2%(w/w) peat amended microcosms in which there was an 1 1.6% loss (Figure

5.5). A culture was enriched fiom al1 three of these replicates, and will be discussed in

section 5.10.

The nutrient and 2%(w/w) peat arnended microcosms of the Aroclor 1260 microcosms

showed a significant PCB loss of 5.5% (Figure 5.5). It is possible that the peat organisms

were responsible for a portion of this loss, but the process was not as efficient due to

cornpetition fkom indigenous soi1 organisms which were stimulated by the nutrients.

Time (days) I 1 -t abiotic control + biotic controf 1 ! +autoclaved peat amended + peat amended

Figure 5.5: Aroclor 1260 degradation observed in 50g microcosrns incubated at

5C (buking agent study).

In the 4kg biopile study, PCB degradation was decreased by adding bullcing agents to

nutrient amended soils (Figure 5.6). Without bulking agent amendment, PCB losses of

18% were observed. l%(w/w) Pearlite addition was slightly more inhibitory, at 9.4%

PCB reduction, than l%(w/w) peat (9.6%). A 12.5% PCB loss was observed in the

1 %(w/w) arctic bluegrass amended microcosms. The Aminoplast was the most

inhibitory of the bulking agents; at a concentration of 0.2%(w/w) a reduction of only

1.6% was observed.

nutrient Pest pearlite bluegrass arninop hst

Figure 5.6: Aroclor 1260 degradation observed in 4kg biopiles incubated for six months at 20C (bulking agent study).

Peat amendment to fertilized soils increased Aroclor 1260 degradation fiom 4% to 14%

in the 2.4kg biopile study. Bark chips were also an effective bulking agent, and PCB

losses of 16% were observed. Chitin is a major byproduct of the shellfish industry, and

can be used to improve microbial activity by acting as a support matrix, retaining

moisture, counteracting acidification and providing nutrients. When crushed crab shells

were combined with peat moss, average PCB removals were increased to 17%. Due to

the errors associated with sampling, analysis. and variability between replicates,

conclusive statements regarding the relative effectiveness of the bulking agents cannot be

made.

-10 I biotic nutrient peat bark peat and

control amended chitan . . . . . -. - - - -- - - - --p. - . . - . .

Figure 5.7: Aroclor 1260 degradation observed in 2.4kg biopiles incubated for eleven months ai 20C (bulking agent study).

44

A discrepancy was seen between the two parallel microcosms in the peat study. Peat

addition (2%(w/w)) inhibited mineralization in the 2-CB experiment, whereas

degradation was improved by peat amendment in the Aroclor 1260 experiment. Absence

of mineraiization however, does not infer that PCB is not being degraded to benzoic and

aliphatic acid intemediates. By stimulating organisms capable of the upper metabolic

pathway, resource availability to the organisms capable of the lower metabolic pathway

may have decreased, resuiting in the decrease in mineralization.

PCB degradation was decreased by peat addition (1 %(w/w)) in the 4kg biopile study.

The fieshly excavated soil used in this study was supporting a healthy indigenous

cornmunit- capable of cometabolizing the more highly chlorinated congeners while

degrading the existing organic matter. Addition of peat af5ected the existing

environmental conditions of the soil, having a negative impact on the original microbiai

composition. The soil used in the microcosm study, which was stored at 4*C for eight

months, would have been supporting a different consortia of microorganisrns. The peat

may have induced the PCB degrading ability of these less effective organisms whereas

the existing organic matter could not.

The increase in PCB removals observed in the 2.4kg peat arnended biopiles was likely

due to improved moisture retention. This biopile study was not protected from moishue

loss, and the soils became arid shortly into the incubation. The bulking agents would

have greatly lengthened the time during which the bactena were active.

5.5 Soi1 phase inducer studies

The results discussed in this section focus on two microcosm studies (2-CB

mineralization and Aroclor 1260 degradation), the two biopiie studies (4kg and 2.4kg).

Although biphenyl and lowly chlorinated congeners may be degraded by bactena

possessing the bph gene, the more highly chlorinated congeners are fortuitously

cometabolized by enzymes which are induced by a chernical analog. Biphenyl has

traditiondly been used as the inducer in laboratory studies, but the ubiquitous nature of

the bph gene combined with the rarity of biphenyl in the environment suggests another

compound must be the intended target of the enzyme system. It has been proposed that

plant produced phenolic compounds may be the naturai inducer. Both biphenyl and plant

produced phenolic compounds were tested for their ability to improve PCB

biodegradation.

