United stales
Department of
Commerce
United States
Environmental Protection
Agency
National Oceanic and Atmospheric Administration
Environmental Research Laboratories
Seattle WA98115
Office of Energy, Minerals, and EPA-600/7-78-148
Industry ju|y 1978
Washington DC 2O460
Research and Development
Microbial Degradation
of Petroleum
Hydrocarbons
Interagency
Energy/Environment
R&D Program
Report
z
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1 Environmental Health Effects Research
2 Environmental Protection Technology
3 Ecological Research
4 Environmental Monitoring
5 Socioeconomic Environmental Studies
6 Scientific and Technical Assessment Reports (STAR)
7 Interagency Energy-Environment Research and Development
8 "Special" Reports
9 Miscellaneous Reports
This report has been assigned to the INTERAGENCY ENERGY-ENVIRONMENT
RESEARCH AND DEVELOPMENT series. Reports in this series result from the
effort funded under the 17-agency Federal Energy/Environment Research and
Development Program These studies relate to EPA's mission to protect the public
health and welfare from adverse effects of pollutants associated with energy sys-
tems. The goal of the Program is to assure the rapid development of domestic
energy supplies in an environmentally-compatible manner by providing the nec-
essary environmental data and control technology. Investigations include analy-
ses of the transport of energy-related pollutants and their health and ecological
effects; assessments of, and development of, control technologies for energy
systems; and integrated assessments of a wide range of energy-related environ-
mental issues.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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MICROBIAL DEGRADATION OF
PETROLEUM HYDROCARBONS
BY
Dr. D.W.S. Westlake
Department of Microbiology
University of Alberta
Edmonton, Alberta
Canada T6G 2E9
Dr. F.D. Cook
Department of Soil Science and Microbiology
University of Alberta
Edmonton, Alberta
Canada T6G 2E9
Dr. A.M. Jobson
Alberta Research Council
Edmonton, Alberta
Canada T6G 2C2
Prepared for the MESA (Marine Ecosystems Analysis) Puget Sound
Project, Seattle, Washington in partial fulfillment of
EPA Interagency Agreement No. D6-E693-EN
Program Element No. EHE625-A
EPA Project Officer: Clinton W. Hall (EPA/Washington, D.C.)
NOAA Project Officer: Howard S. Harris (NOAA/Seattle, WA)
This study was conducted
as part of the Federal
Interagency Energy/Environment
Research and Development Program
Prepared for
OFFICE OF ENERGY, MINERALS, AND INDUSTRY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
May 1978
UNITED STATES
DEPARTMENT OF COMMERCE
Juanita M. Kreps. Secretary
NATIONAL OCEANIC AND
ATMOSPHERIC ADMINISTRATION
Richard A. Frank Administrator
Environmental Research
Laboratories
Wilmot N. Hess. Director
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Completion Report Submitted to
PUGET SOUND ENERGY-RELATED RESEARCH PROJECT
MARINE ECOSYSTEMS ANALYSIS PROGRAM
ENVIRONMENTAL RESEARCH LABORATORIES
by
UNIVERSITY OF ALBERTA
EDMONTON, ALBERTA
CANADA T6G 2E9
This work is the result of research sponsored by the Environmental
Protection Agency and administered by the Environmental Research
Laboratories of the National Oceanic and Atmospheric Administration.
The Environmental Research Laboratories do not approve, recommend,
or endorse any proprietary product or proprietary material mentioned in
this publication. No reference shall be made to the Environmental
Research Laboratories or to this publication furnished by the Environmental
Research Laboratories in any advertising or sales promotion which would
indicate or imply that the Environmental Research Laboratories approve,
recommend, or endorse any proprietary product or proprietary material
mentioned herein, or which has as its purpose an intent to cause directly
or indirectly the advertised product to be used or purchased because of
this Environmental Research Laboratories publication.
ii
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FOREWORD
Substantially increased petroleum tanker traffic, pipeline transport
and refining operations are anticipated in the region of Northern Puget
Sound and Strait of Juan de Fuca with the Alaskan pipeline in operation.
To assess the potential future environmental impact arising from these
activities the Puget Sound Energy-Related Project, contracted the University
of Alberta to undertake a study on the "Microbial Degradation of Petroleum
Hydrocarbons" in Northern Puget Sound and Strait of Juan de Fuca. This
study was supported by U.S. Environmental Protection Agency "pass-through"
funds administered by the NOAA Marine Ecosystem Analysis Program. This
report presents the results of the study.
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ABSTRACT
The responses to Prudhoe Bay oil of the microbial populations present
in water column, beach and sediment samples representative of the diverse
marine shoreline environments found in the northern Puget Sound and Juan de
Fuca areas were investigated under laboratory conditions. The presence and
activity of oil-degrading microbial populations were monitored by determining
changes in the n-alkane components of the saturate fraction of oil by gas
chromatography. Samples were enriched in a nitrogen, phosphorus-deficient
synthetic medium with Prudhoe Bay oil as sole carbon source. Under these
conditions no degradation of Prudhoe Bay oil was observed after either 28
days incubation at 8°C (typical ambient temperature conditions for water in
this area) or 14 days incubation at 30°C. However when nitrogen and phos-
phorus were added to the medium, all sites yielded bacterial populations
capable of utilizing the n-alkanes of Prudhoe Bay oil. The rate of utiliza-
tion was faster at 30°C than at 8°C. Under laboratory conditions approxi-
mately one-third of the total weight of Prudhoe Bay oil was lost by weathering
(i.e. physical-chemical processes), one-third by mineralization due to
microbial activity and one-third left as a residue. No yeasts or fungi were
isolated using these procedures.
Seasonal variations in oil-degrading capabilities were compared by
calculating a "degradative capacity index" (D.C.I.). The index describes the
status of the n-alkane profile of the saturate fraction of degraded, partially
degraded and undegraded Prudhoe Bay oil. If all samples studied contained
microorganisms which completely utilized the n-alkanes and isoprenoids present
in an oil a D.C.I, value of four would be obtained. If none of the samples
contained such microorganisms the D.C.I, would be zero. Water column samples
showed the greatest seasonal variation in their D.C.I, values, being the
lowest in the winter (i.e. January). Beach samples were quite constant in
their ability to yield bacteria capable of utilizing saturate components.
Inter-tidal sediment samples showed some seasonal variability with
regards to the oil-degradative capacity.
Indigenous bacterial populations from sediment, beach and water column
samples primarily consisted of Gram-negative bacteria. Oil-degrading popula-
tions contained relatively high proportions of members of the Gram-negative
genera Pseudomonas and Flavobacterium with varying proportions of Gram-
positive coryneforms. There was no apparent relationship between sample type
or site and the generic composition of bacterial mixtures capable of degrading
Prudhoe Bay 011.
iv
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Initial studies on the rate of oil degradation using a radioisotope
technique also are discussed.
Sediment samples were stored up to six weeks and water column samples
for twelve days without loss of ability to degrade Prudhoe Bay oil.
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CONTENTS
Foreword iii
Abstract iv
Figures vii
Tables viii
Acknowledgement xi
1. Introduction 1
2. Conclusions 4
3. Recommendations 6
4. Materials and Methods 8
5. Results 16
6. Discussion 42
References 46
Appendices
A. Microbiological Media 48
B. Counting!4co2 50
C. Sulfide-generating Data 51
D. Auxiliary Tables 53
vi
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FIGURES
Number Page
1 Map of sample site locations ............... 9
2 Typical 6.C. profiles of Prudhoe Bay oil ......... 12
3 Op rate of release from water column samples - March
Pt. Rd. and West Beach (Deception Pass) .......... 31
4 C02 rate of release from sediment sample - March Pt. Rd. 33
5 14C02 rate of release from mixed sediemnt sample -
Jamestown and Ft. Worden ................. 34
6 Effect of Prudhoe Bay oil on growth of bacteria with and
without nitrogen and phosphorus at 20°C - March Pt. Rd.
and West Beach (Deception Pass) .............. 35
7 Effect of storage temperature on total viable numbers of
bacteria in sediment - March Pt. Rd. and West Beach
(Deception Pass) ..................... 40
vii
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TABLES
Number
1 Latitudes and longitudes of sample sites 10
2 G.C. profile of Prudhoe Bay oil after incubation with water
column samples at 8°C 17
3 G.C. profile of Prudhoe Bay oil after incubation with sedi-
ment samples at 8°C 18
4 G.C. profile of Prudhoe Bay oil after incubation with beach
samples at 8°C 19
5(A) Summary of oil-degrading bacterial activity at 8°C in water
column, sediment and beach samples 20
5(B) Summary of oil-degrading bacterial activity at 8°C at sample
sites 20
6 Viable bacterial counts from beach and sediment samples
(April, 1977 survey) 22
7 G.C. profile of Prudhoe Bay oil after incubation with beach
and sediment samples at 30°C 23
8 Effect of temperature and incubation time on viable bacterial
counts and on G.C. profile of Prudhoe Bay oil incubated with
sediment samples - March Pt. Rd. and Deception Pass (Rosario
Beach) 24
9 Effect of temperature on generic composition of bacterial
populations in water column and sediment samples - West Beach
(Deception Pass) 25
10 Effect of nitrogen and phosphorus concentration on G.C.
profiles after incubation with water column samples at 20°C -
March Pt. Rd. and West Beach (Deception Pass) 26
11 Effect of nitrogen and phosphorus concentration on G.C.
profiles after incubation with combined water column samples
at 20°C - E. Fidalgo Rd. and Neah Bay 28
vi ii
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Number Page
12 Effect of oil concentration on growth and G.C. pattern of
saturate fractions of Prudhoe Bay oil 29
13 Weathering and mineralization data at 8°C and 20°C - March
Pt. Rd. and Rosario Beach 30
14 Generic composition of bacterial populations from beach
samples before and after enrichment at 8°C and 20°C - March
Pt. Rd 36
15 Generic composition of bacterial populations from sediment
samples before and after enrichment at 8°C and 20°C - West
Beach (Deception Pass) 37
16 Generic composition of bacterial populations from water
column samples before and after enrichment at 8°C and 20°C -
West Beach (Deception Pass) 38
17 Generic composition of bacterial populations from sediment
samples before and after enrichment at 8°C and 20°C - False
Bay, San Juan Island 39
Cl Incidence of sulfide-generating bacteria in sediment samples. 51
C2 Incidence of sulfide-generating bacteria in beach samples . . 52
Dl pH of water column samples 53
D2 Temperature of water column samples 54
D3 Dissolved oxygen content of water column samples 55
D4 Salinity of water column samples 56
D5 G.C. profiles after incubation with water column samples at
8°C 57
D6 G.C. profiles after incubation with sediment samples at 8°C . 58
D7 G.C. profiles after incubation with beach samples at 8°C. . . 59
D8 Effect of temperature, incubation time and sample type on
G.C. profiles of Prudhoe Bay oil after incubation at 8°C or
20°C 60
D9 Available phosphorus content of beach, sediment and water
column samples 61
D10 Suspended solids content of representative water column
samples 62
ix
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Number Page
Dll Generic composition of bacterial populations from beach
samples (April, 1977) ' 63
D12 Generic composition of bacterial populations from sediment
samples (April, 1977) 64
D13 Generic composition of bacterial populations from water
column samples after enrichment at 8°C and 20°C - March Pt.
Rd 55
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ACKNOWLEDGEMENTS
We wish to thank Mr. D. Horsfield, Miss. J. Foght and Mr. R. Phillippe
for technical assistance. The efforts of Mr. P. Fedorak in development and
application of the computer data processes used in this study are gratefully
acknowledged. Dr. Adolph Snow at the ARCO Harvey Technical Center provided
the Prudhoe Bay oil used in these experiments.
xi
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SECTION 1
INTRODUCTION
The Straits of Juan de Fuca/northern Puget Sound area have experienced
a continuous increase in oil-tanker and refinery activities over the past
decades. The hydrocarbon demands of the Pacific Northwest and the rest of
the United States coupled with the completion of the Alyeska Pipeline
indicate that the scope of oil-related activities will continue to increase
in the future. As a result of this accelerated activity there is an
increased risk of an oil spill taking place.
The ability of bacteria, yeast and fungi (i.e. microorganisms) to
utilize hydrocarbons as sources of energy and cell carbon is a characteristic
which is widespread in microorganisms but seldom found in other forms of
life (1). This capability together with the ubiquitous distribution of
microorganisms in aquatic and terrestrial environments provides a biological
system which contributes to the natural clean-up of oil spilled in the
environment. Information on the distribution and factors affecting the
activities of oil-degrading microorganisms in the marine environment has been
the subject of recent reviews (2-4). However since no data is available for
the Strait of Juan de Fuca/northern Puget Sound area the primary objective
of this study was to investigate the distribution and factors affecting the
activity of oil-utilizing microorganisms under the environmental conditions
prevalent in this area. Studies were designed to obtain information on
the effect of geographic variation, seasonal variation and proximity to oil
sources on the activity, distribution and types of oil-degrading microbial
flora found in this area.
Microorganisms are intimately involved in the maintenance of the
biological food chain. An understanding of factors, e.g. oil, affecting
the presence and activities of microorganisms is essential to the under-
standing and management of natural ecosystems. Therefore a secondary
object of this study was to investigate the effect of oil on the indigenous
bacterial flora of water, sediments and beaches representative of the
diverse types of marine environment found in this area.
