Marine Pollution Bulletin 139 (2019) 381–389

Contents lists available at ScienceDirect

Marine Pollution Bulletin

journal homepage: www.elsevier.com/locate/marpolbul

Inorganic nutrients have a significant, but minimal, impact on a coastal T

microbial community's response to fresh diluted bitumen
⁎
Alice C. Ortmann , Susan E. Cobanli, Gary Wohlgeschaffen, Peter Thamer, Claire McIntyre,
Jennifer Mason, Thomas L. King
Center for Offshore Oil, Gas and Energy Research, Department of Fisheries and Oceans Canada, Bedford Institute of Oceanography, 1 Challenger Drive, Dartmouth, NS B2Y
4A2, Canada

A R T I C LE I N FO A B S T R A C T

Keywords: Microbes capable of degrading hydrocarbons in oil are present in low abundances in coastal waters, but quickly
Biodegradation respond to oil following a spill. When estimating potential biodegradation rates in the laboratory, high con-
Diluted bitumen centrations of inorganic nutrients are often added to prevent nutrient limitation. In this study, we tested the
Dilbit short term response of coastal microbes to fresh diluted bitumen under varying nutrient conditions in a cold
Nutrient limitation
water regime. Total hydrocarbon concentrations changed minimally over five days; however, oil composition
Coastal waters
changed over time and the abundance of microbes increased in all treatments. Addition of phosphate, with or
without nitrogen, resulted in rapid changes in community composition, but after three days treatments no longer
differed. Nutrients were never depleted in any treatment suggesting that, even at low inorganic nutrient con-
centrations, microbial communities can quickly respond to hydrocarbons following a spill.

1. Introduction
Extraction and transportation of bitumen from oil sands in north
central Canada is expected to increase by 53% between 2016 and 2030
(CAPP, 2017). Bitumen is a semi-solid material that is diluted with
lighter hydrocarbons (diluted bitumen) to enable transport using pi-
pelines to the coast, where it can be transferred to tankers for export
(reviewed in Crosby et al., 2013). Because of the relatively few studies
of diluted bitumen, there is concern regarding the fate, behaviour and
effects of this material if it were to spill, either from a pipeline leak,
during transfer at a port or due to a tanker accident at sea. As a com-
posite of light and heavy oils, diluted bitumen weathers quickly in-
itially, but then undergoes minimal change over a longer time frame
(King et al., 2014).
Physical, chemical and biological processes, collectively known as
weathering, change the physical properties and chemical composition
of the oil immediately following a spill. Degradation of the oil includes
oxidation of compounds either driven by light (photooxidation), che-
micals or microbial activity (biodegradation). Biodegradation is mainly
due to the actions of specific Bacteria, which can break down the oil
(Hazen et al., 2016; Head et al., 2006). Some of the compounds in oil
can be completely degraded to CO2 through the actions of microbial
consortia. Other compounds can be modified and transformed into new
compounds that are not present in the original oil, but may dissolve
more easily in water. Rates and extent of all degradation processes will
vary depending on the oil type and environmental conditions (e.g.
Bagby et al., 2017; Hori et al., 2014; Owsianiak et al., 2009). Following
the Deepwater Horizon spill in the Gulf of Mexico, biodegradation ra-
pidly removed gases and alkanes in the deep water plume (Redmond
and Valentine, 2012; Valentine et al., 2010), with significant biode-
gradation occurring along shorelines (Kostka et al., 2011; Mortazavi
et al., 2013). However, heavier oils, including diluted bitumen, have
higher contributions from resins and asphaltenes that are more resistant
to biodegradation (Prince et al., 2003). Twenty years after the Arrow
spill of Bunker C fuel oil in Chedabucto Bay, Nova Scotia, petroleum
residues ranging from only slightly weathered to highly weathered
could be detected on the shores of the bay (Vandermeulen and Singh,
1994). Even in 2016, oil could still be detected just below the surface of
the cobble beach of Black Duck Cove in Chedabucto Bay (MacDonald,
2017).
Few studies have investigated the ability of the microbial commu-
nity to degrade diluted bitumen. Using microbes enriched from the
Kalamazoo River sediment, which was exposed to diluted bitumen
following a pipeline spill in 2010, high rates of biodegradation were
measured for alkanes and polycyclic aromatic hydrocarbons (PAHs) at
25 °C, with slower rates and less overall degradation measured at 5 °C

⁎
Corresponding author.
E-mail address: Alice.Ortmann@dfo-mpo.gc.ca (A.C. Ortmann).

https://doi.org/10.1016/j.marpolbul.2019.01.012
Received 4 July 2018; Received in revised form 2 January 2019; Accepted 6 January 2019
Available online 12 January 2019
0025-326X/ Crown Copyright © 2019 Published by Elsevier Ltd. All rights reserved.