Significant mineralization was not observed in any of the inducer amended microcosms:

biphenyl, quercetin or c i ~ a m i c acid (Figure 5.8). The inducer concentrations used in

this study were probably toxic to the target organisms. Biphenyl, which has been s h o w

by many researchers in the field to effectively stimulate mesophillic PCB biodegradation

at IOOOppm, was toxic even at 1 OOppm. This introduces an additional challenge to the

operation of an unsanirated bioremediation option such as landfamiing or biopiling in

cold climates. The presence of the inducer may result in the inhibition of the

psychotrophic organisms capable of mineralizing the intermediate products.

Time (days)

- - pp - - - - -- - - - - .- - -

+ abiotic control + biotic control + 1000ppm BP l

+100pprn BP + 1 OOOppm cin. acid + 1 OOOppm quercetin

Figure 5.8: 2-CB mineralization observed in 20g microcosms incubated at 5C

(inducer study).

In the parallel Aroclor 1260 shidy, a 4.5% loss of PCB was observed in the nutrient and

biphenyl amended soils (Figure 5.9).

Time (days)

1 + abiatic control 1 + biotic control + 1000ppm biphenyl

Figure 5.9: Aroclor 1260 degradation observed in 50g microcosms incubated at

5C (inducer study).

The results of the 4kg biopile inducer study indicated that two phenolic cornpoumis,

coumarin and quercetin, both found in local plants may stimulate PCB reductions in peat

amended soils (Figure 5.10). Average reductions of 14% were observed in both these

treatments relative to a 9.6% reduction in soils arnended with peat only. Reproducibility

in the quercetin amended microcosms was excellent, and a t-test indicated a 80%

probability that quercetin amendment increased degradation by 3%. Reproducibility in

the cournarin amended biopiles was not as good, where a 6.3% standard deviation was

observed. A t-test indicated a 74% probability that cournarin amendment increased

degradation by 2.1%. Biphenyl addition did not enhance biodegradation to a greater

degree than peat amendment alone.

peat only

15 -

.- CI Cu 'C3

L 10 - 01

d 5

O nutrient and biphenyl quercetin cournarin

Figure 5.10: Aroclor 1260 degradation observed in 4kg biopiles incubated for

six months at 20C (inducer study).

Other researchers in the field have found that bipheny 1 (Harkness et al., 1 992; Yagi et al.,

1980) or phenolic compounds (Donnelly et al., 1994) can greatly stimulate PCB

degradation in aqueous culture. Although both Aroclor 1260 studies indicated that

amendrnent with inducers such as biphenyl or phenolic compounds may slightly

stimulate PCB degradation in soil, the minenlization microcosm study suggests that this

treatrnent may impede the lower metabolic pathway.

5.6 Aqueous phase inducer study

A pure culture was used in this study to degrade Aroclor 122 1 in aqueous medium. As

naringin and arbutin (two phenolic compounds assessed in this study) have sugar

moieties, glucose and rhamose were added !O the medium to obtain a final molar

concentration of 1 : 1 : 1 of rharnose: glucose: inducer in each culture vessel.

Results of the aqueous inducer study indicated that. at the concentrations used, biphenyl

and the phenolic compounds do not stimulate Aroclor 122 1 degradation as well as sugar

amendrnent alone. PCB degradation was found to be greatest in the biotic controls

(biomass, medium, sugars, and Aroclor 122 1) where up to 53% reductions were

observed.

Biphenyl, naringin, or arbutin amendment decreased the extent of PCB biodegradation

relative to cultures incubated with only sugar and PCBs. Removais decreased linearly as

inducer concentrations were increased (Figure 5.1 1). Arbutin was the Ieast inhibitory,

reducing removals by 0.84 % for each additional mmol/l; naringin reduced PCB removals

by 2.27 %/(mmol/l). In the concentration range tested, the inhibitory effects of biphenyl

were particularly pronounced (7.55 %/(mmol/l)). As this culture was enriched on

biphenyl, it is doubtfd that the observations were due to any toxic effects. A more

plausible expianation is that at these high concentrations (above the water solubility)

biphenyl may be preferentially rnetabolized by the microorganisms. Because biphenyl is

much more biodegradable than its chlorinated counterpart, the bactena may be

consuming it as a carbon source while only minimaily affecting the PCBs.