Studies on the enumeration of hydrocarbon-utilizing microorganisms
have been concerned with their incidence as a fraction of the normal hetero-
trophic population (5). The problems encountered in such enumeration studies
have been investigated by Walker and Colwell (6). Such experimentation has
shown that oil-or hydrocarbon-polluted areas have higher levels of hydrocarbon-
utilizing microorganisms than adjacent unpolluted areas. In general, these
studies have been carried out using a pure hydrocarbon, such as an n-alkane
1
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e.g. hexadecane as carbon source, the hypothesis being that the ability to
grow on a pure n-alkane is related to the ability to grow on a whole crude
oil. However data are available which show that the ability to grow on pure
hydrocarbons does not necessarily correlate well with an ability to grow on
•:rude oil even though the n-alkane component is present in that oil. For
example, fungi are noted for their ability to grow on n-alkanes (7), although
few studies are published on their ability to grow on oils. Davies and
Westlake (unpublished results) determined the ability of such fungal isolates
including Cladosporium resinae, and new fungal isolates obtained by a crude
oil enrichment technique, to utilize crude oils. A very poor correlation
was observed between utilization of an n-alkane (i.e. tetradecane) and the
ability to grow on crude oils containing n-alkanes. Information (8-10) also
is in the literature showing that the chemical composition of crude oil can
influence the microbial and thus the chemical responses observed. In order
the consider the interaction of different compounds present in oil, studies
on the distribution of oil-degrading microorganisms should be carried out
using a whole crude oil typical of those which might be spilled in the area.
The measurement of the microbial growth by monitoring the increase in
cell mass or numbers when a complex material such as oil is used as sub-
strate does not yield information as to what compound(s) is(are) supporting
growth. Growth can be monitored indirectly by following changes in the
concentration of the substrate(s) present (11). Of the four major components
of crude oil i.e. asphaltenes, saturates, aromatics and the polar N.S.O.
(nitrogen, sulfur, oxygen) - containing molecules only the n-alkanes (and
to a lesser extent the isoprenoids) of the saturate fraction (8) and the
mono-, di- and tri-ring compounds of the aromatic fraction (12, 24) are
readily degraded by microorganisms. Changes in the content of the n-alkanes
and the isoprenoids can be readily detected by gas chromatography (G.C.).
While the aromatic compounds can be resolved by G.C. their resolution depends
on a more rigorous pre-treatment of the oil than is required for monitoring
changes in the n-alkanes and isoprenoids. The approach used in these studies
to assess the distribution and factors affecting the activity of oil-
degrading microorganisms was based on an enrichment technique utilizing crude
oil (Prudhoe Bay) as sole carbon and energy source. Changes in the n-alkane
and isoprenoid components of recovered oil were monitored by G.C. and used
as an indicator of oil-degrading microbial activity.
Weathering, i.e. the loss of volatile components by physical-chemical
processes, and mineralization, i.e. the conversion of organic carbon to C02»
are natural processes by which oil may be removed from the environment.
While the volatile components contain the most toxic components of oil, they
also contain some of the more biodegradable compounds, and thus their loss
will affect the amount and types of microorganisms which will grow on oil.
The weathering also can result in the formation of structures, e.g. tar balls,
which are resistant to microbial attack. Thus the interaction between these
processes can affect the rate at which oil is removed from the environment
by natural processes. The relationship between these two processes and loss
of Prudhoe Bay oil in water column and sediment enrichments was determined
under laboratory conditions using a gravimetric procedure. Intial experi-
ments also were carried out on the application of a technique using the rate
of '4C02 liberation from 14c-hexadecane-"spiked" Prudhoe Bay oil as an
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indicator of the rate of n-alkane removal.
The activity of oil-utilizing microorganisms has been shown to be
controlled by temperature (13-15) and by the nutrient status, especially
nitrogen and phosphorus content (15-18, 23), of the environment. Thus the
effects of these parameters on the activity of microbial oil-degrading
populations also were investigated in the Strait of Juan de Fuca/northern
Puget Sound area.
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SECTION 2
CONCLUSIONS
The results of the survey for the presence of oil-degrading micro-
organisms in water columns, beach and sediment samples representative of the
marine environments found in the northern Puget Sound, San Juan Islands and
the southern shore of the Strait of Juan de Fuca indicate their ubiquitous
presence in these areas. Microbial psychrotrophic (cold-tolerant) and
psychrophilic (cold-requiring) populations will predominate as the tempera-
tures of these marine environments are in the range of 5°C to 20°C.
The enrichment procedures used resulted in the isolation of mixed
populations of psychrotolerant, Prudhoe Bay oil-degrading bacteria. No yeasts
or fungi were observed under these experimental conditions. The oil-degrading
bacterial populations obtained primarily consisted of members of the Gram-
negative genera Pseudomonas or Flavobacterium and the Gram-positive coryneform
groups. The proportion in which members of these groups occurred varied but
was not related to a particular sample type, sample site or enrichment con-
dition e.g. temperature. However in one sample, sediment from False Bay,
San Juan Island, members of the Alcaligenes genus comprised the majority of
the Gram-negative bacteria found on the oil-degrading population. All of these
taxonomic groups have been reported to contain oil-and/or hydrocarbon-
degrading organisms. Members of these groups also were present in unenriched
indigenous bacterial populations.
Oil-degrading activity of these bacterial populations, as measured by
determining the degradation patterns of the n-alkane and isoprenoid profiles
of recovered oil, increased with increasing temperature. Activity however
was observed only when sample materials were incubated with a nutrient
supplement containing nitrogen and phosphorus. The level of nitrogen present
was more critical in determining the activity observed than was the level of
phosphorus. If an oil spill occurs in this area, then the residual oil left
after physical-mechanical processes have been used to recover oil would only
be rapidly degraded if nitrogen and phosphorus were provided,theoretically
in an oleophilic form. Under these conditions the ambient temperature, not
the nutritional status of the environment - would become the rate-limiting
parameter. Similarly previous laboratory studies reported that nitrogen
and phosphorus levels controlled the rate of oil degradation on northern
soils. The application of fertilizer containing nitrogen and phosphorus to
actual spills in that area accelerated the rate of oil degradation and the
rate of revegetation of such sites.
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Under laboratory conditions used, approximately 1/3 of the weight of
Prudhoe Bay oil was lost by weathering, 1/3 lost by mineralization leaving a
residue of 1/3 of the original weight of oil. Similar values were obtained
at both 8°C and 20°C.
The bacterial population in "pristine" areas such as Rosario Beach
and West Beach (Deception Pass Park) which contain low levels of organic
matter and are relatively free of oil-industrial activities, is more sensitive
to added oil than that present in organic matter-rich areas like March Pt. Rd.,
E. Fidalgo Rd. and the refinery site (Cherry Point) which are also near oil-
industrial activities. The growth responses of oil-degrading populations
from these two environments, while of a different order of magnitude, are
similar. Initial studies on the rate of n-alkane utilization indicate that
water column bacterial populations from sites near oil-industrial activities
have faster degradation rates than observed in water column samples from
"pristine" areas. Sediment samples from these two types of environment
however gave similar rates of n-alkane degradation.
Sediment samples also can be stored for up to 6 weeks without loss
of oil-degrading capabilities. Water column samples have been stored for twelve
days at 8°C without loss of oil-degrading capabilities although significant
increases in viable populations occurred.
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SECTION 3
RECOMMENDATIONS
The results of this initial survey indicate that the Pt. Partridge
Park and the E. Fidalgo sites represent the extremes of the marine environment
studied regarding bacterial oil-degrading capability. These two sites should
be the subject of an intensive investigation. The nature of the indigenous
bacterial populations present, the rate of oil-degradation and the effect of
oil on the indigenous bacterial populations should be investigated. Samples
from these sites should be limited to water column and beach material since
sediment samples (as defined in this study) represent a continuously changing
transition zone between water column and beach materials. All samples should
be analyzed for nitrogen, phosphorus, organic carbon, oils and greases and
oil-degrading capability. Representative samples should also be subjected
to enrichment procedures which would select for oil-degrading yeast and fungi.
The effect of nutritional conditions - the nitrogen-phosphorus levels used
and presence of iron or yeast extract - should also be investigated on samples
representative of these two areas. Sampling times must be scheduled so as to
confirm the seasonal trend noted for water column samples. These studies
should be complemented by parallel investigations with Port Angeles area as
a site representative of industrial activity (particularly areas of known
hydrocarbon content) and Freshwater Bay as being representative of a
"pristine" control. Studies to date have centered on the use of Prudhoe Bay
oil as carbon and energy source. As the refineries at Anacortes and Cherry
Point are receiving other oil and as it has been clearly shown that oils
differ in their biodegradability characteristics (and their toxicity), the
study of oil-degrading activity at the above sites should also be measured
against oils which are presently being transported in this area.
As many beaches in this area consist of rocks and cobble stones the
ability of microbial populations in these areas to respond to oil should be
investigated. Similarly a study of the ability of plant material e.g. kelp
to harbor oil-degrading microorganisms and the effect of oil on their
epiphytic microbial flora should be undertaken.
The presence and effect of oil on sub-surface microbial populations
should be studied. In particular a comparison should be undertaken of sub-
surface water samples from the Strait of Juan de Fuca (which should have a
low sediment content) with high sediment-containing samples from waters
affected by the Fraser orSkagit River drainage systems. Similarly, subtidal
sediments from hydrocarbon-polluted areas and pristine areas should be
investigated. The degradation rate of oil in sediment material low in
oxygen should be studied, as the long term effects of oil pollution will be
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expressed in this environment.
Consideration should be given to the evaluation of the effect of
generic composition of oil-degrading populations on the rate and chemical
changes brought about in oil. In particular, there is a need for the develop-
ment and acceptance of a taxonomic classification system which if adopted
would make possible meaningful comparisons between results obtained in
different laboratories.
Comparisons of rates of oil-degradation should be expressed on a sur-
face basis as well as on a per gram and/or ml basis as particle contents from
different sites will have different surface areas depending on sediment
composition. All experiments should be carried out under psychrophilic condi-
tions i.e. around 4°C to 8°C as this represents the rate-limiting minimum for
this parameter in this area.
An integrated "in situ" study involving chemists, microbiologists and
biologists at a selected site to determine the environmental effect of oil
spills on the total ecosystem should be undertaken. The data provided not
only would outline the effect of an oil spill but would also possibly permit
the development of procedures to minimize oil's effect on the marine environ-
ment in this area.
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SECTION 4
MATERIALS AND METHODS
Sample Sites
The sites used in this study are shown in Figure 1, and their
locations (latitude and longitude) described in Table 1. The environmental
codes characterizing each sample site are available in NOAA's computerized
data bank. Sites were chosen to represent the diverse environmental
situations encountered in this study area and on the basis of their access-
ibility. Whenever tide and terrain permitted, three samples - a water
column, a sediment and a beach sample - were obtained at each site. All
materials were transported in cooler chests containing sufficient ice packs
to keep the temperature at 10°C ± 5°C.
Water samples were obtained at approximately 30 to 45 cm depth using
a wide mouth 4 litre plastic bottle. Microbial, physical and chemical
examinations were carried out on a sample which was obtained after rinsing
the bottle three times with the water to be examined. Large volumes of water
for laboratory use were taken and transported in sterile 4 litre bottles.
In this survey sediment samples refer to the sand which was saturated
by tidal action (mid to low intertidal) and beach samples refer to sand above
the detritis (upper intertidal) left by spring tidal action. Two hundred
to five hundred grams of surface composite (1 cm depth) sediment or beach
materials from approximately a 0.5 square meter area were transferred using
sterile tongue depressors to sterile 250 ml centrifuge bottles. Beach and
sediment samples were stored at -20°C unless otherwise indicated.
Samples were obtained from sites as indicated in this report in April,
July, September, November, 1977 and January, 1978.
Microbiological Survey and Activity Studies
The chemical composition of all microbiological media used in this
study is reported in Appendix A. An enrichment technique using a mineral
medium with a neutral pH and Prudhoe Bay oil as sole carbon source was used
to screen for the presence of oil-degrading microorganisms. The effect of
nutrition (i.e. nitrogen and phosphorus levels) on oil-degrading activities
of such populations was investigated using this technique.