A.C. Ortmann et al.
(Deshpande et al., 2018). In contrast, a coastal microbial community
that had not previously been exposed to diluted bitumen exhibited slow
degradation rates, and minimal degradation was measured at ~18 °C
over 13 days (King et al., 2014). The difference in these estimated rates
could be due to previous exposure or the ability of freshwater microbial
communities to degrade diluted bitumen more effectively than marine
communities; however, inorganic nutrient concentrations were also
significantly different between the two experiments. With the microbes
enriched from the freshwater sediments, high concentrations of ni-
trogen and phosphorous were used in the experiments, while the ex-
periment using the coastal microbial community included no additional
nutrients above what was naturally present in the seawater. Because oil
is dominated by carbon, nitrogen and phosphorous are thought to limit
the microbial response (Atlas and Hazen, 2011), thus the low de-
gradation rates measured for the marine community may be due to low
availability of inorganic nutrients.
A short term experiment was carried out to quantify the impact of
inorganic nutrients on degradation that may occur immediately fol-
lowing a spill of diluted bitumen in coastal waters. Fresh diluted bi-
tumen, along with a natural coastal microbial community was added to
artificial seawater with low concentrations of nitrate, ammonium and
phosphorous and samples were collected over a five day period. Other
treatments included addition of inorganic nutrients alone, or in com-
bination, to determine if total degradation rates could be increased.
While both abiotic and biological degradation would be occurring in
the flasks, inorganic nutrients are most likely to impact biological
processes, without affecting physical or chemical weathering of the oil.
The short time frame was chosen to characterize what might happen in
the first few days following a spill. Given that in an actual spill situa-
tion, water movement will introduce nutrients and dilute contaminants,
a longer incubation was not undertaken. Additionally, the short time-
frame alleviates bottle affects associated with long incubations of small
volumes. Previous studies also suggest that diluted bitumen rapidly
weathers, with most of the changes occurring in the first few days (King
et al., 2014). The experiment with the Kalamazoo sediments also de-
termined that most of the biodegradation occurred within the first
10 days, even at 5 °C (Deshpande et al., 2018), therefore, much of the
degradation that is likely to occur should be observed in the first few
days following a spill. The experiment was also carried out at low
temperature to reflect winter conditions.
2. Materials and methods
2.1. Sample collection and experimental set-up
Seawater was collected near the shore of Bedford Basin, Dartmouth,
Nova Scotia in December 2016. At the time of sampling, temperature,
salinity and dissolved oxygen (DO) were measured using a Professional
Plus 2030 handheld probe (YSI). Seawater was taken to the Bedford
Institute of Oceanography, where cells were concentrated over 47 mm
0.22 μm Durapore® (polyvinylidene fluoride) filters (EMD Millipore).
One litre of seawater was reduced to 0.2 L using gentle vacuum
(5 in. Hg) in 15 separate autoclaved filter holders. Cells were re-sus-
pended gently using disposable Pasteur pipettes. The 0.2 L from each of
the filter holders were combined and diluted to a final volume of 15 L in
artificial seawater (ESAW) at a salinity of 27 (Berges et al., 2001;
Harrison et al., 1980). The recipe was modified to reduce the con-
centrations of nitrate, ammonium, phosphate and silicate (Table 1,
control and dilbit). Approximately 2 h passed between collection of
seawater and dilution of cells in ESAW.
The degradation experiment included 7 treatments: a control with
no additions to confirm that manipulations did not kill the microbial
community, diluted bitumen only (dilbit) and diluted bitumen with
added nitrate, ammonium, phosphate, nitrate + phosphate or ammo-
nium + phosphate. For each treatment, 18 baffle flasks were prepared
using the ESAW containing the natural microbial community. Each
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Marine Pollution Bulletin 139 (2019) 381–389
Table 1
Mean initial concentrations of nutrients in the original seawater and in the
seven treatments in ESAW. Standard deviations are in parentheses for the tri-
plicate samples.