Vanillic acid, cinnamic acid, and coumarin did not exhibit a linear relationship between

PCB removals and concentration (Figure 5.12). At concentrations of 1.63 mmolA,

vanillic acid and cinnamic acid had little effect on the extent of PCB removal relative to

cultures incubated without an inducer (53%); coumarin only slightly decreased removals

at this concentration (49%). Disappearance of PCB changed little as the inducer

concentration was raised to 3.25 mmoV1. At higher concentrations however, these

compounds significantly impeded biodegradation, lowering PCB removals to about 35%.

This behavior is indicative of a toxic concentration between 3.25 mmoM and 4.88

m o l A .

O 1 O 1 2 3 4 5

lnducer concentration ( m U i )

+ biphenyl + naringin - arbutin --A-, abiotic control

Figure 5.1 1 : Linear inhibition of Aroclor 122 1 degradation due to biphenyl, arbutin, and naringin addition in aqueous culture incubated for 35 days at SC.

O 1 2 3 4 5 lnducer Concentration ( m V i )

+ vanillic acid + cinnamic acid --+ cournarin .- abiotic control

Figure 5.12: Non-linear inhibition effects of vanillic acid, cinnarnic acid and coumarin on the degradation of Aroclor 122 1 in aqueous culture incubated for 35 days at 5C.

Although very little research has focused on the ability of plant-produced phenolic

compounds to induce aerobic PCB biodegradation, work with mesophillic bacteria in

pure culhue has indicated that some phenolics are effective at stirndating the metabolic

pathway (Fletcher et al., 1995; Domeliy et al., 1994).

As this shuiy was designed with the major objective to assess the inductive capabilities of

several phenolic compounds, sampling kvas innequent and detailed kinetic data are not

available. However, the process appears to be rapid with over 90% of the observed

degradation occurring withùi the first 10 days (Figure 5.13). Results at the low and high

inducer concentrations exhibited similar dynamics.

h 60

I

4 4 t

9

O 5 10 15 20 25 30 35 Time (day)

r i + abiotic control +- biotic control 1 --A-. biphenyl arnended - coumarin amended !

Figure 5.13 : Aroclor 122 1 removd kinetics for the controls and two inducer amended (3.25 mmol/L) liquid cultures. Incubation was at SC.

5.7 Preferentiai degradation of specific congeners

Aerobic degradation rates differ for each congener due to steric hindrance (Focht, 1995).

As biphenyl dioxygenase is believed to attack the biphenyl skeleton at the bond between

the ortho- and meta- carbons, chlorine substitution at one of these sites will impede

biodegradation. The congeners of each sample were separated in a gas chromatography

column and quantified using an electron capture detector. Calculation of the PCB

concentration was based on six prominent peaks.

Congener specific degradation was observed in the aqueous phase ArocIor 122 1 study.

Peak 1 and 2 are monochlorinated biphenyls, whereas the others are dichlorinated

biphenyls. Almost complete removai of the congeners indicated by peaks 1 and 3 was

achieved (Figure 5.14). Although peaks 2,4 ,5 , and 6 did not show significant

reductions, if biologicai activity can be maintained after depletion of the more easily

degraded congeners, it is possible that the more recalcitrant congeners will also be

subsequently metabolized.

Peak number

O abiotic control biotic control biphenyl arnended

Figure 5.14: Preferentid depletion of specific congeners of Aroclor 122 1 was observed in the aqueous phase inducer study. Each peak number corresponds to a different congener. Incubation was at SC for 35 days.

Preferential metabolization of specific congeners was also observed in the Aroclor 1260

degradation microcosms. Figure 5.15 illustrates the relative abundance of the six

congeners used to calculate PCB concentration standardized to the congener responsible

for peak 6. These congeners contain five to seven chiorine atoms, and chlorination

increases nom peak 1 through peak 6. The highest degradation was observed in peak 1

(the Ieast chiorinated congener), and the degree of degradation decreased as the

chlorination of the congeners increased.

.à C 0.9 ..

0.8 - t

0.7.- O

0.6 . - O 0.5 - -

0-4 - -

0.3 .. 0.2 .-

[II 0.1 .-

a standard Amclor 1260 rnetabolued Aroclor 1260 !

Figure 5.15 : Preferential depletion of specific congeners of Aroclor 1260 was observed in the autoclaved peat amended microcosm. Each peak nurnber corresponds to a different congener. Incubation was at SOC for six months.