The activity of water column material was studied by setting up "in
situ" duplicate enrichments consisting of two hundred ml of water and 0.2 ml
8
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49*
45'
30
15
48°
Vancouver
Island
CANADA
UNITED STATES
Cherry Point
Victoria
of Juan de Fuca
Angeles
Figure 1. Map showing location of sample sites used in this study (see Table 1 for description)
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TABLE 1. SAMPLING LOCATIONS
Site No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
Description
Birch Bay State Park
Between Atlantic Richfield and
Mobil Docks (refinery site)
Beach Access of Sucia Road
Samish Island Public Beach
2 miles east Jet. 20W and East
March Point Road
Mud Flats 2 miles from Jet. East
March Point Road and East Fidalgo
Roads
Rosario Beach3
Point Partridge Park
Entrance Dungeness State Park
Clallam Bay
Pillar Point
Salt Creek Recreation Campground
Port Angeles (Below Red Lion Inn)
Fort Worden State Park
Jamestown
Neah Bay
Lopez Island - Shoal Bightb
San Juan Island - South Beachb
Orcas Island - Crescent Beachb
Deception Pass State Park -
West Beach
Orcas Island - Terrill Beachb
San Juan Island - False Bayb
Latitude (N)
485405
485108
485124
483450
482930
482930
482510
481330
480935
481 540
481251
481000
480710
480845
480831
482337
482737
482723
484141
482408
484244
482915
Longitude (W)
1224610
1224500
1 224349
1223235
1223320
1223550
1223935
1224605
1230900
1241720
1 240603
1234217
1232525
1224525
1230623
1243021
1224906
1230013
1225351
1223946
1225252
1230352
only sampled in April and July, 1977; site 20 used for September,
November, 1977 and January, 1978 surveys
bonly sampled in September, 1977 survey
10
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of Prudhoe Bay oil in 500 ml screw-capped Erlenmeyer flasks. One of the
duplicate flasks received 2.0 ml of a nitrogen-phosphorus supplement. To
permit aeration the screw-caps were replaced in the laboratory with sterile
foam plugs prior to incubation on a rotary shaker. Ten grams of beach or
sediment material were removed aseptically in a laminar flow hood and added
to 500 ml Erlenmeyer flasks each containing 200 ml of sterile artificial sea
water medium and 0.2 ml Prudhoe Bay oil. Duplicate flasks were prepared for
each sample, one of which received a nutrient supplement of nitrogen and
phosphorus, and all flasks were incubated in a rotary shaker. Unless other-
wise specified all rotary shakers were run at 250 rpm and had an eccentricity
of 3.8 cm. The temperature of incubation varied with experiments. After
a suitable incubation period the oil was recovered and the pentane-soluble
fraction subjected to gas chromatographic (G.C.) analysis. Activity was
determined by comparing the G.C. profile of the saturate fraction of
recovered oils with that of undegraded Prudhoe Bay oil subjected to the same
recovery procedures. Typical G.C. profiles obtained by this technique and
used in evaluating bacterial oil-degrading activity are shown in Figure 2.
A "degradative capacity index" was calculated for each sample material by
numerically rating a completely degraded n-saturate fraction (i.e. C) equal to
4; presence of residual isoprenoids (i.e. I) equal to 3; partial reduction of
n-alkanes profile (i.e. P) equal to 2; selective removal of n-alkanes, C]2 to
Cig in carbon content (i.e. S) equal to 1; no degradation of n-alkanes (i.e.
NJ equal to 0; summing the numerical values and dividing by the number of
samples.
Microbial Numbers
The spread plate technique using a basal marine agar was used in all
these studies for determining total viable count. All dilution blanks used
contained an artificial sea water salt solution at pH 7.3. Plates were dried
before using by incubating for 2-3 days at 22°C and were stored at 4°C in
sealed plastic bags. Plates used in the field were transported, inverted, in
sealed plastic bags. Five replicate plates were prepared of each dilution
and viable counts are reported as the average (±) one standard deviation.
Microbial counts from water column samples are reported per ml and beach and
sediment data per gram of dry material.
Isolates for taxonomic studies were obtained by grouping colonies
growing in five plates of the dilution used for enumeration as to colonial
color and morphology. Colonies representative of each type in all surveys
were checked for purity by streaking on basal medium agar. The April isolates
were examined for cellular morphology, Gram reaction, oxidase test and were
compared as to their ability to grow in glucose tubes under aerobic and
anaerobic conditions. The presence or absence of flagella on these isolates
was determined by examining negatively-stained (1% phosphotungstic acid)
preparations using an electron microscope. The isolates from the later
surveys were examined for cellular morphology, Gram reaction, oxidase reaction,
catalase reaction, and the utilization of glucose and lactose under aerobic
and anaerobic conditions. Motility was determined either by examining wet
mounts or by directly observing flagella with the electron microscope
technique previously described.
11
-------
Ma water — N.P
n-C«
Figure 2. Typical G.C. profile of saturate fraction of Prudhoe Bay oil
before and after growth of enrichment populations (N=no
degradation; S=selective metabolism, n-alkanes Ci2 to Cig;
P=partial removal, n-alkane peaks still discernible; I=only
isoprenoids remaining; C=complete utilization of n-alkanes
and isoprenoids).
12
-------
Type cultures of isolates were kept by storage at 4°C on basal marine
agar in sealed plastic bags. Some oil-degrading populations were maintained
by monthly transfer in the enrichment medium containing Prudhoe Bay oil.
Physical and Chemical Measurements
All instruments were calibrated before and after each sample trip in
addition to the usual adjustments made prior to each measurement.
The acidity of water column samples was determined using a portable
Radiometer pH meter (Model #29) which was standardized using a Radiometer
(51001) buffer system. Salinity and water column temperature were measured
in the field using a Yellow Springs SCT meter (Model #33). The salinity
function of this instrument was calibrated according to the manufacturer's
specifications (i.e. with a standard solution of KC1 in distilled water).
.Beach temperatures were obtained using a standard laboratory thermometer
(0-50°C scale). Dissolved oxygen content of water column samples was obtained
using a Yellow Springs Oxygen meter (Model #52) which was calibrated against
a dropping mercury electrode system at the City of Edmonton's Goldbar
Wastewater Treatment Plant analytical control laboratory.
Visual observations of habitat parameters were recorded and submitted
as part of the computer data bank but are not included in this report.
The dry weights of beach and sediment samples were determined by
accurately weighing, in triplicate, 1 to 2 gram samples and drying for
72 hours at 100°C. The average difference between the wet and dry weights
was used to calculate a correction factor for reporting results on a dry
weight basis. The weight of suspended material in water column samples was
obtained by filtering aliquots through glass fibre filters (Reeve Angel)
and weighing after drying to constant weight at 104°C.
The weight of oil lost due to weathering (inoculum consisted of
sediment or water sterilized by autoclaving) and/or mineralization was
determined by recovering oil from cultures using a chloroform extraction
procedure and evaporating to dryness. This was followed by extraction of
the residue with benzene, filtration of the soluble fraction, evaporation
of the benzene and weighing of the dried residue. The sterility of the cul-
tures used for measuring weathering was checked throughout the experiment
using a plate count technique.
Oil was recovered from cultures for gas chromatography by a flotation
technique. Cultures were cooled in an ice-water bath, and acidified to
pH < 1 to minimize emulsion formation. Five ml of n-pentane were added
and the culture flasks were stoppered with rubber bungs previously washed
with pentane and benzene. The inner surfaces of the flasks were gently
rinsed with the pentane layer and then the contents mixed for 25 minutes
on a rotary shaker at 200 rpm. The oil-pentane mixture was recovered by
replacing the solid stopper with one containing a short fine bore glass tube
(3 mm internal diameter) and a larger bore (5 mm internal diameter) glass
tube reaching the bottom of the flask. Tap water was slowly added via the
larger tube so that the pentane-oil mixture slowly floated to the top of the
13
-------
Erlenmeyer flask and was recovered via the fine bore tube using a microlitre-
hypodernric syringe. If a sample was not immediately run on the gas chroma-
tograph, it was left in the acidified state and held at 4°C until the oil
could be extracted and immediately analyzed.
The n-alkane and isoprenoid content of the pentane-soluble fraction
of recovered oils was obtained on a Van'an Aerograph Model 1740 gas chromato-
graph. This instrument is equipped with a flame ionization detector and two
6.1 m x 0.32 cm stainless steel columns packed with 3% SE 30 ultra-phase on
Chromosorb W (AE-DMCS), 80/100 mesh. The carrier gas used was N2 at a flow
rate of 15 ml/min. The temperature program consisted of a 2 min holding
period at 50°C, then the temperature was increased at a rate of 10°/min from
50°C to 300°C and program completed by a period of 20 min at 300°C.
The oxygen content of sealed incubation mixtures (used for radioactive
studies) was determined using a Varian Model 700 gas chromatograph equipped
with a thermal conductivity detector. Oxygen was resolved from other gases
using a 3.7 m x 0.95 cm aluminum column packed with molecular sieve 13X
(40/60 mesh). Helium was used as a carrier gas at a flow rate of 32 ml/min
and the oven temperature was maintained at 60°C. Oxygen content was calcu-
lated using an air reference sample and comparing peak heights.
The methods used for nitrogen and phosphorus analyses are described
in Methods of Soil Analysis (19). The ammonium and nitrate nitrogen content
of samples was analyzed by a micro-Kjeldahl technique. Beach and sediment
samples were dried and screened through a 2 mm sieve. The material passing
through the sieve was divided into two samples: one was extracted with
sodium bicarbonate (0.5M) and the other with an ammonium fluoride (0.03N)-
dilute sulfuric acid (0.03N) solution. The soluble phosphorus content of
these extracts and water column samples was determined by a colorimetric
procedure utilizing an ammonium molybdate-ascorbic acid reagent.
Radioactivity Determinations
The liberation of 14C02 from the Prudhoe Bay oil "spiked" with either
n-[l-14C]-hexadecane Or n-[!4C]-octadecane was used in studies aimed at
determining the rate of oil degradation by marine microorganisms. Activity
was measured using 125 ml Erlenmeyer flasks containing 20 yl of "spiked" oil
and 25 ml of aqueous solution representing water column or 1:5 dilutions of
beach or sediment material. The Erlenmeyer flasks were modified so that a
small plastic cup containing a 0)2 trapping agent (either phenethylamine or
Hyamine hydroxide) could be lowered into the air phase via the neck of the
flask. A side port was added to the flask so that an acidifying agent
(0.5 ml 4N H2S04) could be injected into the flasks to stop growth and
release CO?. After a further 60 min incubation period the 12*C02 content of
the trapped 0)2 was determined in a toluene-based PPO/POPOP scintillation
fluid using a Nuclear Chicago scintillation counter. When phenethylamine was
used asthe C02 trapping agent, radioactivity was measured in an Isocap Model
300 (unrefrigerated) scintillation counter. Chemiluminescence problems were
encountered when Hyamine hydroxide was used for trapping 0)2 so when this
absorbent was used, a Mark I scintillation counter (refrigerated) was used
for determining the amount of radioactivity. Some of the problems
14
-------
encountered in the application of this technique to rate studies are discussed
in Appendix B.
Data Management
All the data collected or obtained by the physical-chemical and
microbiological methods used in this study together with the characteristics
of sample sites and types are available from the N.O.D.C., Washington, D.C.
15
-------
SECTION 5
RESULTS
Survey for Activity of Oil-utilizing Bacteria
The physical-chemical parameters temperature, dissolved oxygen content
and salinity of water column samples are presented in Tables Dl, D2, D3 and
D4 respectively. The pH of the majority of samples ranged from 6.6 to 8.3
and thus is suitable for normal heterotrophic bacterial growth. The temp-
eratures which range from a low of 4.5°C in January, 1.978 to a high of 16.0°C
in September, 1977 would support psychrotrophic bacterial populations.
Sufficient dissolved oxygen was present at all sampling times to support the
presence of aerobic bacterial species. The salinity of April and September
samples was in the range of 28 to 30 ppt with the lowest value being recorded
in January which could have resulted from heavy December rains in this area.
The degradation states as shown by the G.C. profiles of the saturate
fraction of Prudhoe Bay oil recovered after 28 days incubation at 8°C with
water column, sediment and beach samples supplemented with added nitrogen
and phosphorus are presented in Tables 2, 3 and 4 respectively. The pattern
of n-alkane utilization observed varied both with sample site and sampling
time. The results of similar experiments without nutrient supplementation
are presented in Tables D5, D6 and D7. No degradation of the n-alkanes of
Prudhoe Bay oil was observed without nutrient supplementation. The
degradative patterns as a function of sample type and time of sampling are
summarized in Table 5A, in terms of a "Degradative Capacity Index". The
greatest variation in the degree of n-alkane utilization was observed with
water column samples, the lowest value being found in January and highest
in September samples. The lowest oil degrading activity for beach and
sediment samples was observed in the September sampling. The beach samples
showed the smallest variation in n-alkane utilization patterns observed over
the three sampling periods. The slight activity found in non nutrient-
enriched beach samples in September (Table D7) could be due to natural
nutrient enrichment and/or the higher temperatures which occurred in this
material during the summer months. The data in Table 5B show the summary
of oil-degrading ability as a function of sample site. The greatest oil-
degrading capability was observed in those sites closest to oil refinery
activity i.e. the refinery site near Cherry Point and the E. Fidalgo Rd.
site near Anacortes refineries, with slightly lower values being found at
Pt. Angeles and Neah Bay. These outer sites are characterized by having
a relatively high organic matter content and being subjected to chronic
low levels of oil pollution (personal communication, Dr. D. Brown, NOAA
Analytical Laboratories).