Treatment NO3− (μM) NH4+ (μM) PO4−3 (μM) N:P
Bedford Basin Water 6.70 6.30 1.01 13.24
Control 3.43 (0.20) 6.63 (1.75) 3.52 (0.35) 2.91
Dilbit 3.71 (0.43) 6.16 (0.35) 3.72 (0.07) 2.70
Nitrate 241.17 (3.63) 5.71 (0.31) 3.52 (0.43) 70.18
Ammonium 4.27 (0.12) 301.18 (9.27) 3.40 (0.39) 89.97
Phosphate 3.39 (0.41) 6.98 (1.40) 41.24 (2.43) 0.26
Nitrate + phosphate 244.58 (2.63) 5.58 (0.61) 41.68 (2.17) 6.01
Ammonium + phosphate 3.99 (0.08) 310.95 (7.01) 40.05 (3.32) 7.87
flask received 100 mL of ESAW + microbes, 2 mL of nutrient solutions
or distilled water, and 15 μL of fresh Access Western Blend (AWB), a
bitumen product diluted with condensate (dilbit). The controls received
no AWB or additional nutrients. The concentrations of nitrate, ammo-
nium and phosphate in the ESAW were chosen to represent the lower
concentrations detected in the Bedford Basin over a 20-year time series
(Li, 2014). The added nitrate and ammonium resulted in the nitrate and
ammonium concentrations being similar to that in Bushnell Haas
media, a common media used for isolating hydrocarbon degrading
bacteria (Bushnell and Haas, 1941), although at one half the total ni-
trogen concentration in the Bushnell Haas media, which contains both
nitrate and ammonium. These concentrations were ~35 times higher
than measured in the Bedford Basin water. Phosphate was added to
maintain the N:P ratio from the Bedford Basin, but were determined to
be ~40 times higher than in Bedford Basin, resulting in concentrations
of 20% of that found in Bushnell Haas media. Baffle flasks for each
treatment were all prepared one at a time, resulting in a staggering of
the start times for each treatment. After addition of water, nutrients and
oil, flasks were capped and sealed with Teflon tape.
Following preparation of each treatment, three flasks were sacri-
ficed. The remaining flasks were stored in an incubator set to 4 °C until
being transported to a shipping container equipped with orbital sha-
kers, a small heater to prevent freezing, an air temperature probe and
three temperature data loggers (HOBO® pendants) submerged in
100 mL of water. Low light was used during sampling otherwise in-
cubations occurred in the dark. Shakers were set to 150 rpm to ensure
rapid mixing of the flasks. Three flasks for each treatment were
equipped with a Presens O2 optical sensor. A Fibox 4 handheld probe
was used to measure O2 concentrations in the headspace of the flasks
immediately after the treatment flasks were prepared and sealed,
45 min after the last treatment was placed in the shipping container (3 h
after the first treatment was finished) and then every day for the
duration of the experiment (t1–t5). For analysis, the measurement
made 0.75–3 h post set up was considered the beginning of the ex-
periment (t0). O2 was measured by taking three consecutive measure-
ments for each flask. Air temperature and barometric pressure were
input into the Fibox 4 system prior to collection of data. The system was
previously calibrated using air and N2 (0 mg L−1 O2). Triplicate mea-
surements were averaged for each flask and time point and readings
were converted to mg L−1. After measuring O2, three flasks from each
treatment were collected each day and processed as described below.
Flasks with O2 sensors were sacrificed on the final day of the experi-
ment after measuring O2.
2.2. Subsampling incubations
Flasks were sampled with an autoclaved, 15 cm long stainless steel
needle connected to an all-plastic syringe. A 30 mL sample was ex-
tracted, and ~1 mL was ejected into a microfuge tube. From this vo-
lume, a 980 μL aliquot was added to 20 μL of 25% EM grade glutar-
aldehyde in a cryovial. After 10 min on ice, the fixed sample was flash
A.C. Ortmann et al.
frozen in liquid N2 and stored at −86 °C until prokaryotes were
quantified by flow cytometry. The remaining volume in the syringe was
filtered through a 0.2 μm polycarbonate filter using an acid washed
polypropylene filter holder. The filtrate was collected in an acid cleaned
plastic vial and frozen at −20 °C for analysis of inorganic nutrients. The
filter was removed, folded in half and placed in a cryovial. The filter
was flash frozen and stored at −86 °C for community diversity analysis
by DNA sequencing. The remaining 72 mL in each flask was carefully
poured into a 150 mL amber glass bottle. 10 mL of dichloromethane
(DCM) was added to the flask, rinsing the needle. The DCM was then
swirled around the flask to dissolve any dilbit stuck to the sides and
poured into the amber bottle. The bottles were sealed, shaken and
stored at 4 °C until they were extracted to analyze hydrocarbons.