5.8 Mineralization of PCB

The CO2 which was monitored in the mineralization study was likely not produced by the

same organisms responsible for opening the aromatic ring. It is believed that three

groups of bactena are required for the aerobic mineralization of PCBs: organisms which

achially degrade the PCB by opening an aromatic ring producing a chlorobenzoic acid

and a chlorinated 5-carbon carboxylic acid (Focht, 1995), and a different group of

organisms to mineralize each of these intermediates (Hemandez et al., 1995). Production

of CO2, the parameter measured in the rnineralization study, was effected by the latter

two groups of organisms. A compound-supplied commensal relationship exists between

the PCB degrading organisms which complete the upper metabolic pathway and the two

rnineralizing species which complete the lower metabolic pathway. The end products of

the first organisms serve as carbon and energy sources for the other two. Although

microcosrns which exhibited I4co2 production must have contained active PCB

degrading populations, the absence of 14c0, production does not infer inactivity of PCB

degrading organisrns. Muieralization of lowly chlorinated PCBs by Arctic soil

microorganisms has been observed by other researchers (Mohn et al., 1997).

The upper pathway products of 2-chiorobiphenyl degradation are 2-chlorobenzoic acid

and acetate, and the acetate is much more biodegradable than the c h l o ~ a t e d benzoic

acid. Al1 12 carbon atoms in the 2-chlorobiphenyl were I4c. Assuming a total

mineralkation of the acetate intermediate, and no mineralization of the c hlorinated

benzoic acid intermediate, a maximum minerakation of 42% would be observed. It is

possible that the 2-CE3 was approaching total conversion, and the entire 30%

mineralization observed in the most effective microcosms originated solely fkom the

acetate intermediate. Support for this hypothesis would require GC analysis to show the

accumulation of 2-chloro benzoic acid.

5.9 Soi1 texture

Observations of soil texture were recorded for the Aroclor 1260 microcosm study which

was run in parallel to the minerakation study.

Five days into the incubation, it was observed that the soil in the nutrient and the nutrient

and biphenyl arnended microcosms had formed aggregates. Aggregate size in nutrient

amended soils was about 3mm in diameter and in nutrient and biphenyl amended soils

was about 4mm in diameter. M e r 1.5 months, the soil in these two microcosms

appeared much more moist than observed on day five, and aggregate size had

approximately doubled. After 5 months of incubation, the aggregates in the nutrient and

biphenyl amended rnicrocosms had dried into bnttle pellets which crurnbled upon

vigorous agitation. The aggregates in the nutrient arnended microcosms had also dried

somewhat, but the aggregates maintained their integrity upon agitation.

The texture of both peat amended soil microcosms remained constant throughout the

experiment: the soils remained loose and moist as well as exhibiting homogenous

conditions throughout the individual microcosms.

After 5 months of incubation, soils in both the abiotic and biotic controIs had formed

small (-2mm) dry aggregates which were destroyed upon shaking.

From these observations, it can be inferred that the nutrients were hygroscopic, resulting

in aggregate formation which can reduce oxygen availability at the center of the

conglomerate. This effect is counteracted by a suitable buking agent. The use of a

b u h g agent can also improve moisture retention over long incubations.

5.10 Microbial populations

By monitoring prominent bacterial species which are indigenous to the soil, and

detennining how these species can be selectively emiched by various amendments, a

strategy c m be devised to improve contaminant degradation. The spread plate technique

was used to analyze microbial composition in the Aroclor 1260 degradation microcosm

study. Microbial counts were performed after 1.5 and 5 months of incubation. The

resdts are sumrnarized in Table 5.2.

M e r incubating the month 1.5 plates for less than two weeks, pale yellow circular

convex colonies had developed on the plates prepared korn the biotic controls, nutrient

amended soils and the nutrient and peat amended soils. The biotic control contained the

organism at a concentration of 1 . 2 ~ 1 04cfu/g soil; the other two contained 1 . 5 ~ 1 06cfdg

soil. After 3 months of incubation, the plates prepared fkom both the abiotic controls and

the stenle peat amended microcosms had developed orange circular convex colonies as

well as white filamentous colonies which elongated radially and unifomily fiom their

origin. Although the plates prepared nom the biphenyl and nutrient amended

microcosms displayed poor reproducibility, the pale yellow circular colonies were present

as well as both species observed in the autoclaved soils.

Table 5.2: Relative populations of soil microorganisms d e r 1.5 months and 5 months of

incubation at 5 ' ~ (Aroclor 1260 microcosm shidy).