16
-------
TABLE 2. G.C. PROFILE OF PENTAME E/TRACT OF RECOVERED PRUDHOE BAY OIL
AFTER 28 DAYS INCUBATION AT fc*C WITH WATER COLUMN SAMPLES
SUPPLEMENTED WITH NITROGEN AND PHOSPHORUS
Site
Birch Bayb
Refinery Site
Sandy Pt. (Sucia Rd.)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passb
Pt. Partridge^
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Salt Creekb
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
P
S
I
I
S
c
c
S
P
_d
P
P
P
I
S
-
-
-
-
-
-
G.C. Profile3
Sept. '77
P
C
I
I
I
I
I
I
C
C
P
P
-
P
P
P
c
c
I
c
c
Jan. '78
N
I
P
N
S
S
N
S
N
P
S
I
-
P
S
S
-
-
-
—
aN=no degradation; S=selective metabolism, n-alkanes C-j2 to C-jg; P=partial
removal, n-alkane peaks still discernible; I=only isoprenoid peaks
remaining; C=complete utilization of n-alkanes and isoprenoids.
*>State Park
conly sampled in September, 1977
dno sample
17
-------
TABLE 3. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL
AFTER 28 DAYS INCUBATION AT 8°C WITH SEDIMENT SAMPLES
SUPPLEMENTED WITH NITROGEN AND PHOSPHORUS
Site
Birch Bayb
Refinery Site
Sandy Pt. (Sucia Rd.)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passb
Pt. Partridge^
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Salt Creekb
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Islandc
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
I
I
I
I
I
I
I
S
I
_d
I
P
-
I
I
-
-
-
-
-
-
G.C. Profile3
Sept. '77
N
P
S
S
-
P
I
N
N
P
N
I
-
I
P
I
P
N
P
N
P
Jan. '78
S
-
P
S
I
C
N
N
I
S
-
I
-
-
S
I
-
-
-
-
-
aN=no degradation; S=selective metabolism, n-alkanes C-\2 to CIQ; P=partial
removal, n-alkane peaks still discernible; I=only isoprenoid peaks
remaining; C=complete utilization of n-alkanes and isoprenoids.
bState Park
conly sampled in September, 1977
dno sample
18
-------
TABLE 4. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL
AFTER 28 DAYS INCUBATION AT 8°C WITH BEACH SAMPLES
SUPPLEMENTED WITH NITROGEN AND PHOSPHORUS
Site
Birch Bayb
Refinery Site
Sandy Pt. (Sucia Rd. )
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passb
Pt. Partridge
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Salt Creekb
Pillar Pt.
Clal! am Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
I
C
I
S
I
C
I
I
p
_d
I
I
-
I
I
-
-
-
-
-
-
G.C. Profile9
Sept. '77
S
I
P
P
I
I
P
P
N
C
P
I
-
P
N
C
S
I
I
N
P
Jan. '78
S
C
I
S
-
C
I
S
I
p
-
I
-
-
p
I
-
-
-
-
—
aN=no degradation; S=selective metabolism, n-alkanes C^ to C^g; P=partial
removal, n-alkane peaks still discernible; I=only isoprenoid peaks
remaining; C=complete utilization of n-alkanes and isoprenoids.
bState Park
Jjonly sampled in September, 1977
dno sample
19
-------
TABLE 5. SUMMARY OF OIL-DEGRADING BACTERIAL ACTIVITY
(A) In Water Column, Sediment
Nitrogen
Phosphorus
Sample Supplementation
Water +
Sediment +
Beach +
and Beach Samples Respectively
Degradative Capacity
April '77 Sept. '77
2.28 3.05
0 0
2.78& 1.47
0 0
2.92b 2.10
0 0
AT 8°C
Index3
Jan. '78
1.2
0
1.8
0
2.5
0
(B) At Sample Sites
Site
Birch Bayc
Refinery Site
Sandy Pt. (Sucia Rd.)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Pass0
Pt. Partridge0
Ft. Worden0
Jamestown
Dungeness
Pt. Angeles
Degradative
Capacity
Index3 Site
1.44 Salt Creek
3.00 Pillar Pt.
2.32 Clallam Bay
1.67 Neah Bay
2.42 Lopez Island
3.11 Shoal Bight
2.33 San Juan Islandd
1.34 South Beach
1.89 False Bay
2.50 Orcas Islandd
1.86 Crescent Beach
2.67 Terrill Beach
Degradative
Capacity
Index9
_e
2.57
1.67
2.67
2.00
2.34
2.60
1.00
2.34
aa value of 4.0 would be obtained if all samples in a survey completely
utilized the n-alkanes and isoprenoids in Prudhoe Bay oil. (See Materials
and Methods)
bvalues of 3.15 and 3.46 for sediment and beach samples supplemented with
nitrogen and phosphate incubated for 14 days at 30°C
cState Park
°only sampled in September, 1977
eonly one water column sample obtained at this site (April, 1977)
20
-------
Bacterial Numbers and Oil-degrading Activity
A comparison of the bacterial counts of the April sediment and beach
samples with the degradative pattern observed (Table 6) indicates that there
is no positive relationship between viable bacterial counts and degree of
utilization of n-alkane components of Prudhoe Bay oil. Viable bacterial
populations of water column samples ranged from 10^ to 10^ cells per ml for
"pristine" areas like Pt. Partridge Park to 105 to 106 for waters adjacent
to oil refinery activities such as the refinery and E. Fidalgo Rd. sites.
A ten-fold increase in viable population was observed in water column samples
shipped by air from sites examined.
Factors Affecting Oil Degradation
(a) Temperature
The effect of incubation temperature on the utilization of Prudhoe
Bay oil by microorganisms present in the beach and sediment samples obtained
in the April survey is shown by comparing data in Table 7 (i.e. at 30°C)
with those in Tables Sand 4 (i.e. at 8°C). A much greater activity is
observed at the higher temperature as the "Degradative Capacity Index" for
the sediment and beach samples incubated at 30°C is 3.15 and 3.75 respectively
compared with 2.78 and 2.92 for the 8°C incubations. The 30°C data in this
experiment also was obtained with only one-half (i.e. 14 days) the incubation
time of that used at 8°C.
The effect of incubation temperatures on changes brought about in
the n-alkane components of Prudhoe Bay oil with sediment from March Pt.
Rd. (i.e. Anacortes refinery area) and the Deception Pass sites (Rosario
Beach - a "pristine" environment) as a function of incubation time is shown
in Table 8. The rate of utilization of n-alkanes, as observed earlier,
increases as the incubation temperature is raised. The microbial populations
in the sediment from the refinery area (March Pt. Rd.) bring about more rapid
changes in the n-alkane profile than do the populations in sediment from
Rosario Beach. A more detailed study (Table D8) shows the changes in total
viable count at 8°C and 20°C and the accompanying changes in the G.C.
profile of recovered oil, confirming the previous observations.
The effect of incubation temperature on the generic components of the
indigenous bacterial populations in water column and sediment samples from
West Beach (Deception Pass) is presented in Table 9. Differences between
the percentage generic incidence within water column and sediment samples
and between temperatures of incubation within the same sample material are
readily discernible. However no relationship between generic composition and
differences in rate of oil utilization is apparent.
(b) Nitrogen and Phosphorus Levels
The effect of the level of nitrogen and phosphorus supplementation on
the degradative capacity of water column samples from March Pt. Rd. and
West Beach (Deception Pass) is shown in Table 10. The results indicate that
the degradative pattern obtained 1s more sensitive to nitrogen deprivation
21
-------
TABLE 6. VIABLE BACTERIAL COUNTS OF BEACH AND SEDIMENT SAMPLES
OBTAINED IN APRIL, 1977 (28 DAYS INCUBATION at 8°C)
Site
Sediment
Beach
Birch Bayb
Refinery Site
Sandy Pt. (Sucia Rd.)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passb
Pt. Partridge
Ft. Wordenb
Dungeness
Pt. Angeles
Pillar Pt.
Clallam Bay
16.6 ± 6.2 (I)C 59 ± 22.0 (I)
9.9 ± 1.9 (I) 2.8 ± 0.4 (C)
1.0 ± 0.04 (I) 106 ± 15.7 (I)
126 ± 21.6 (I) 25.6 ± 4.9 (S)
52 ± 7.1 (I) 13.2 ± 2.1 (I)
161 ± 17.4 (I) 17.8 ± 3.8 (C)
35
12.9 ± 2.0 (I)
7.0 ± 0.7 (S)
± 11.5 (I)
0.6 ± 0.01 (I)
5.6 ± 1.6 (I) 25.0 ± 1.7 (P)
33.6 ± 5.5 (I) 65 ± 10.1 (I)
62 ± 1.5 (P) 21.8 ± 3.0 (I)
106 ± 21.2 (I) 168 ± 38.3 (I)
0.7 ± 0.0 (I) 61 ± 12.4 (I)
aaverage ± one standard deviation
bstate Park
C6.C. profile (S=selective metabolism, n-alkanes C-\2 to CIQ; P=
partial removal, n-alkane peaks still discernible; I=only
isoprenoid peaks remaining; C=complete utilization of n-alkanes
and isoprenoids).
22
-------
TABLE 7. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED
PRUDHOE BAY OIL AFTER 14 DAYS INCUBATION AT
30°C WITH BEACH AND SEDIMENT SAMPLES3
G.C. Profileb
Site
Birch Bayc
Refinery Site
Sandy Pt. (Sucia Rd.)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passc
Pt. Partridge0
Ft. Wordenc
Dungeness
Pt. Angeles
Pillar Pt.
Clallam Bay
Sediment
I
I
I
P
C
I
C
P
P
C
C
C
I
Beach
C
C
-
C
C
C
C
I
I
C
C
C
I
asamples taken in April survey (nitrogen and phosphorus added)
bP=partial removal, n-alkane peaks still discernible; 1=
only isoprenoid peaks remaining; C=complete utilization
of n-alkanes and isoprenoids
estate Park
sample
23
-------
TABLE 8. EFFECT OF TEMPERATURE AND INCUBATION TIME ON VIABLE BACTERIAL
COUNTS AND ON THE G.C. PROFILE OF RECOVERED PRUDHOE BAY OIL
INCUBATED WITH SEDIMENT3 FROM MARCH PT. RD. AND DECEPTION
PASS (ROSARIO BEACH)
March
Pt.
Rd.
Rosario Beach
viable
bacterial
Temperature
8°C
14°C
20° C
30° C
Time
(days)
4
7
15
29
4
7
15
29
4
7
15
29
4
7
15
29
G.C.
profileb
N
_d
S
I
N
N
P
I
N
I
I
C
I
I
I
C
count
x!06/gc
10.
16.
26.
7.
19.
39
47.
11.
56
116
34
245
121
70
39
6 ± 1.4
-
3 ± 2.6
2 ± 1.7
5 ± 0.8
2 ± 2.5
± 6.6
9 ± 5.1
5 ± 1.7
± 12.4
± 9.2
± 6.2
± 34.2
± 14.7
± 15.9
± 7.2
G.C.
profile0
N
N
N
P
N
N
S
C
N
N
P
-
I
I
I
-
viable
bacterial
CO
xlO
0.8
1.8
7.4
66
3.1
75
127
4.5
44
285
233
166
154
unt
6/gc
± 0.0
± 0.1
± 0.4
± 14.3
± 0.3
-
± 15.6
± 29.3
± 0.4
± 6.1
± 79.3
-
± 43.8
± 12.5
± 38.4
-
asites sampled in July, 1977
t>N=no degradation; S=selective metabolism, n-alkanes C]2 to C-jg; P=
partial removal, n-alkanes still discernible; I=only isoprenoid peaks
remaining; C=complete utilization of n-alkanes and isoprenoids
^average ± one standard deviation
"no sample
24
-------
TABLE 9. EFFECT OF INCUBATION TEMPERATURE ON GENERIC COMPOSITION OF
BACTERIAL POPULATIONS IN WATER COLUMN AND SEDIMENT SAMPLES
(SEPT., 1977) FROM WEST BEACH (DECEPTION PASS)
Genus
A1 call genes
Bacillus
coryneforms
Cytophaga
Flavobacterium
Pseudomonas
Vibrio
Unidentified0
Non- transferable
co1oniesd
Water
8°C
3.2 (l)a
3.8 (1)
4.1 (3)
44.4 (6)
24.4 (3)
4.1 (1)
15.0 (1)
0.9 (2)
% Generic
Column
20°C
_b
9.4 (3)
0.8 (1)
26.7 (5)
25.6 (4)
37.6 (2)
Composition
Sediment
8°C
18.5 (1)
11.1 (1)
9.3 (2)
3.8 (1)
27.8 (4)
20.4 (2)
9.3 (1)
20°C
10.8
30.0
39.2
13.3
6.7
(3)
(3)
(3)
(2)
(1)
anumber of colonial types in parentheses
bno colonies appeared on dilution plate used to assess population
composition
csimilar to either Alea11 genes or Pseudomonas spp. with the exception
that isolates were capable of non acid-generating, anaerobic growth
dcolonies would not grow on transfer
25
-------
TABLE 10. EFFECT OF NITROGEN AND PHOSPHORUS CONCENTRATION
ON THE 6.C. PROFILE3 OF THE N-SATURATE FRACTION
OF RECOVERED PRUDHOE BAY OIL AFTER 14 DAYS
INCUBATION AT 20°C WITH WATER COLUMN SAMPLES
FROM MARCH PT. RD. AND WEST BEACH (DECEPTION PASS)b
Phosphorus
March Pt. Rd. 2N
2N
Nc
Nitrogen 0.1N
0.01N
0
West Beach
(Deception Pass)
2N
Nc
Nitrogen 0.1N
0.01N
0
C
C
C
P
N
C
s
C
s
N
Nc
C
-
S
N
N
C
N
S
N
N
0.1N
C
I
C
P
N
P
C
I
P
S
0.01N
C
C
C
P
N
I
P
I
P
N
0
P
P
P
P
N
S
S
P
N
N
aN=no degradation; S=selective metabolism, n-alkanes Ci2 to
Cig; P=partial removal, n-alkane peaks still discernible;
I=only isoprenoid peaks remaining; C=complete utilization
of n-alkanes and isoprenoids
"samples obtained in November, 1977
Cnormal levels used in all enrichment and survey studies
equivalent to 350 ygNper ml and 180 yg P per ml
26
-------
than to low phosphorus supplementation. Similar results were obtained in a
study using a combined sediment sample from E. Fidalgo Rd. and Neah Bay
(Table 11). This effect is supported by the results of the chemical analysis
of samples for nitrogen and phosphorus (Table D9). Phosphorus was detected
in all samples tested whereas nitrogen levels were below the sensitivity of
the method used in the survey. Increasing the nitrogen and phosphorus levels
beyond those normally used in the survey resulted in a more rapid change in
the physical appearance of such cultures when compared to those receiving a
normal nutrient supplement.