To quantify prokaryote cell numbers, cryovials were thawed in a
water bath at 37 °C. Samples were diluted 1:10 in Tris-EDTA (TE,
pH = 8) and incubated at room temperature for 10 min with 5 μL of a
1:200 dilution of SYBR Green (Lonza) (Ortmann et al., 2012) and run
on MED for 3 min through a FACSCalibur flow cytometer (BD Bios-
ciences) equipped with a 488 nm laser. For quality control, 1 μm
fluorescent beads were added to each sample. The total number of cells
was determined by gating the signals in a FL1 (green) vs. side scatter
(SSC) plot and converted to cells mL−1 using the run time and the ca-
librated flow rate. Inorganic nutrients were measured using a SEAL
Analytical AA3 continuous segmented flow autoanalyzer using standard
methods. Detection limits were determined for each run and only va-
lues above the detection limit were considered non-zero. NO3− was
calculated as the difference between NO2− + NO3− and NO2−. DNA
was extracted from filters using the QIAGEN MagAttract PowerWater
DNA/RNA Kit run on a KingFisher Duo Prime (Thermo) as per the
manufacturer's instructions, with the initial bead-beating in 2 mL tubes
on a Disruptor Genie (Scientific Industries). The V4–V5 region of the
16S rRNA gene was amplified (Walters et al., 2016) and sequenced on
an Illumina MiSeq (300 + 300 bp) by the Integrated Microbiome Re-
source at Dalhousie University, Halifax, NS.
Raw demultiplexed FASTQ files were analyzed following the
workflow described in the Microbiome Helper SOP (Comeau et al.,
2017). Briefly, paired reads were stitched together using PEAR (Zhang
et al., 2014) and filtered to remove low quality short reads. Remaining
sequences were screened to identify and remove chimeras using VSEARCH
(Rognes et al., 2016) and then processed via the open reference picking
pipeline in QIIME (Caporaso et al., 2010) using SORTMERNA (Kopylova
et al., 2012) and SUMACLUST (Mercier et al., 2013) to identify operational
taxonomic units (OTUs) at 97% similarity. The OTU table was then
filtered to remove OTUs representing < 0.1% of the total reads
(Bokulich et al., 2013). Because samples were incubated in the dark, the
OTU table was filtered to remove photosynthetic taxa including phylum
Cyanobacteria, order Chromatiales and family Rhodospirillaceae, all of
which decreased over the course of the experiment. The filtering step
removed 119 Cyanobacteria OTUs representing 7.9% of the sequences,
8 Chromatiales OTUs representing 0.04% of the sequences and 29
Rhodospirillaceae OTUs representing 0.4% of the sequences. Ad-
ditionally, taxa identified as mitochondria were removed (12 OTUs,
0.1% of the sequences) to retain only sequences associated with Bac-
teria and Archaea. Finally, the OTU table was normalized to a final
sequencing depth of 12,100 sequences per sample. Due to low sequence
numbers, three samples were omitted from further analysis. Ten sepa-
rate rarefied OTU tables were generated to estimate sample diversity
metrics with one being randomly selected for community structure
analysis. For each OTU table, Shannon diversity and Phylogenetic dis-
tance (PD) were calculated. The mean of the ten estimates was averaged
to generate a single diversity estimate for each sample. Community
structure was measured by calculating a Bray-Curtis dissimilarity ma-
trix for one randomly selected OTU table. FASTQ files are available
through the NCBI SRA database (accession number SRP145477).
Samples for quantifying total petroleum hydrocarbons (TPH) were
placed on a Wheaton R2P roller (Wheaton, Millville, NJ) at 9 rpm for
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Marine Pollution Bulletin 139 (2019) 381–389
18 h before the DCM layer was removed, weighed and evaporated using
an N-Evaporator (Organomation, Berlin, MA). Samples were diluted to
a final volume of 10 mL with DCM and stored at −20 °C. After con-
centration to 1 mL, extracts (1 μL) were injected and analyzed by an
Agilent 7890B GC connected to a flame ionization detector (FID) and
quantified using an 8 point calibration curve generated from the stock
AWB (Cole et al., 2007; King et al., 2015). Raw data were exported as
text and spectra were aligned using SpecAlign (Wong et al., 2005) with
the average spectrum generated from all sample extracts using peak
matching. Spectra were trimmed to retain data from 5 to 20 s and im-
ported into R for analysis. The remaining sample in DCM was ex-
changed into hexanes and purified using a modified solid-phase ex-
traction method (EPA, 1996). These extracts were analyzed using an
Agilent 6890 GC coupled to an Agilent 5973N MS operated in the se-
lective ion monitoring mode (SIM). Specific n-alkanes (C10 to C35),
PAHs and alkylated PAHs were quantified using retention time
matching to a seven-point calibration curve for identification (EPA,
2007). Values were standardized to the concentration of 17α(H),
21β(H)-hopane, a conservative biomarker (Prince et al., 1994; Venosa
et al., 1997). Hydrocarbon analysis was not carried out for control
samples.