(+) = 10' colony forming units per gram soil (*) = 1 o6 colony forming units per gram soi1

Treatment t= 1.5 months t=5 months

abiotic control orange circ. (+)

filamentous (+)

biotic control yellow circ. (+) orange circ. (+)

stenle peat amended orange circ. (+) yellow circ. (+)

filamentous (+)

nutrient arnended yellow circ. (*) yellow circ. (*)

nutrient and peat amended yellow circ. (*) yellow circ. (u)

filamentous (+)

nutrient and biphenyl amended yellow circ. (+) yellow circ. (++)

orange circ. (+)

filamentous (+)

Plates were also prepared after 5 months of incubation Two of the three plates prepared

from the abiotic control rnicrocosms were sterile, whereas those prepared kom the biotic

controls developed orange circular colonies corresponding to 1.3~10' cfdg soil. Al1 four

of the other microcosms were supporting pale yellow circular convex colonies to varying

degrees: the autoclaved peat amended microcosrns contained 1 . 8 ~ 1 O' cm, the nutnent

amended soi1 contained 1 . 0 ~ 1 o6 cWg, the nutrient and peat amended soils contained

6.1 x 10' cfidg, and the nutrient and biphenyl amended microcosms contained

4.5 x 1 05cfdg. The agar plates prepared from the nutrient and peat amended microcosm

also developed white filamentous colonies.

Scintillation vials used in the initial dilution for the month 5 biomass analysis, containing

5g soi1 and 15ml water, were allowed to incubate under static conditions at room

temperature for three weeks. M e r this time al1 three replicates prepared fkom the sterile

peat amended microcosm had become turbid; no other vids had developed turbidity. A

pure culture was successfully isolated on tryptic soy broth agar plates, and was tentatively

identified using Rapid MT@ test strips as Pseudornonas cepacia, a known PCB

degrader. The species was not culturable on the minimal medium with biphenyl agar

plates. This was probably due to some nutritional requirement which was absent.

Although both the soil and peat used in these microcosms were autoclaved twice, sterility

was not achieved. This would indicate that either the culture is able to enter a resting

stage which is more resistant to extreme heat than the vegetative cell, or the soil matrix

provided enough protection for a few bacteria to survive the autoclaving process.

Pseudornonas does not f o m spores and is not a particdarly resilient genus, casting doubt

on the accuracy of the identification. The absence of the species fiom the autoclaved soil

microcosms would indicate that it originated fiom the peat moss. This is consistent with

the expectation that the peat rnoss would have been supporting a community of organisms

capable of performing rùig cleavage.

The peat amended microcosms showed less reduction in PCB concentration (5.5%)

relative to the autoclaved peat amended microcosrns (1 1.6%). Cornpetition by non-PCB

degrading organisms may have resulted in a reduction of resources available to the

aromatic degraders. This hypothesis is supported by the plate counts which indicate that

the indigenous soil organisms were over three times denser in the non-autoclaved peat

amended microcosms than the autoclaved peat arnended microcosms.

The plate counts indicated that there were only half as many of the most prominent

organism, which formed pale yellow circular colonies, in the biphenyl amended soil than

in the nutrient amended soils which showed no significant reductions in PCB

concentration. This observation suggests that these organisrns were not responsible for

the observed degradation. Organisrns which formed white filarnentous colonies were

indigenous to the soil, and the addition of biphenyl possibly stimulated these organisms

resulting in the degradation.

The white filamentous growth observed in the autoclaved soils as well as the soils

amended with either autoclaved peat, non-autoclaved peat or biphenyl may have been

partiy responsible for the observed degradation. Although the species was not

conclusively identified, it appeared to be an actinomycete: a comrnon filamentous soi1

organisrn. This organism was indigenous to the soi1 as evîdenced by its presence in both

the abiotic control and the biphenyl amended microcosm. The fact that no PCB

degradation was observed in the control can be explained by the absence of an inducer. It

is of primary importance that al1 three microcosms which showed significant losses

contained a substance which could act as an inducer in the cometabolism of PCB (Figures

5.5 and 5.9).

5.1 1 Soil pH

Change in soi1 conditions, such as pH, can affect the ability of bactena to proliferate and

perform metabolic functions. Psychrotrophic organisms are especially sensitive to

variations in environmental conditions.

Aroclor 1260 microcosms were analyzed for soil pH after 5 months. Average values with

standard deviations are presented in Table 5.3. Addition of 2%(w/w) peat moss reduced

pH to about 5.6, corresponding to a hydrogen ion concentration an order of magnitude

above that of the biotic control. Biphenyl addition also resulted in a slight acidification

of the soil.

Table 5.3: Soil pH of Arocior 1260 microcosms after 5 months of incubation at 5'C.

Initial pH=7.0.