(c) Oil Concentration
The data presented in Table 12 indicate that oil concentration has an
effect on the rate of utilization of the n-alkanes present in Prudhoe Bay oil
particularly at 8°C. The beach samples from the "pristine" Rosario site used
in this experiment were not as effective in yielding psychrotrophic oil-
degrading bacteria as was the sample from the refinery site, i.e. March Pt. Rd.
Rate of Oil Utilization
Gravimetric data on the rate of weight loss of Prudhoe Bay oil, under
laboratory conditions, are presented in Table 13. Approximately 30 to 34%
of the oil was lost due to "weathering" (i.e. physical, chemical processes),
35% due to mineralization (i.e. bacterial action) and approximately 30 to
35% of the original weight of oil was left as a residue. Further incubation
of up to 48 days did not result in any significant changes in the loss of
oil due to "weathering" or mineralization. Similar experiments were carried
out with sediment samples from these two sites. However, while similar
patterns were observed, considerable variation in replicates was noted.
As the gravimetric procedure is not very sensitive, a radioactive
technique was used which measured the rate of 1^C02 evolution from Prudhoe
Bay oil "spiked" with carbon-14 labelled hexa- or octadecane. The results
obtained with the water column samples from March Pt. Rd. and West Beach
(Deception Pass) are presented in Figure 3. Considerable scattering was
observed particularly with the sample from March Pt. Rd. This was attributed
in part to the presence of a high content of suspended material (Table D10)
of the March Pt. Rd. sample and the failure during sub-sampling of this
material to keep the particles in suspension. A rate of 0.42 and 0.12 mg
hexadecane was removed per litre per day during the linear phase of 14r,02
evolution for March Pt. Rd. and West Beach (Deception Pass) water column
samples supplemented with nitrogen and phosphorus. A similar experiment
tFigure Dl) with non nutrient-supplemented water samples yielded initial rates
of I4C02 release corresponding to the utilization of 0.008 mg and 0.060 mg of
hexadecane per litre per day. However the G.C. profile of the substrate
fraction of recovered oils from the non-nutrient supplemented samples revealed
no detectable changes in the n-alkane profile of recovered oil although 30,000
DPM were recovered from the West Beach (Deception Pass) sample. The re-
utilization of produced 14C02 is indicated in both experiments but only with
nutrient supplementation does enough growth take place to markedly change the
n-alkane profile of recovered oil. The results from a similar experiment
using sediment material from March Pt. Rd. and West Beach (Deception Pass)
27
-------
TABLE 11. EFFECT OF NITROGEN AND PHOSPHORUS CONCENTRATIONS ON
THE G.C. PROFILE3 OF THE N-SATURATE FRACTIONS OF
PRUDHOE BAY OIL AFTER 14 DAYS INCUBATION AT 20°C
WITH SEDIMENT (SAMPLE WAS A MIXTURE OF SEDIMENT FROM
E. FIDALGO RD. AND NEAH BAY)b
4N
4N
2N
NC
Nitrogen 0.1N
0.05N
0.01N
0
C
C
I
C
P
N
S
2N
I
C
C
C
I
S
S
NC
C
C
C
C
-
N
-
Phosphorus
0.1N
P
I
C
I
P
N
S
0.05N
I
I
I
I
P
S
N
0.01N
I
I
I
I
P
N
N
0
P
S
P
P
N
N
N
aN=no degradation; S=selective metabolism, n-alkanes Ci2 to
Cig; P=partial removal, n-alkane peaks still discernible;
I=only isoprenoid peaks remaining; C=complete utilization of
n-alkanes and isoprenoids
bsamples obtained in September, 1977
cnormal levels in all enrichment and survey studies equivalent
to 350 yg N per ml and 180 yg P per ml
28
-------
TABLE 12. EFFECT OF OIL CONCENTRATION ON GROWTH AND G.C.a PATTERN OF
SATURATE FRACTIONS OF RECOVERED PRUDHOE BAY OIL.
Temperature
(days
"i nrnha t "inn \
Beach Samples0
Oilb
March Pt. Rd.
G.C.
_ j
profile *10bu
8°C
(29)
20°C
(14)
500
1000
2000
4000
8000
500
1000
4000
8000
C
I
S
N
N
C
C
C
C
40
56
67
87
111
40.1
124
730
3350
± 4.7
± 8.1
± 9.7
± 19.0
± 29.5
± 8.9
± 21.5
± 8.3
± 509
G.C.
Rosario
profile Xl0b
N
-
N
N
N
C
-
C
P
16.5
70
187
278
550
1360
1390
± 1.6
_e
± 11.0
± 13.7
± 18.9
± 129
—
± 115
± 414
aN=no degradation; S=selective metabolism, n-alkane Ci2 to C]g; P=
partial removal, n-alkane peaks still discernible; I=only isoprenoid
peaks remaining; C=complete utilization of n-alkanes and isoprenoids
bconcentration to nearest 10 mg/500 ml culture medium
csamples obtained in July, 1977
daverage viable count/ml ± one standard deviation
esample lost
29
-------
GO
O
TABLE 13. WEATHERING AND MINERALIZATION OF PRUDHOE BAY OIL-WATER COLUMN SAMPLES3
FROM MARCH PT. RD. AND ROSARIO BEACH SUPPLEMENTED WITH NITROGEN AND
PHOSPHORUS INCUBATED AT 8°C AND 20°C
Time
(days)
5
7
8
13
14
20
21
27
28
March
.0
•o
2!
>-P C"
O-K A-
fO
2
_d
31.8
-
-
34.0
-
33.0
-
30.4
8°C
Pt. Rd.
o
T3
0)
N
** (O
l_
0)
—
0.1 (N)e
-
-
6.3 (I)
-
23.4 (C)
-
39.0 (C)
Rosario Beach
^^J ^O
(!) (1)
t- N
QJ •!-
•(-> »< (C
(O l-
0)
-------
80
14co2
60
DPM
40
X103
20
0
e
o •'
°
t
march / *\ / ,
pt.rd./ \
; \ •
./ \ /
/ \ 0
I \ '
. / . x '.
i . .' v#
* -' \
/ . ••"'' -\
/ -' °
/• -'deception
f / pass o
1 iii 1 i 1 i I
04 8 12 16
DAYS
Figure 3. Rate of ^C02 release from Prudhoe Bay oil containing
hexadecane incubated with water column samples from March Pt.
Rd. and West Beach (Deception Pass) supplemented with nitrogen
and phosphorus (DPM=disintegrations per minute).
31
-------
are presented in Figure 4. A rate of 3.4 mg hexadecane removed per kg of
sediment per day during the linear phase of 14C02 evolution for March Pt. Rd.
and West Beach (Deception Pass) sediment samples was calculated.
The results presented in Figure 5, using a slightly different radio-
activity technique, show a rate of 14C02 liberation from 14c-octadecane
containing Prudhoe Bay oil of 1.1 mg octadecane per kg sediment per day
(mixed sample from Jamestown and Ft. Worden sites after two months' storage
at -20°C). The plot represents the average of five replicates plus or minus
one standard deviation and shows an acceptable degree of reproducibility.
Effect of Oil on Indigenous Population
The effect of Prudhoe Bay oil on the growth of indigenous bacterial
populations of water column samples from March Pt. Rd. and West Beach
(Deception Pass) with and without nutrient supplementation at 20°C is shown
in Figure 6. While both populations show a "diauxie" effect (two successive
growth cycles separated by a lag phase) the Deception Pass sample also shows
an inhibition of the total viable population. Gas chromatographic analysis
of the recovered oil shows that only the March Pt. Rd. sample yielded a
residual Prudhoe Bay oil which had no n-alkane or isoprenoid components.
The nutrient-supplemented West Beach (Deception Pass) sample also would have
yielded a similar result on longer incubation.
The effect of Prudhoe Bay oil and nutrient supplementation on the
generic composition of enrichment populations from March Pt. Rd. beach,
West Beach (Deception Pass) sediment and water column and sediment from
False Bay, San Juan Island is presented in Tables 14,15, 16 and 17 respectively.
The generic composition of the bacterial populations present in beach and
sediment samples (April, 1977 survey) and in the water column samples
(September, 1977) from March Pt. Rd. is presented in the Appendix in Tables
Dll, D12 and D13 respectively. All oil-enriched populations contain a
significant content of either coryneforms or Pseudomonas spp.
Storage of Samples and Oil-degrading Capability
The data presented in Figure 7 show the changes in viable cell count
for sediment samples from March Pt. Rd. and West Beach (Deception Pass) stored
at different temperatures. All samples after 6 weeks storage were still
capable of bringing about the complete utilization of the n-alkane fraction
of Prudhoe Bay oil. Changes were noted in the colonial characteristics of
the viable population as the proportion of pigmented colonies decreased after
storage at 4°C. Similar viability patterns were observed, although of a
different magnitude, with both sediment sample types. Growth took place in
both samples stored at 4°C with slight decreases being observed between the
1st and 2nd week of storage at -20°C and -60°C.
Although water column samples increased in viable cell number during
transit such samples after a further 12 days storage at 4°C still retained
the capability of utilizing the n-alkanes and isoprenoids present in Prudhoe
Bay oil.
32
-------
00
so
40
DPM
90
x*3
LO
CO
20
10
march
pt. rd
•I
• x •
I • \ .
' -\
• \
-4
40
30
DPM
20f-
x,63
10
deception
pass
4
DAYS
2 4
DAYS
Figure 4. Rate of C0« release from Prudhoe Bay oil containing C-hexadecane incubated with
sediment samples from March Pt. Rd. and West Beach (Deception Pass) supplemented with
nitrogen and phosphorus.
-------
40 -
14,
CO2
30
DPM
20
X10"
10
O 1
23
DAYS
Figure 5. Graph of time of incubation vs net dpm ± 1 standard deviation
from sediment (mixed sample from Jamestown and Ft. Worden
taken in January, 1978).
34
-------
10
8
CELLS
10
PER ML
10
march pt. rd.
(* n.P)/ o N
M
deception pass
/
/
i
i
i
i
8
DAYS
N
Figure 6. Effect of Prudhoe Bay oil on the growth (20°C) of bacteria
present in March Pt. Rd. and West Beach (Deception Pass) water
column samples (Nov., 1977) with and without nitrogen, phosphorus
supplementation. (March Pt. Rd. •---«+N.P., -N.P.; West
Beach (Deception Pass)o——o+N.P., -N.P.). Description of
6.C. profile, n-alkane components, saturate fraction of recovered
oil; N=no change, C=complete utilization of n-alkanes and
isoprenoids.