2.3. Data analysis
The control flasks were used to confirm that the manipulation of the
samples had not inhibited cell activity and were not included in the
analysis of the various parameters. The effect of nutrient treatment on
prokaryote abundance, Shannon diversity, PD and TPH was determined
by applying two-way ANOVAs to test the effects of treatment, time and
the interaction term. Post hoc tests were carried out using a control
group, with comparisons to either t0 or the Dilbit treatment following
the Holm-Šídák method (SigmaPlot 13, Systat Software, Inc.). For TPH,
concentrations at each time point were divided by the average con-
centration at t0 for each treatment to compensate for differences in the
starting TPH concentrations between treatments. ANOVA analysis was
then carried out on the relative TPH concentrations. After alignment,
trimming and background subtraction, GC-FID spectra were analyzed
using Principle Components Analysis (PCA) in R (R Core Team, 2017)
using the rda function with scaling in the VEGAN package (Oksanen et al.,
2018). O2 concentrations were analyzed using a two-way repeated
measures ANOVA. Post hoc tests were carried out with Dilbit and t0 as
the control groups. For inorganic nutrients, 1-way ANOVAs or Kruskal
Wallis analyses were carried out for each treatment and nutrient se-
parately to identify changes over time. This was due to the large dif-
ferences in starting concentrations among treatments. The initial mi-
crobial community in the seawater was compared to the microbial
communities in the experimental flasks at t0 using the SIMPER routine
in Primer (Primer-E, Auckland). Taxa contributing at least 2% to the
differences between communities were identified. The effects of treat-
ment and time on community structure were identified using 2-way
ANOSIM analysis in Primer based on the Bray-Curtis dissimilarity ma-
trix. For pairwise comparisons, communities were considered sig-
nificantly different when the R2 value was > 0.3 and the p-value
was < 0.05. Changes in community structure were visualized with
Principle Correspondence Analysis (PCO). The rarefied OTU table was
imported into STAMP (Parks et al., 2014) to identify taxa that differed
between the two groups of treatment identified by ANOSIM for each
time point. OTUs were collapsed into taxonomic groups at the lowest
level possible, which for most OTUs meant identification to the family
level. For each time point, the two groups were compared using Welch's
two sided t-test with a Benjamini Hochberg FDR correction. Taxa with
corrected p-values < 0.05 and > 1% difference between mean abun-
dances were considered significantly different. Taxa that changed over
time were analyzed in STAMP for each ANOSIM group and the control
treatment using ANOVA with Tukey-Kramer post hoc tests and the
Benjamini Hochberg FDR. Corrected p-values < 0.05 and η2 effects
A.C. Ortmann et al.
sizes of > 0.9 were identified taxa with significant change in abundance
over time. For clarity, mean relative abundances of abundant taxa were
plotted for each group at each time. Abundant taxa were defined as
those with a mean relative abundance of ≥0.5%. To estimate de-
gradation rates, linear regressions of the natural log of the sum of the
alkanes, alkylated PAHs or PAHs normalized to hopane were carried
out against time for each treatment. Significant slopes were determined
using a two-tailed t-test with p < 0.1.
3. Results
The temperature of the seawater at collection was 6.1 °C with a
salinity of 26.27, slightly lower than the ESAW media. DO was mea-
sured at 9.48 mg L−1, compared with 11–12 mg L−1 at t0 (Fig. S1).
ESAW was stored in a 4 °C incubator prior to setting up the experiment,
minimizing the temperature difference. Samples for prokaryote abun-
dance, inorganic nutrients and bacterial community structure were
collected prior to filtering to characterize parameters in the original
seawater. The initial prokaryote abundance in the seawater was
7.61 × 105 cells mL−1, while estimates for all treatments at t0 averaged
2.66 × 105 cells mL−1 ± 6.02 × 104. Thus, the density of prokaryotes
at the beginning of the experiment was only ~35% of the in situ con-
centrations.
At t0, nitrate was lower in the control and Dilbit treatments com-
pared to in situ values, while phosphate was higher and ammonium was
similar to in situ values. Nutrient additions resulted in significantly
higher nitrate, phosphate or ammonium, depending on the treatment
(Table 1). The N:P ratio was lower than in situ for all treatments except
for the nitrate-only and ammonium-only additions.
Microbial diversity in the flasks was similar to that in the seawater.
PD averaged 51.7 ± 1.5 in the flasks compared to 49.2 in the seawater,
while Shannon diversity in the flasks averaged 7.16 ± 0.04, with 7.16
measured in the seawater. The structure of the microbial communities
was slightly different in the flasks compared to the initial seawater
based on SIMPER analysis using the Bray-Curtis dissimilarity metric
(average dissimilarity = 21.21%). The 7 OTUs contributing the most to
the differences between samples included 4 OTUs with higher average
abundances in the experimental flasks. These OTUs included
Saprospiraceae (flasks = 2.6%, seawater = 1.4%) and
Flavobacteriaceae (Bacteroidetes) (flasks = 4.5%, seawater = 3.3%)
and two Rhodobacteraceae (Alphaproteobacteria) OTUs, one of which
was identified as the genus Octadecabacter (flasks = 7.1%, sea-
water = 6.1%) and the other that was only identified to the family level
(flasks = 1.7%, seawater = 0.9%). Two Pelagibacteraceae
(Alphaproteobacteria) OTUs (flasks = 1.8%, seawater = 3.7%) along
with the OCS155 family of the Acidimicrobiales (flasks = 1.5%, sea-
water = 2.3%) had lower abundances in the experimental flasks com-
pared to the whole seawater. Pelagibacteraceae are generally small cells
and may have passed easily through the filter (Rappe et al., 2002).