Treatment pH at 5 months

abiotic control 6.5

biotic control 6.8

sterile peat amended 5.6

nutrient amended 6.6

nutnent and peat amended 5.7

nutrient and biphenyl amended 6.4

Soil pH measurements were ais0 recorded for the 1997 biopiles (Table 5.4). The biotic

control, which was unarnended soil, was near neutrality.

Nutrient addition resulted in a slight acidification @H=6.9) of the soil.

Al1 of the buiking agents resuited in depressing the pH fiom 6.9 to approximately 6.4.

1 %(w/w) peat amendmeq,,. reaulted in a greater degree of acidification than either,

peariite, bluegrass, or aminoplast.

When the potential induces were added to the soils which were already amended with

peat and nutrients, the pH dropped M e r . These soils had a hydrogen ion concentration

one order of magnitude higher than the biotic controls.

Many of the biopiles exhibited a reduction in pH as the soils were incubated. It is

believed that the production of acidic metabolites was the cause. These metabolic

byproducts may be toxic to the organisms, and product inhibition may be a factor in the

cessation of PCB degradation.

Table 5.4: Soi1 pH of 1997 biopiles after 18 days, 3 1 days, and 180 days of incubation at

20°c.

t= 1 8 days t=3 1 days t=180 days

nuirient study

abiotic control 6.9 6.8 7.0

biotic control 7.2 7.0 7.0

nutrient 6.9 6.8 6.5

buking agent study

pearlite+ nutrient 6.6 6.8 5.7

bluegrass+ nutrient 6.5 6.9 5.8

inducer srudy

peat+nutrient+ biphenyl 6.1 5 -9 6.1

peat+nutrient+ quercetin 6.1 6.1 5.4

5.12 Inoculation

There are reports in the literature that the addition of microorganisms which c m degrade

specific recalcitrant compounds like PCB has resulted in the degradation of such

compounds which might othenvise peaist in the environment (Focht and Bnuiner, 1985).

Since the number of specific degraders and hence the biocatalyst concentration is high,

bioaugmentation reduces the arnount of t h e required to treat a contaminant.

The manner in which the inoculum is prepared and introduced on site can have an impact

on the sumival of the microbial cells and hence the arnount of active biocataiyst which is

available to degrade PCB. Because Saglek is a remote site, the feasibility and ease of

various methods of preparing and transporting a viable inoculurn were investigated. Three

inoculurn preparittions were tested: a live culture supported on coarse peat moss during

transportation, a centrifuged culture which was resuspended on site, and a freeze-dried

culture which was resuspended on site.

A 14 1 % degradation of kocior 1260 was obse~ed in the uninocuiated contml(Figure

5.16). Inoculation widi the resuspended lyophilized culture increased degradation to 23 * 0.1%. The concentrated live suspension and peat immobilized cells did not result in

increased removals, 15 3% and 13 7% respectively. It is likely that the peat

immobilized culture and the concentrated [ive suspension did not survive weii during

transportaiion. ï h i s would have resulted in a lower ce11 number in the inocula relative to

the revived lyophilized preparatïon.

nutrient and Wat concentrated tyophilized peat oniy imbilized Que suspension cutture

Figure 5.16: Aroclor 1260 degradation observed in 2.4kg biopiles incubated for

eleven months at 20C (inoculurn study).

5.1 3 Moisture-nutrient-peat optimization

This rnicrocosm study was designed to obtain the optimum concentrations of water,

nutrients and peat moss for scale-up in a landfm application. The optimum operating

conditions were found to be at a moisture content of 40% the water holding capacity of

the soil, 1 %(w/w) peat moss, and a nutrient concentration between 0.1 and 0.4 g N k g soil

at an N:P ratio of 5: 1 (Figure 5.17).

peat O to 2%

nutrients O to 0.5 g/kg

Figure 5.17: Results of optimization study. The CO-

ordinate system in (b) refen to the 3-D diagram in (a) of a cube going into the page. The face of the cube represents treatments where nutrients were not added, whereas the back of the cube represents treatments which received 0.5g Nkg soil. The surface at the bottom of the cube represents treatments which received no peat, whereas the surface at the top represents treatments which received 2% peat. The left surface of the cube represents treatments which were 20% moist, whereas the nght surface of the cube represents treatments which were 60% moist. Numbers refer to percent Aroclor 122 1 degradation.

Within the range tested in this study, nutrient addition stimulated PCB degradation at al1

combinations of peat concentration and moisîure content. This result contradicts the

negative impact of nutrient addition observed in the studies discussed in Section 5.3. The

reason for this discrepancy is likely the difference in the N:P ratio. Results of the 2.4kg

biopile study indicated that an N P ratio of 2.5: 1 was preferable to 1 : 1. By further

increasing the ratio to 5: 1, nutrient addition can stimulate PCB depdation even without

peat amendment.