35
-------
TABLE 14. GENERIC COMPOSITION OF MARCH PT. RD. BEACH (SEPT., 1977)
BACTERIAL POPULATIONS BEFORE AND AFTER ENRICHMENT IN THE
PRESENCE OF NITROGEN, PHOSPHORUS AND PRUDHOE BAY OIL AT
8°C and 20°C
% Generic Composition
8°C
enrichment
Genus
Al cali genes
coryneforms
Cytophaga
Flavobacterium
Pseudomonas
Vibrio
Unidentified6
Before3
5.7 (l)c
29.7 (4)
2.3 (3)
58.8 (8)
2.3 (3)
1.1 (1)
Afterb
22.8 (2)
34.8 (5)
12.0 (1)
8.9 (1)
21.5 (6)
20°C
enrichment
Before
_d
40.4 (6)
30.3 (3)
24.9 (6)
After
52.4 (5)
0.3 (1)
42.1 (3)
5.2 (2)
Non-transferable - - 4.3 (1)
colonies'
abefore enrichment (i.e. original sample)
"nitrogen, phosphorus and Prudhoe Bay oil present
cnumber of colonial types in parentheses
dno colonies present on dilution plate used to assess population
composition
esimilar to either Alcall genes or Pseudomonas spp. with the exception
that they were capable of non acid-generating anaerobic growth
'colonies which would not grow on transfer
36
-------
TABLE 15. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN SEDIMENT
SAMPLES (SEPT., 1977) FROM WEST BEACH (DECEPTION PASS)
BEFORE AND AFTER ENRICHMENT IN THE PRESENCE OF NITROGEN,
PHOSPHORUS AND PRUDHOE BAY OIL
% Generic
8°C
enrichment
Genus
Al call genes
Bacillus
coryneforms
Cytophaga
Flavobacterium
Pseudomonas
Vibrio
Unidentified6
Non- transferable
colonies*
Before9
18.5 (l)c
11.1 (1)
9.3 (2)
3.8 (1)
27.8 (4)
20.4 (2)
9.3 (1)
Afterb
5.2 (1)
12.7 (2)
-
8.0 (1)
50.9 (2)
7.1 (2)
16.0 (4)
™
Composition
20°C
enrichment
Before
_d
10.8 (3)
30.0 (3)
39.2 (3)
13.3 (2)
6.7 (1)
After
31.7 (4)
-
35.5 (1)
32.8 (4)
"
abefore enrichment (i.e. original sample)
^nitrogen,phosphorus and Prudhoe Bay oil
cnumber of colonial types in parentheses
dno colonies present in dilution plate used to assess population
composition
esimilar to either Alcall genes or Pseudomonas spp. with the exception
that isolates were capable of non acid-generating anaerobic growth
fcolonies would not grow on transfer
37
-------
TABLE 16. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN WATER
COLUMN SAMPLES (SEPT., 1977) FROM WEST BEACH (DECEPTION
PASS) BEFORE AND AFTER ENRICHMENT ON THE PRESENCE OF
NITROGEN, PHOSPHORUS AND PRUDHOE BAY OIL
% Generic
8°C
enrichment
Genus
Al cali genes
coryneforms
Cytophaga
Flavobacterium
Pseudomonas
Vibrio
Unidentifiede
Non- transferable
colonies^
Before3
3.2 (l)c
3.8 (1)
4.1 (3)
44.4 (6)
24.4 (3)
4.1 (1)
15.0 (1)
0.9 (2)
Afterb
_d
8.7 (3)
5.8 (3)
48.0 (2)
0.7 (1)
39.8 (5)
Composition
20°C
enrichment
Before
9.4 (3)
0.8 (1)
26.7 (5)
25.6 (4)
37.6 (2)
After
0.4 (1)
34.8 (3)
19.5 (3)
45.3 (5)
^before enrichment (i.e. original sample)
bnitrogen, phosphorus and Prudhoe Bay oil present
cnumber of colonial types in parentheses
dno colonies present in dilution plate used to assess population
composition
esimilar to either Alcaligenes or Pseudomonas spp. with the exception
that isolates were capable of non acid-generating, anaerobic growth
^colonies would not grow on transfer
38
-------
TABLE 17. GENERIC COMPOSITION OF BACTERIAL POPULATIONS IN SEDIMENT
(SEPT., 1977) FROM FALSE BAY, SAN JUAN ISLAND BEFORE AND
AFTER ENRICHMENT IN THE PRESENCE OF NITROGEN, PHOSPHORUS
AND PRUDHOE BAY OIL AT 8°C AND 20°C
Genus
Al call genes
coryneforms
Cytophaqa
Flavobacterium
Pseudomonas
Vibrio
Unidentified6
Non- transferable
colonies^
% Generic
8°C
enrichment
Before3
19.0 (l)c
0.3 (1)
1.1 (1)
47.1 (2)
2.5 (2)
2.2 (1)
24.4 (5)
3.4 (1)
Afterb
34.8 (2)
16.6 (3)
19.1 (2)
3.5 (1)
26.1 (1)
Composition
20°C
enrichment
Before
18.0 (1)
_d
0.1 (1)
63.6 (3)
4.0 (3)
4.9 (2)
9.4 (1)
After
56.1 (3)
5.4 (2)
1.8 (2)
37.8 (6)
abefore enrichment (i.e. original sample)
bnitrogen, phosphorus and Prudhoe Bay oil present
cnumber of colonial types in parentheses
dno colonies present on dilution plate used to assess population
composition
esimilar to either Alcall genes or Pseudomonas spp. with the exception
that colonies were capable of non acid-generating, anaerobic growth
fcolonies would not grow on transfer
39
-------
io8
CELLS
10
6
PER
ML
10
-60c
I
deception pass
»\
-20c
• »
h*« • • • * "
"••••- •*• .- 60c
*••...
Figure 7.
0246
WEEKS
Effect of storage temperature on total viable numbers of
bacteria in sediment (Nov., 1977) from March Pt. Rd. and
West Beach (Deception Pass).
40
-------
Incidence of Sulfide-generating Bacteria
The distribution of sulfide-generating bacteria in the beach and
sediment samples analyzed during this survey is presented in Tables Cl and
C2. The generation of sulfide from sediment populations was not affected
by the addition of sulfite to the medium. The beach samples yielded more
positive enrichments when sulfite was present, particularly in samples
obtained during the September survey.
41
-------
SECTION 6
DISCUSSION
The marine environment of this area of the Pacific northwest represents
a wide diversity of conditions. Beaches range from cobble rocks to fine sands
low in organic matter, to back bays where beaches and sediments are high in
organic matter. Each type of environment can be found in all areas of the
survey. Some sites are subject to heavy wave action, others to industial
activities, while some exist in a more "pristine" situation. Consequently
there was a need for a large number of sample sites in this initial survey
to adequately characterize the response of the microbiological flora to oil.
The temperature of a natural environment is a critical physical
parameter controlling microbial growth and activities. The microbial flora
of this area will be psychrotrophic in character as the temperatures at the
sample sites examined ranged from 5°C to 20°C (beach temperatures were not
reported but were in general approximately five degrees warmer than reported
water column values). The pH, dissolved oxygen and salinity of water column
samples were characteristic of an environment which would support the growth
of a typical marine psychrotrophic, heterotrophic flora.
As long as temperature is in a range which will allow microbial life,
the nutritional status of an environment, in particular the nitrogen and
phosphorus levels, will be the key parameter in controlling microbial hetero-
trophic growth if a utilizable carbon source e.g. oil is introduced to that
environment. The oil-degrading activities observed in this survey confirm
this: without the addition of nitrogen and phosphorus, the indigenous populaton
was not able to significantly modify the n-alkane components of Prudhoe Bay
oil under the experimental temperature conditions surveyed. The oil-degrading
populations were more sensitive to nitrogen levels than to phosphorus levels.
Increasing the temperature of incubation of nutritionally-supplemented samples
resulted in a more extensive utilization of the n-alkanes and isoprenoids of
Prudhoe Bay oil. Thus, factors controlling the rate of oil-utilization by
bacterial populations studied in this survey are as previously reported for
other marine environments (2-4).
The microbial oil-degrading populations obtained by the enrichment
procedures used consisted solely of bacteria. The isolation of oil-degrading
yeast and fungi would require the application of selective environmental
pressures such as low pH. Such conditions would limit the growth of oil-
degrading bacteria and allow the growth of oil-degrading yeast and fungi.
42
-------
Differences were observed in the psychrotrophic oil-degrading capability
of beach, sediment and water column samples obtained at each site as well as
between sites. Differences were observed with similar materials obtained
at the three different sampling times, i.e. April, September and January, used
in this study. However at least one sample type from a given site always
yielded bacterial populations capable of degrading n-alkanes and isoprenoids
present in Prudhoe Bay oil under psychrotrophic conditions. The greatest
variation in oil-degrading capacity was observed with water column samples
from a high degradative index value of 3.05 in September to a low of 1.2 in
January. In contrast, the oil-degrading capacity of beach samples was rela-
tively consistent ranging from an index of 2.10 to 2.92. More samples have
to be examined before the significance of these differences can be ascertained.
The oil-degrading capabilities of bacterial populations obtained from sediment
samples varied more than those from beach samples but less than populations
from water columns. This is probably a result of the beach materials
providing a more stable environment, as in soil, in contrast to the highly
variable, sensitive water column environment. The sediment, as defined
in this study, is considered as a transition zone between a stable beach and
a sensitive water environment.
As reported in the literature (3), there was a positive relationship
between oil-related industrial activities in the area, and the oil-degrading
capability of the indigenous population. Consistently high values for oil-
degrading activity were observed for all materials obtained from the refinery
site and from E. Fidalgo Rd.
The environmental parameters observed at March Pt. Rd. and Rosario
Beach sites represent two extreme conditions. The former site is near oil
refineries and high in organic matter while the latter represents a "pristine"
site low in organic matter. Therefore water column and sediment samples
from these sites were used in "weathering" and mineraliation studies of Prudhoe
Bay oi1.
The weight of Prudhoe Bay oil lost due to "weathering" under laboratory
conditions represents 30% of the weight of oil and is independent of sample
source, at least for March Pt. Rd. and Rosario Beach materials. A further
30 to 35% by weight, can be lost due to "mineralization" by microbial activities,
This gravimetric approach because of the few number of samples used in this
study did not reveal any significant differences in the rate of oil utilization
between March Pt. Rd. and Rosario Beach materials. The data reported were
obtained with water column samples and no major problems were encountered
with reproducibility of oil recovery. The values reported for weathering
are similar to those noted by Atlas and Busdosh(20). However, similar exper-
iments using sediments from these two sites, while supporting the observations
reported, showed wide variation in reproducibility of replicates. This is
attributed to material in the sediments interfering with the extraction of
residual oil. In subsequent studies the West Beach (Deception Pass State Park)
site was used in place of Rosario Beach because of the presence of more uniform
particle size sand at West Beach.
The use of a more sensitive technique involving the measurement of the
rate of release of 14C02 from l4C-hexa-or 14C-octadecane-"spiked" Prudhoe
43
-------
Bay oil revealed a more rapid rate of release from water column samples from
March Pt. Rd. than from West Beach (Deception Pass). These rates represent
the total n-alkane metabolized in "spiked" Prudhoe Bay oil. A similar study
using sediment samples obtained from these two sites revealed no difference
between the rates of 14C02 release. Considerable difficulties were
encountered in the initial application of this method (see Appendix B) and
the results have to be considered as only being indicative of the potential
of this technique. The rates reported are considerably higher than those
previously observed (21-22) for n-alkanes and may be a result of the presence
of larger microbial populations in the water column samples used. In addition,
the use of 14c-"spiked" oil versus pure hydrocarbons (21-22) could result
in the differences in activities observed. As it has been noted (23) that the
disappearance of n-alkanes from G.C. profiles of the saturate fraction of oil
occurs prior to the period of rapid weight loss in this fraction (at least
in Boreal soils) the relationships between the different rates observed with
water column samples from these sites and weight loss of the different
fractions of oil should beinvestigated.
Differences were noted in the response to oil of bacterial populations
from West Beach (Deception Pass) and March Pt. Rd. The addition of oil to
nutritionally-enriched water column samples results in an intial decrease of
viability with the population from West Beach but not with that from March
Pt. Rd. However both populations show a "diauxie" type of growth curve
where the second growth phase of the March Pt. Rd. sample was related to
n-alkane utilization. It is probable that further incubation of the West
Beach (Deception Pass) sample also would have shown n-alkane utilization.
Similarly a slow, more variable response of sample material from West Beach
was noted in the effect of nitrogen and phosphorus levels on Prudhoe Bay oil
utilization (Table 10).
The generic composition of indigenous bacterial populations present
in unamended water column, sediment and beach samples consists primarily of
Gram-negative rods of either the Pseudomonas or Flavobacterium genera with
an occasional sample showing a high proportion of members of the Acinetobacter
genus. A few samples contained members of the Gram-positive (to Gram-variable)
coryneform group. Samples from two locations in the April survey, Clallam Bay
and Pillar Pt., contained a relatively high content of members of this group
which could be related to low salinity of the water column, due to extensive
underground seepage of freshwater. Members of the Pseudomonas and Flavo-
bacterium genera and the coryneform group also appear in varying proportions
in almost all oil-degrading enrichments. The only oil-degrading pdpulation
which differed was the one obtained from the sediment of False Bay, San Juan
Island, which contained a high proportion of A1call genes spp. Member of all
these groups are recognized as containing hydrocarbon-degrading species (9).
The significance of the population composition and oil-degrading capability
is not known. Such populations will retain their oil-degrading capability
for one or two transfers on non hydrocarbon-containing medium e.g. marine
agar and therefore, the possibility exists of resolving these mixed populations
into axenic cultures and studying their individual roles in the degradation of
oil. The temperature at which the growth and enrichments were carried out
affected the composition of the population obtained, but no definite trends
were apparent (9). More samples of a similar type must be examined to
44
-------
determine whether trends exist as a result of temperature and sample type
and/or site differences.
It is not possible to carry out a detailed comparison of the generic
composition of populations from different sample types from West Beach
(Deception Pass) and March Pt. Rd., because tidal conditions during the samp-
ling period precluded the possibility of obtaining all three sample types
from March Pt. Rd. Unfortunately, the dilution also was missed for the water
column sample from this site which would have yielded the water column popu-
lation for comparative purposes.
The differences in population composition of sediments from low organic
matter-containing environments such as West Beach and organically rich
environments (False Bay, San Juan Island) are reflected as well in differences
in total viable population (i.e. West Beach and March Pt. Rd.). However
a study of the stability of the oil-degrading capability following various
sample storage conditions revealed no differences between the populations.