Some marine Acidimicrobiales may also be small enough to pass
through the filter (Ghai et al., 2013). The four most dominant OTUs
were the same in both the flasks and the seawater and included three
Rhodobacteraceae, including two Octadecabacter spp., and Flavo-
bacteriaceae.
3.1. Control treatment
In the control treatment, O2 concentrations in the headspace re-
mained relatively constant over time with an increase at t3, corre-
sponding to a decrease in incubation temperature. Cell numbers in-
creased over the experiment, with a final average abundance of
2.66 × 106 cell mL−1 ± 7.64 × 105, higher than in the initial sea-
water. During the experiment, the concentration of inorganic nutrients
changed little, with the exception of ammonium, which increased ~3
fold between t2 and t3. Microbial community diversity decreased in the
control flasks over time, with PD showing a gradual decrease from
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Marine Pollution Bulletin 139 (2019) 381–389
Fig. 1. Abundance of prokaryote cells in the different treatments over time.
Maximum cell density in nitrate + phosphate occurred at t3, while maximum
densities for ammonium + phosphate occurred at t4.
52.2 ± 0.7 to 28.6 ± 2.4. Shannon diversity decreased from t0 to t2
and then remained relatively constant through to the end of the ex-
periment. Oleispira spp., Glaciecola spp., Sediminicola spp. and
Colwelliaceae increased significantly in the control flasks compared to
t0. Thirty-three taxa decreased significantly over the five days.
3.2. AWB and Nutrient additions
Analysis of O2 concentrations in the headspace identified significant
differences over time (p < 0.001) and among treatments (p < 0.001),
but the interaction term was not significant. Generally, O2 decreased
from t0 to t2, but increased at t3 before decreasing by t5 (Fig. S1).
Holm-Šídák post hoc tests indicated that all time points were sig-
nificantly different from t0. All nutrient treatments except Nitrate were
determined to have significantly lower O2 concentrations compared to
the Dilbit treatment.
Cells increased in all treatments over the duration of the experi-
ment, with abundances in several treatments exceeding
2.0 × 106 cells mL−1 (Fig. 1). Abundances in the nitrate + phosphate
treatment peaked at t3 and decreased through the remainder of the
experiment. In the ammonium + phosphate flasks, abundances peaked
at t4. The other treatments appeared to still be increasing at the end of
the experiment. Two-way ANOVA of cell abundances identified a sig-
nificant interaction between time and treatment (p < 0.001). Holm-
Šídák post hoc tests indicated that for dilbit and ammonium, cells did
not grow significantly until after t2, as neither t1 nor t2 were sig-
nificantly different from t0. For all other treatments, t0 and t1 were not
significantly different, but t2–t5 were higher than t0 suggesting a
shorter lag period. Significantly higher cell densities were detected at t2
(nitrate + phosphate), t3 (nitrate + phosphate) and t4 (ammo-
nium + phosphate) compared to the dilbit treatment.
Inorganic nutrients differed due to treatment by design, so 1-way
ANOVAs were carried out for each treatment. Even where concentra-
tions were low, nutrients were never depleted during the experiment
(Fig. 2). In the dilbit treatment, nitrate decreased between t0 and t1,
but then increased with t1 significantly lower than t4 and t5
(p = 0.008). Phosphate showed no change over time (p = 0.085), but
similar to the controls, ammonium increased between t2 and t3, with
significantly higher concentrations throughout the end of the experi-
ment (p < 0.001).
For the nitrate treatment, neither nitrate (p = 0.538) nor phosphate
(p = 0.288) showed change over time, but ammonium increased be-
tween t1 and t2, with significantly higher concentrations from t3
through t5 relative to t0 and t1 (p < 0.001). The ammonium treatment
had a similar nitrate pattern as dilbit alone, decreasing by t1 and then
increasing at t4 and t5 (p = 0.002). A Kruskal-Wallis ANOVA on ranks
indicated significant differences in phosphate concentrations due to
A.C. Ortmann et al. Marine Pollution Bulletin 139 (2019) 381–389

Fig. 2. Concentrations of inorganic nutrients in the flasks over time with values in μM. The top panel includes treatments where additional nutrients were added. The
bottom panel includes treatments where concentrations began at the concentration in the ESAW.