As water content was increased from 20% to 40%, the effectiveness of the process also

increased. Above this moishue however, aerobic metabolism decreased. This was likely

due to aggregate formation resulting in oxygen limitanon at the center.

Peat addition did stimulate the bioremediation process, but an intermediate optimal value

was also observed for this parameter. Above l%(w/w) peat moss, the PCB degradation

decreased, possibly due to the acidification effect of this humic material.

6. Results: Anaerobic studies

6 . 1 Introduction

Four anaerobic studies were conducted. They are: an anaerobic biopile study, an

inoculation test, a srnall scale deoxygenation study, and a large scale deoxygenation

study. As the reductive dechlorination process requires very low redox potentials

(-400mV), manipulating the soil conditions by stimulating oxygen scavenging aerobes

was a major focus of these studies. Other investigations included the stimulation of

indigenous anaerobic microorganisms and survival of an introduced anaerobic PCB

degrading consortia.

The biopile study consisted of four distinct preparations each prepared in triplkate. The

biopiles consisted of 3kg sahirated soi1 with arnendments, and were incubated for six

months at 20°C. Redox potentiais were measured and PCB losses analyzed.

The inoculation test consisted of two 8kg soil systems saturated with water, one of which

was arnended with nutrients and carbon. After two weeks of incubation to stimulate

oxygen scavenging organisms, the amended soi1 was inoculated with a PCB

dechlorinating consortia. The inoculation test soils were incubated for five months at

20'~. Redox potentials were measured before inoculation and after the six month

incubation. A qualitative PCB analysis was performed to determine if dechlorination had

occurred.

The smail scale deoxygenation study consisted of seven preparations each prepared in

duplicate. The preparations contained 1 kg of subsaturated soi1 arnended with various

concentrations of nutrients and carbon. Tirne to oxygen depietion was determined using

oxygen sensitive indicator strips.

A large scale deoxygenation study was conducted in 1.2m x 1.2m x 1.2m wooden crates

filled with subsaturated soils. The soi1 at Saglek is being excavated and stored in this

type of crate unti1 a remediation technique is available. Redox potential was measured

afker 1.5 rnonths of incubation. During the incubation, the crates were stored on Antenna

Hill, and were exposed to daily temperature fluctuations.

6.2 Redox potential manipulation

After six months of incubation, redox potentials of the biopiles had dropped significantiy

fiom the original value of about 5mV (Table 6.1). Aithough the observed redox

potentials were not low enough to support PCB-dechlorinating consortia, the results

hdicate that, with M e r optimization, it may be possible to attain the desired

environmental conditions.

Table 6.1 : Redox potentials of anaerobic biopiles after 5 months of incubation. Initial

redox potential = 5mV.

Treatment Redox potentid (mv)

control -33 & 10

nutrient -40 i 12

nutrient + carbon -42 k 10

nutrient + carbon + sedirnent -24 & 6

After incubating the inoculation test vessels for two weeks, the redox potential of the

amended soils had dropped to -175mV; the control soi1 remained at a value of -5rnV.

M e r five months of incubation, the redox potentials of the soils in both vessels was

measured at -25mV. The observed increase in redox potentiai between the measurements

taken at two weeks and five months may be due to carbon limitation. Initially, the easily

degradable carbon was quickly consumed by aerobic organisrns resulting in low redox

potentials. When the aerobic organisms depleted the biodegradable carbon metabolic

activity slowed down, and the rate of oxygen diffusion into the vessels overcame the rate

of oxygen depletion. A more intricate culture vesse1 with proper seals and a gas trap

could potentially maintain low redox potentials.

The small scale deoxygenation preparations were monitored for tirne to oxygen depletion

using treated paper which turned fiom blue to white in the absence of oxygen. Oxygen

depletion was rapid (<4 hours) regardless of amenciments as shown in Table 6.2.

Table 6.2: Summary of arnendments added to each treatment in the lkg soi1

deoxygenation study and the time to oxygen depletion.

Treatment Time

control 2 hours

low nutrient

no carbon

low carbon

medium carbon

high carbon

2.5 * 0.5 hours

2 hous

3.5 * 0.5 hours

3 * 0 hours

hi& nutrient

low carbon 2.5 k 0.5 hours

medium carbon 2.5 * 0.5 hours

After 1.5 months incubation the five treatrnents which were amended with a carbon

substrate had developed black, red, and white patches. The black and red patches are

indicative of sulfate and iron reduction respectively; both of these microbial processes

require very low redox potentials. These observations suggest that it may be possible to

obtain environmental conditions conducive to PCB dechlorination while contaminated

soils are in storage.