Similar growth responses were observed although at different levels. Both
populations lost a proportion of the pigmented population at 4°C but this was
not reflected in changes in oil-degrading capability, as shown by the methods
used in this study.
The survey of the incidence of sulfide-generating bacteria in the beach
and sediment samples analyzed in this survey indicates their presence at all
sites examined. The activity of these microorganisms is undoubtedly limited
by the lack of suitable organic matter for growth. Such material would be
provided by the products of the aerobic degradation of oil spilled in this
area (Jobson, Cook, Westlake, unpublished results).
45
-------
REFERENCES
1. Walker, J.D., R.R. Colwell and L. Petrakis. Degradation of Petroleum
by an Alga, Prototheca zopfii. Applied Microbiol., 30:79-81, 1975.
2. McKenzie, P. and D.E. Hughes. Microbial Degradation of Oil and Petro-
chemicals in the Sea. In: Microbiology in Agriculture, Fisheries and
Food. F.A. Skinner and J.6. Carr, eds. Academic Press, 1976. pp. 91-108.
3. Colwell, R.R. and J.D. Walker. Ecological Aspects of Microbial Degrada-
tion of Petroleum in the Marine Environment. In: Critical Reviews in
Microbiology, A.I. Laskin and H. Lechevalier, ed. C.R.C. Press, 1977.
pp. 423-445.
4. Karrick, N.L. Alteration in Petroleum Resulting from Physico-chemical
and Microbial Factors. In: Effects of Petroleum on Arctic and Sub-
Arctic Marine Environments, D.C. Mai ins, ed. Academic Press, 1977.
pp. 235-279.
5. Mulkins-Phillips, G.J. and J.E. Stewart. Distribution of Hydrocarbon-
utilizing Bacteria in Northwestern Atlantic Waters and Coastal Sediments.
Can. J. Microbiol., 20:955-962, 1974.
6. Walker, J.D. and R.R. Colwell. Enumeration of Petroleum-degrading
Microorganisms. Applied Environ. Microbiol., 31:198-207, 1976.
7. Klug, M.J. and A.J. Markovetz. Utilization of Aliphatic Hydrocarbons
by Microorganisms. Adv. Microbial Phys., 5:1-43, 1971.
8. Jobson, A., F.D. Cook and D.W.S. Westlake. Microbial Utilization of
Crude Oil. Appl. Microbiol. 23:1082-1089, 1972.
9. Westlake, D.W.S., A. Jobson, R. Phillippe and F.D. Cook. Biodegrad-
ability and Crude Oil Composition. Can. J. Microbiol., 20:915-928, 1974.
10. Walker, J.D., L. Petrakis and R.R. Colwell. Comparison of the Biodegrad-
ability of Crude and Fuel Oils. Can. J. Microbiol., 22:598-602, 1976.
11. Westlake, D.W.S. Heterotrophic Activity Determination by Substrate
Dissappearance. In: Native Aquatic Bacteria, Enumeration, Activity and
Ecology, R.R. Colwell and'j.W. Costerton, ed. A.S.T.M. Publ. 1978.
12. Walker, J.D. and R.R. Colwell. Biodegradation Rates of Components of
Petroleum. Can. J. Microbiol., 22:1209-1213, 1976.
46
-------
13. Atlas, R.M. Effect of Temperature and Crude Oil Composition on
Petroleum Biodegradation. Appl. Microbiol., 30:396-403, 1975.
14. Gibbs, G.E., K.B. Pugh and A.R. Andrews. Quantitative Studies on Marine
Biodegradation of Oil. II. Effect of Temperature. Proc. Roy. Soc.
(London), B:83-94, 1975.
15. Ward, D.M. and T.D. Brock. Environmental Factors Influencing the Rate
of Hydrocarbon Oxidation in Temperate Lakes. Appl. Environ. Microbiol.,
31:764-772, 1976.
16. Atlas, R.M. and R. Bartha. Degradation and Mineralization of Petroleum
in Sea Water: Limitation by Nitrogen and Phosphorus. Biotech. Bioeng.
14:309-318, 1972.
17. Jobson, A., M. McLaughlin, F.D. Cook and D.W.S. Westlake. Effects of
Amendments on the Microbial Utilization of Oil Applied to Soil. Appl.
Microbiol., 27: 166-171, 1973.
18. Gibbs, G.F. Quantitative Studies on Marine Biodegradation of Oil.
I. On Nutrient Limitation at 14°C. Proc. Roy. Soc. (London), B:84-94,
1975.
19. Black, C.A., ed. Methods of Soil Analysis. Part II. Chemical and
Microbiological Properties. Amer. Soc. Agronomy (pub), Madison, Wise.
1965.
20. Atlas, R.M. and M. Busdosh. Microbial Degradation of Petroleum in the
Arctic. In: Proceedings of the Third International Biodegradation
Symposium, J.M. Sharpley and A.M. Kaplan, ed. Appl. Sci. Publ., London.
pp. 79-86, 1976.
21. Lee, R.F. and C. Ryan. Biodegradation of Petroleum Hydrocarbons by
Marine Microbes, J.M. Sharpley and A.M. Kaplan, ed. Appl. Sci. Publ.,
London, pp. 119-125, 1976.
22. Robertson, B., S. Arhelger, P.J. Kinney and O.K. Button. Hydrocarbon
Biodegradation in Alaskan Water. In: The Microbial Degradation of Oil
Pollutants, D.G. Adhearn and S.P. Meyers, Ed. Louisiana State University,
Baton Rouge, La. Publ. No. LSU-SG073-01. pp. 171-184, 1973.
23. Westlake, D.W.S., A.M. Jobson and F.D. Cook. In situ Degradation of
Oil in a Soil of the Boreal Region of the Northwest Territories. Can.
J. Microbiol., 24:254-260, 1978.
24. Rubenstein, I., O.P. Strausz, C. Spyckerelle, R.J. Crawford and
D.W.S. Westlake. The Origin of the Oil Sand Bitumens of Alberta:
A Chemical and a Microbiological Simulation Study. Geochimica et
Cosmochimica Acta, 41:1341-1353, 1977.
47
-------
APPENDIX A
Microbiological Media Used in this Study
(i) Artificial Seawater Solution
Nad 23.40 g
KC1 0.75 g
MgS04-7H20 7.00 g
Distilled water 1000 ml
pH 7.3
(ii) Basal Marine Agar (for viable cell counts)
proteose peptone (Difco) 1 g
yeast extract (Difco) 1 g
agar (Difco purified) 20 g
artificial sea water 1000 ml
pH adjusted to 7.5 before autoclaving
final pH 7.3
(iii) Media for Taxonomy
April Survey
(a) Board and Holding Medium (Modified)
Solution A - NH4H2P04 0.5 g
K2HP04 0.5 g
yeast extract (Difco) 0.5 g
Agar (Difco purified) 5.0 g
Bromthymol blue 0.08 g
distilled water 1000 ml
Solution B - glucose 10 g
distilled water 100 ml
Solution A and B sterilized separately, mixed, and dispensed 2 ml/
13x100 mm test tube.
(b) Oxidase reagent - 1% solution of N,N,N',N'-tetramethyl-p-
phenylenediami ne-di hydrochlori de
September and January Survey and other Taxonomic Studies
(a) Modified 0-F Medium
Solution A - NaCl 23.40 g
KC1 0.75 g
MgS04-7H20 7.00 g
yeast extract 1.00 g
48
-------
Bromthymol blue 0.03 g
agar (Difco-purified) 15.0 g
distilled water 750 ml
Solution B - glucose or lactose 10 g
distilled water 200 ml
Solution C - NH4H2P04 0.50 g
K2HP04 0.40 g
distilled water 50 ml
The pH of all solutions was adjusted to 7.15 before autoclaving:
solutions were cooled, A and B mixed, and solution C then added
and the mixture dispensed into Petri plates.
(b) Oxidase reagent - as per April survey
(c) Catalase reagent - 3% H202 (freshly prepared)
(iv) Butlin's medium for sulfide-generating bacteria
(a) Butlin's
NH4C1 1.0 g
N32S04 2.0 g
MgS04-7H20 0.02 g
Sodium lactate 1.5 ml (60% syrup)
yeast extract 1.0 g
K2HP04 0.5 g
distilled water 1000 ml
Ten mis dispensed per 18x150 mm test tube and two iron finishing
nails added per tube for poising the medium before autoclaving.
(b) Butlin's plus sulfite medium prepared as in (a); media sterilized,
cooled and 3 drops of a freshly prepared sterile (filtration)
solution of 10% N32S03 added.
(c) Saline Butlin's - prepared in artifical sea water
(v) Nitrogen and phosphorus-containing nutrient solution
10% K2HP04 420 ml
10% KH2P04 180 ml
NH4N03 60 g
pH adjusted to 7.3 with ION NaOH and used at the rate of 1 ml/100 ml of
natural or artificial sea water medium.
49
-------
APPENDIX B
Comments on the 14C-radioisotope Procedure
Initial studies using phenethylamine as a C02-trapping agent, PPO/POPOP
in toluene as a fluor, and an unrefrigerated Isocap 300 scintillation counter
resulted in a large variation in the results obtained with replicate samples.
The substitution of Hyamine hydroxide as the trapping agent resulted in a
chemiluminescence problem which could not be overcome even by refrigeration
or the addition of a few drops of glacial acetic acid or carbon tetrachloride
to scintillation vials before counting. Reproducible results have been
obtained using Carbo-Sorb II as a trapping agent and Monophase-40 as the fluor.
Using this system any chemiluminescence can be eliminated by reprogramming
the Mark I scintillation counter or using instruments programmed to make such
adjustments automatically.
50
-------
APPENDIX C
TABLE Cl. INCIDENCE OF SULFIDE-GENERATING BACTERIA IN SEDIMENT SAMPLES
Formation of black preci'pitatec
April September January
1977 1977 1978
Site
Birch Bayb
S04 SO?7S04 SO/f
+ + +
SOf/SOC SO/f
+
SQf/SQf
+
Refinery Site + + + +
Sandy Pt. (Sucia Rd.) + + + +
Samish Island (Public Beach) + + + +
March Pt. Rd. + + NS NS
E. Fidalgo Rd. + + + +
Deception Passb - + + +
Pt. Partridgeb
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island
Shoal Bight
San Juan Island
South Beach
False Bay
Orcas Island
Crescent Beach
Tefrill Beach
+
NS
+
+
-
+
NS
NS
NS
NS
NS
NS
+ +
+ +
NS +
+ +
+ +
+ +
+ +
NS +
NS +
NS +
NS +
NS +
NS +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ + +
+ NS NS
+ NS NS
+ NS NS
+ NS NS
+ NS NS
+ NS NS
a+=formation of black precipitate; -=no precipitate formed after 3 weeks
incubation at room temperature; April samples - normal Butlin's medium;
September and January samples - saline Butlin's (See Appendix A).
bState Park
NS=not sampled
51
-------
TABLE C2. INCIDENCE OF SULFIDE-GENERATING BACTERIA IN BEACH SAMPLES
Formation of black precipitate8
April September January
1977 1977 1978
Site S04 SO.f/SO/r SOA S037SO/T S0a~
Birch Bayb + + - + + +
Refinery Site ++-+++
Sandy Pt. (Sucia Rd.) + + + +
Samish Island (Public Beach) + + - + +
March Pt. Rd. ++-+++
E. Fidalgo Rd. ++_+_+
Deception Passb - + - +
Pt. ?artridgeb - + + + + +
Ft. Wordenb + + + +
Jamestown NS NS + + +
Dungeness - + + + + +
Pt. Angeles + + + +
Pillar Pt. + - + NS NS
Clallam Bay - + +
Neah Bay NS NS + +
Lopez Island
Shoal Bight NS NS + NS NS
San Juan Island
South Beach NS NS + NS NS
False Bay NS NS + + NS NS
Orcas Island
Crescent Beach NS NS + + NS NS
Terrill Beach NS NS + + NS NS
a+=formation of black precipitate; -=no precipitate formed after 3 weeks
incubation at room temperature; April samples - normal Butlin's medium;
September and January samples - saline Butlin's (see Appendix A)
bState Park
NS=not sampled
52
-------
APPENDIX D
TABLE Dl. pH WATER COLUMN SAMPLES
Site
Birch Bay3
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Road
E. Fidalgo Road
Deception Pass3
Pt. Partridge9
Ft. Worden3
Jamestown
Dungeness
Pt. Angeles
Salt Creek3
Pillar Point
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
8.1
8.0
7.7
7.7
7.7
8.3
7.8
7.9
8.2
_b
8.1
8.2
8.9
8.3
7.5
-
-
-
-
-
-
PH
Sept. '77
7.9
7.7
7.6
7.9
7.5
7.4
7.8
7.8
8.1
8.2
8.1
8.0
-
7.8
7.8
8.0
8.1
7.7
7.3
7.9
7.9
Jan. '78
7.7
7.7
6.9
7.7
6.9
7.7
8.0
7.6
7.6
7.1
7.5
6.6
-
7.8
8.1
6.8
-
-
-
—
3State Park
"not sampled
°only sampled in September, 1977
53
-------
TABLE D2. TEMPERATURE - WATER COLUMN SAMPLES
Temperature (°C)
Site
Birch Baya
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Pass3
Pt. Partridge3
Ft. Wordena
Jamestown
Dungeness
Pt. Angeles
Salt Creek
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
9.9
9.3
8.3
12.6
12.6
8.3
10.5
10.5
12.0
_b
12.0
9.7
9.0
12.0
9.0
-
-
-
-
-
-
-
Sept. '77
16.0
16.0
14,0
12.0
12.5
13.5
11.0
11.0
12.0
14.0
16.0
11.0
-
11.2
11.0
12.0
12.8
11.0
10.0
10.0
15.0
12.0
Jan. '78
6.2
4.5
5.5
6.2
6.3
10.5
11.5
7.0
9.8
9.3
7.5
8.3
-
8.4
9.3
9.0
-
-
-
-
-
-
JState Park
"not sampled
conly sampled in September, 1977
54
-------
TABLE D3. DISSOLVED OXYGEN CONTENT - WATER COLUMN SAMPLES
Site
Birch Baya
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passa
Pt. Partridge3
Ft. Worden3
Jamestown
Dungeness
Pt. Angeles
Salt Creek3
Pillar Pt.
