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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
National Library Bibliothèque nationaIe du Canada
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The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts f?om it Xi la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.
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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