time (p = 0.022); however, Tukey post hoc pairwise tests did not detect
differences among time points. The ammonium treatment, although
starting with much higher ammonium concentrations, also had in-
creases between t2 and t3, resulting in significantly higher concentra-
tions at t3, t4 and t5 compared to t1 (p = 0.004). The phosphate
treatment had no significant change in concentrations of phosphate
(p = 0.631) or ammonium (p = 0.118), but nitrate concentrations at t5
were significantly higher than at t0 (p = 0.010). No change in the
phosphate concentration was detected in the nitrate + phosphate
treatment (p = 0.631). Both nitrate and ammonium increased in this
treatment with all time points significantly higher than t0 for nitrate
(p = 0.002) and t2 through t5 significantly higher than t0 and t1 for
ammonium (p < 0.001). The ammonium + phosphate treatment had
significantly higher nitrate concentrations at t5 compared to t1
(p = 0.031), but neither phosphate (p = 0.754) nor ammonium
(p = 0.115) showed changes over time.
The diversity of the microbial community decreased throughout the
experiment in all treatments (Fig. 3). Two-way ANOVA indicated a
significant interaction between time and treatment for both PD and
Shannon diversity (pPD = 0.001, pShannon < 0.001). Post hoc tests in-
dicated that the diversity in phosphate, nitrate + phosphate and am-
monium + phosphate was significantly lower at t1 and t2 compared to
the dilbit treatment indicating a faster decrease compared to the nitrate
and ammonium treatments which did not differ from the dilbit
treatment. By t3, there were no longer significant differences among
treatments. Looking at overall community structure, there was a clear
shift in all treatments over time. Two-way ANOSIM analysis using the
Bray-Curtis similarity metric identified significant differences between
all pairs of time points (Global R = 0.892, p = 0.001,
ppairwise = 0.464–1.00, p = 0.001). Treatment was also significant
(Global R = 0.499, p = 0.001), but not all pairwise comparisons were
significant resulting in two groups of samples. Dilbit, nitrate and am-
monium samples formed one group (D-N-A), while phosphate, ni-
trate + phosphate and ammonium + phosphate (P-NP-AP) made up
the second group. The most obvious difference in these groups was at t1
and t2, where the two groups formed separate clusters (Fig. 4). By t3,
the difference in the two groups was reduced, although differences in
specific taxa could be detected at t3 and t4. Using STAMP, six taxa were
determined to be significantly different between P-NP-AP and D-N-A at
t1. In the P-NP-AP group, Colwelliaceae, Glaciecola spp., Sediminicola
spp. and Rhodobacteraceae averaged 2.6, 4.8, 1.2 and 2.7% higher,
respectively, when compared to the D-N-A group (Fig. 5). In contrast,
Pelagibacteriaceae and Flavobacteriaceae were 2.5 and 1.5% higher,
respectively, in the samples without additional phosphate. By t2, only
two taxa were found to be significantly different, with Rhodobacter-
aceae 7.4% higher in the phosphate group, while Flavobacteriaceae was
1.1% higher without phosphate. Two taxa were significantly different
between groups at t3, with 4.6% more Colwelliaceae in treatments

Fig. 3. Bacterial and archaeal diversity decreased over time, with significantly lower diversity in phosphate, nitrate + phosphate and ammonium + phosphate
treatments compared to dilbit at t1 and t2. Shannon diversity (A) and Phylogenetic distance (B) show the same patterns.

385
A.C. Ortmann et al.
Fig. 4. PCO plot of bacterial and archaeal communities over time based on the
Bray-Curtis dissimilarity metric. Colours indicate time points from dark (t0) to
light (t5). Treatments are grouped based on ANOSIM results, which groups
treatments with additional phosphate together (P-NP-AP) separate from the
other treatments (D-N-A).
without additional phosphate and 6.8% more Glaciecola spp. in the
additional phosphate treatments. At t4, Colwelliaceae and Rhodo-
bacteraceae were 5.3 and 5.0% higher, respectively, in the treatments
without phosphate. Glaciecola spp. was 8.9% higher with phosphate.
Averaged across groups, Rhodobacteraceae was the most abundant taxa
in all treatments at all times, except for the P-NP-AP samples at t5,
where Glaciecola spp. was higher on average. In all of the oiled treat-
ments, Colwelliacea, Octadecabacter spp., and the Gammaproteo-
bacteria genus HTC (family HTCC2188) rounded out the five most
abundant taxa. Of all the taxa that changed significantly among time
points (nD-N-A = 34 and nP-NP-AP = 41), only three showed significant
increases over time. Those taxa were Colwelliaceae, Rhodobacteraceae
and Glaciecola spp. (Fig. 5). These three taxa have been associated with
hydrocarbon degradation. Other abundant taxa linked with hydro-
carbon degradation included Gammaproteobacteria in the HTCC2188
family and the genus HTCC2207.