Clal! am Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
.Crescent Beach
Terrill Beach
April '77
9.3
_b
-
19.6
13.0
13.5
9.0
9.1
13.6
-
9.7
10.6
8.9
10.8
10.2
-
-
-
-
-
-
Do (ppm)
Sept. '77
8.6
8.7
8.2
7.4
6.1
5.3
6.9
7.6
8.2
11.1
8.9
9.2
-
7.2
7.8
8.0
10.5
8.5
6.2
8.8
8.8
Jan. '78
10.7
10.3
10.6
10.4
11.0
10.4
10.6
10.4
10.4
10.1
10.0
9.3
-
11.1
11.8
10.4
-
-
-
-
~
jJState Park
knot sampled
conly sampled in September, 1977
55
-------
TABLE D4. SALINITY - WATER COLUMN SAMPLES
Site
Birch Bay3
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Pass3
Pt. Partridge3
Ft. Worden3
Jamestown
Dungeness
Pt. Angeles
Salt Creek3
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
28.6
29.3
28.0
28.7
28.7
28.5
28.8
29.8
29.5
_b
29.5
28.6
30.7
20.4
20.9
-
-
-
-
-
-
Salinity (ppt)
Sept. '77
26.5
26.0
27.5
27.4
27.2
27.7
30.0
29.4
29.7
29.8
28.5
30.9
-
29.8
30.1
30.0
27.5
28.8
29.4
27.4
28.1
Jan. '78
19.7
20.6
20.8
19.8
16.5
19.5
20.8
21.8
22.2
22.0
20.3
18.8
-
18.3
21.9
20.8
-
-
-
-
-
3State Park
bnot sampled
conly sampled in September, 1977
56
-------
TABLE D5. 6.C. PROFILE OF PENTANEEXTRACT OF RECOVERED PRUDHOE BAY OIL
AFTER 28 DAYS INCUBATION AT 8°C WITH WATER COLUMN SAMPLES
Site
Birch Bayb
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Pass0
Pt. Partridge0
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Salt Creek0
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
N
N
N
N
N
N
N
N
N
_d
N
N
N
N
N
-
-
-
-
-
-
G.C. Profile9
Sept. '77
N
N
N
N
N
N
N
N
N
N
N
N
-
N
N
S
N
N
N
N
N
Jan. '78
N
N
N
N
N
N
N
N
N
N
N
N
-
N
N
N
-
-
-
-
"
aN=no degradation; S=selective metabolism, n-alkanes C12 to
DState Park
°only sampled in September, 1977
dnot sampled
57
-------
TABLE D6. 6.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL
AFTER 28 DAYS INCUBATION AT 8°C WITH SEDIMENT SAMPLES
Site
Birch Bayb
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passb
Pt. Partridgeb
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Salt Creekb
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Island0
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
N
N
N
N
N
P
N
N
N
_d
N
N
-
N
N
-
-
-
-
-
-
G.C. Profile3
Sept. '77
N
S
N
N
N
N
N
N
N
N
N
N
-
N
N
N
N
N
P
N
N
Jan. '78
N
-
N
N
N
N
N
N
N
N
-
N
-
-
N
N
-
-
-
-
-
aN=no degradation; S=selective metabolism, Ci2 to C-jg; P=partial removal
n-alkane peaks still discernible
bState Park
°only sampled in September, 1977
dnot sampled
58
-------
TABLE D7. G.C. PROFILE OF PENTANE EXTRACT OF RECOVERED PRUDHOE BAY OIL
AFTER 28 DAYS INCUBATION AT 8°C WITH BEACH SAMPLE
Site
Birch Bayb
Refinery Site
Sandy Point (Sucia Road)
Samish Island (Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Passb
Pt. Partridgeb
Ft. Wordenb
Jamestown
Dungeness
Pt. Angeles
Salt Creekb
Pillar Pt.
Clallam Bay
Neah Bay
Lopez Island0
Shoal Bight
San Juan Islandc
South Beach
False Bay
Orcas Island0
Crescent Beach
Terrill Beach
April '77
N
N
N
N
N
N
N
N
N
_d
N
N
-
N
N
-
-
-
-
-
-
G.C. Profile9
Sept. '77
-
-
N
N
S
N
N
N
N
S
N
N
-
N
N
N
N
N
N
S
N
Jan. '78
N
N
N
N
-
N
N
N
N
N
N
N
-
-
N
N
-
-
-
-
"
aN=no degradation; S=selective metabolism, n-alkanes
bState Park
conly sampled in September, 1977
dno sample
to
59
-------
TABLE D8. EFFECT OF TEMPERATURE, INCUBATION TIME AND SAMPLE TYPE OBTAINED IN JULY, 1977 ON THE G.C.
PROFILE OF PRUDHOE BAY OIL RECOVERED AFTER INCUBATION AT 8 OR 20°C
a\
o
March Pt. Road
Water Column
Total
bacterial
Sediment
Rosario
Water Column
Total
bacterial
Sediment
Total
bacterial
Total
bacterial
Time G.C. count G.C. count G.C. count G.C.
(days) Profile x!06/mlb Profile x!06/ml Profile x!06/ml Profile x
8°C
20°C
aN=no
still
7
14
21
28
35
42
49
56
5
8
13
20
48
N
I
C
C
C
C
C
C
I
I
C
C
C
degradation;
discernible
0.7 ± 0.05
31 ± 4.6
272 ± 86.6
266 ± 31.9
364 ± 52.4
450 ± 64
550 ± 72
242 ± 17.4
133 ± 17.4
242 ± 30.9
229 ± 27.0
236 ±61.5
4.8 ± 6.9
N
S
S
I
I
C
-
C
I
I
C
C
C
S-select1ve metabolism,
; I=only isoprenoid peaks
13.2 ±
21.0 ±
32 ±
47 ±
55 ±
21.3 ±
-
19.3 ±
287 ±
235 ±
120 ±
18.7 ±
13.5 ±
6.9
8.1
7.5
8.2
7.7
2.1
1.1
14.0
23.5
17.8
17.1
2.14
n-alkanes C]? to C
remaining; C=comp
N
S
C
C
C
C
C
C
I
C
C
C
C
1
3.3±
48 ±
0.3
5.4
440 ±103
408 ±
558 ±
315 ±
310 ±
255 ±
466 ±
470 ±
301 ±
354 ±
185 ±
31.8
35.4
28.3
23.6
35.3
39.1
64
19.1
62.8
24.0
N
N
N
P
P
I
I
I
N
C
C
C
C
3.
3.
22.
116
87
87
137
110
12.
290
320
400
73
count
lOfyml
6 ± 0.4
9 ± 0.2
1 ± 3.0
± 12.5
± 17.8
± 8.4
± 14.3
± 10.2
5 ± 0.5
± 26.5
± 48
± 89
±8.5
gj P=partial removal, n-alkane peaks
ete utilization of n-alkanes and
isoprenoids
^average ± one standard deviation
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TABLE D9. AVAILABLE PHOSPHORUS CONTENT OF BEACH, SEDIMENT AND WATER
COLUMN SAMPLES
Sampling
July '77
Site
March Pt
Rosario
. Rd.
Beach
yg P/ga
Beach Sediment
14. Ob (13.7)c
2.6 (47)
Mg P/mla
Water
0.67
0.10
Sept. '77
March Pt. Rd.
Deception Pass
Clallam Bay
Jamestown
Crescent Beach
(Orcas Island)
False Bay
(San Juan Island)
5.75 (6.38) - 0.16
2.25 (5.38) 1.46 (3.63) 0.04
1.46 (3.38) 2.95 (4.25) 0.04
3.44 (8.75) 4.79 (5.38) 0.07
2.51 (4.00) 2.74 (6.25) 0.03
4.49 (8.25) 28.0 (36.88)
aascorbic acid-ammonium molybdate method
Extracting solution - 0.5M NaHCOa
cextracting solution - 0.03N NH4F +
61
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TABLE D10. SUSPENDED SOLIDS CONTENT
OF REPRESENTATIVE WATER
COLUMN SAMPLES
Si tea
March Pt. Rd.
Deception Pass
Clall am Bay
Jamestown
Crescent Beach
aSampled September,
Suspended solids
mg/1
35.8
6.6
6.3
35.1
12.7
1977
62
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TABLE Dll. GENERIC COMPOSITION OF INDIGENOUS BACTERIAL POPULATIONS
OF BEACH SAMPLE (APRIL, 1977)
Site
Birch Bay3
% Generic
v> to
u E
C S- ro
OJ
O) (O -f-
•»-> C >+-
0 O -.-
-O O O C
O T3 -i- O)
> 3 S_ T3
ID oj .a M-
•— «/> ••- C
u_ o. > ID
38.5 -b - 12.2 37.0 - 12.6
Refinery Site 18.5 18.9 - 10.9 - - 51.8
Sandy Pt. 17.4
(Sucia Rd.)
Samish Island 8.9 36.5
(Public Beach)
March Pt.
E. Fidalgo
Deception
Rd. 8.5
70.4 - 12.1
5.8 9.2 39.6
16.9 13.2 - 63.3
2.7 - - 65.6 19.0 11.8 1.0
Pass3
Pt. Partridge9 15.4
Dungeness
100
15.5 34.1 - 35.4
0.6 3.1 - 75.2 17.8 - 3.1
Clallam Bay - 83.3
Pillar Pt.
42.6 49.2
8.3 6.7 - 1.7
1.8 - 6.2
Pt. Angeles 3.9 - 1.6 22.9 50.6 - 20.8
Ft. Worden3 21.7
7.0 12.0 - 59.1
astate Park
bno colonies present on dilution plate used to assess population
composition
63
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TABLE D12. GENERIC COMPOSITION OF INDIGENOUS BACTERIAL POPULATIONS
OF SEDIMENT SAMPLES (APRIL, 1977)
% Generic Composition
Site
Birch Bay3
Refinery Site
Sandy Pt.
(Sucia Rd.)
Samish Island
(Public Beach)
March Pt. Rd.
E. Fidalgo Rd.
Deception Pass3
Pt. Partridge3
Dungeness
Clallam Bay
Pillar Pt.
Pt. Angeles
Ft. Worden3
to
c
QJ
O1
*t—
^™
IO
o
5
-b
18.
7.
_
-
2.
19.
23.
-
2.
6.
18.
12.
3
2
1
6
0
2
9
4
6
E
3
•r-
10 i-
E 0)
t «0| -M
O Ol O
««- 1C , O >
S- -M (O
O >i •—
0 01 U.
10.5
29.7 - 6.3
6.7
36.8 - 0.7
10.6 - 6.0
2.9 - 57.5
1.4 4.6
9.6 5.1
52.7
1.8 6.3 34.3
5.0
5.8 20.5
15.4 1.4
•o
(/) Q)
fO T-
C <4-
§•!"•
^)
0 C
•0 t- 0»
3 J- T3
=3
14.2 4.8 70.4
18.6 27.1
45.8 - 40.5
10.3 4.7 39.5
42.0 11.2 25.2
22.2 11.8 3.4
58.1 - 16.3
62.3
8.2 - 38.9
16.9 4.9 33.8
6.0 - 82.1
17.3 3.7 34.2
46.7 3.7 20.1
aState Park
bno colonies present on dilution plate used to assess population
composition
64
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TABLE D13. GENERIC COMPOSITION OF MARCH PT.
RD. WATER COLUMN POPULATIONS
(SEPT., 1977) AFTER ENRICHMENT
IN THE PRESENCE OF NITROGEN,
PHOSPHORUS AND PRUDHOE BAY OIL
AT 8°C AND 20°C
% Generic Composition
Genus 8^ 20°C
AT callgenes sp. - 7.3 (2)b
coryneforms 5.7 (2) 0.7 (1)
Flavobacterium sp. 60.2(2) 45.5(3)
Pseudomonas sp. 27.8(3) 39.8(6)
Unidentified0 2.8 (3) 6.8 (1)
Non-transferable 3.4 (1)
colonies^
ano colonies present on dilution plate used
to assess population composition
bnumber of colonial types in parentheses
csimilar to either Alcaligenes or
Pseudomonas spp. with the exception that
isolates were capable of non acid-
generating anaerobic growth
dcolonies would not grow on transfer
65
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