The redox potentials of the soil-filled crates were measured after one month of incubation

by removing a core and inserting a redox probe into core sections fiom various depths.

The average redox potentials of the control and the amended soil were both

approximately -5mV. The well aerated soil had a redox potential of 5mV. By saturating

the soils, it may be possible to further suppress redox potentids.

6 . 3 PCB degradation

The anaerobic biopiles were sarnpled after six months of incubation. A reduction of 13%

was observed in the nutrient and carbon amended biopile (Figure 6.1). This observed ioss

may have been effected by aerobic organisms before the oxygen had been consumed fiom

the headspace, and M e r analysis is required to veriS the route of degradation.

control nutrient nutrient+ nutrient+

carbon carbon+ sediment

Figure 6.1 : Aroclor 1260 losses observed in anaerobic biopiles incubated for six

months at 20C.

6.4 Inoculation

The inoculated vesse1 was supporting a very healthy anaerobic community when sampled

after 5 months of incubation. When the lid was removed, a strong scent of fatty acids

emanated fkom the system, and a thick layer of rusty-brown scum was floating on the

surface of the liquid. The soi1 had become somewhat gray, and the liquid had become

black. Visually, the control system had not changed.

A qualitative andysis indicated that PCB dechlorination had not occurred.

7. Conclusions

These midies were conducted to assess the feasibility of implementing a biologicai

system for the remediation of PCB contarninated soils at the long-range radar facility at

Saglek (LAB-2). The system would involve two stages. An anaerobic dechlorination

step would fust remove some of the meta- and para- substituted chlorine atoms. An

aerobic step could then mineralize the lowly chlorinated, mostly ortho- substituted

biphenyl molecules to water, carbon dioxide, and chloride ions.

Anaerobic studies were conducted on two scaies: treatment of about 5kg of soil, and

treatment of about 3 tonnes of soil. The smaller scale studies indicated that redox

potentials can be significantly reduced solely by oxygen-scavenging microorganisms;

redox potentials were lowered to - 175mV in one study. Further optimization is required

to attain ideai redox potentials of AOOmV. Poor results were obtained in the larger scale

systems where the redox potentials were reduced only to -5mV. The soil in these larger

systems were not saturated, and this is likely the reason for the large discrepancy.

The smailer anaerobic systerns, which contained saturated soil covered to depths of about

1 Ocm by aqueous liquid were particularly effective when a carbon source was provided at

liberai concentrations. In one of these systems an anaerobic culture was successfdly

introoduced, and dinved without any intervention for 5 months.

Aerobic studies were performed on two scales: microcosrns containing less than lOOg

soil, and biopiles containing up to 4kg soil. Several amendments were tested for their

ability to stimulate PCB biodegradation. Loosening the soi1 and maintainhg moisture

was found to improve degradation significantly. Biopiles, arnended only with water,

containing Aroclor 1260 contaminated soil at a concentration of 200ppm exhibited

reductions of 20% during a six month incubation at 2 0 ' ~ . An N:P ratio of at least 5: 1

was required to improve PCB losses. Peat addition did irnprove degradation when added

to stored soils which had undergone undesirable microbial succession, but fiesh soils

supported effective populations which were hindered by the acidincation effect of peat.

Although sorne inducers slightly stimulated the degradation of PCB to chlorobenzoic acid

and chlorinated carboxylic acid, the mineralization of these intemediates was Unpeded

by the presence of those inducers.

The rnicrocosm studies indicated that the optimum operating conditions of a subsaturated

aerobic soil remediation system for Aroclor 122 1 degradation are: a moisture content of

40% of the water holding capacity of the soil, l%(w/w) peat moss, and a nitrogen

concentration of O X g available nitrogen/ kg soil at a N:P ratio of 5: 1.

The three aerobic groups of bactena required for the complete mineralization of lowly

chiorinated PCB are indipnous to the Saglek soil as indicated by the radiolabelled ortho-

chlorobiphenyl study.

Although biostirnulation was achieved in Aroclor 1 260 contaminated soil, the degree of

degradation was not extensive enough to justi@ large scale implementation at the site at

Saglek. Further research is required to opllmize and more thoroughly understand the

process before bioremediation can be reliably applied.

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