TPH concentrations at t0 averaged 120.41 μg mL−1 ± 15.94 (Fig.
S2). Because of this variation, the concentrations were divided by the
average for each treatment at t0 to produce relative TPH concentrations
and two-way ANOVA was carried out using these values. The analysis
detected significant differences due to both time (p < 0.001) and
treatment (p < 0.001), but the interaction term was not significant
(p = 0.461). t2 and t3 had significantly higher relative TPH
Fig. 5. Mean relative abundances of taxa of abundant taxa, defined as those with mean abundances ≥0.5% of the total microbial community. Taxa discussed in the
text are highlighted in the legend. Samples are grouped based on ANOSIM analysis as in Fig. 4. A full list of taxa with mean relative abundances and standard
deviations are provided in Table S1.
386
Marine Pollution Bulletin 139 (2019) 381–389
Fig. 6. Plot of the first two PCA axes from analysis of the GC-FID spectra. Points
are coloured based on time from dark (t0) to light (t5), with shapes indicating
treatments.
concentrations compared to t0. Compared to dilbit alone, neither ni-
trate nor phosphate was significantly different, while ammonium, ni-
trate + phosphate and ammonium + phosphate generally had higher
relative TPH concentrations. Analysis of TPH spectra with PCA in-
dicated a change in the composition of the oil during the experiment.
There was no pattern due to treatment, but samples generally became
more positive along the PCA Axis 1, but more negative on PCA Axis 2,
indicating a change in composition over time (Fig. 6). Measured with
GC–MS, normalized alkylated PAHs and PAHs showed no significant
decrease over the five days, regardless of the treatment. p-Values for the
t-tests for the significance of the slopes ranged from 0.173 to 0.973.
Alkylated PAHs averaged 8.52 ± 0.65 at t0 and 9.12 ± 0.91 at t5
when normalized to hopane. For PAHs, the means were 0.63 ± 0.06 at
t0 and 0.64 ± 0.04 at t5. For most treatments, the slope of the re-
gression for alkanes was not significantly different from zero (p-va-
lues = 0.157–0.329), suggesting minimal degradation occurred. How-
ever, in the nitrate + phosphate and ammonium + phosphate
treatments, slopes were significant (p = 0.068 and p = 0.070, respec-
tively), and the alkane degradation rate for nitrate + phosphate was
determined to be 0.0230 d−1 while the rate for ammonium + phos-
phate was 0.0235 d−1.
4. Discussion
The addition of fresh diluted bitumen to a natural coastal microbial
community resulted in microbial growth and a shift in the community
towards known hydrocarbon degrading organisms. The total amount of
petroleum hydrocarbons did not decrease significantly over the short
A.C. Ortmann et al.
time frame of the experiment, but changes in the profile of the GC-FID
spectra indicated that degradation occurred (Fig. 6). This was sup-
ported by GC–MS analysis in two treatments, nitrate + phosphate and
ammonium + phosphate, where significant decreases in alkanes were
measured over the five days of the experiment. While the patterns ob-
served for TPH and alkanes may appear contradictory, differences in
the methods can explain why this is not true. GC–MS data was stan-
dardized relative to hopane concentrations. Thus, the decrease in al-
kanes is relative to the stable marker, hopane. This is in contrast to the
TPH data, where concentrations are not standardized with a marker,
but represent total extractable hydrocarbons in the sample. Ad-
ditionally, GC–MS is more sensitive than GC-FID. Based on the calcu-
lated degradation rates for alkanes in nitrate + phosphate and ammo-
nium + phosphate treatments, over the course of 5 days, there was a
loss of ~41 ng mL−1 of alkanes against a background TPH concentra-
tion of > 100 μg mL−1.
The degradation rates calculated in this experiment when both ni-
trogen and phosphate was added fell between previous estimates for
dilbit degradation rates (Deshpande et al., 2018; King et al., 2014). The
measured rates were ten-fold higher than the rates observed by King
et al. (2014) with a natural marine microbial community at ~18 °C
without added nutrients. With the addition of dilbit alone, no sig-
nificant decrease in alkanes was observed, suggesting the additional
nitrogen and phosphorous stimulated hydrocarbon degradation. Even
with additional inorganic nutrients, the rates observed here were still
ten-fold lower than the rates observed for incubations of fresh dilbit and
excess inorganic nutrients with microbial communities enriched from
the Kalamazoo River at 5 °C (Deshpande et al., 2018). The main dif-
ference between studies is the prior exposure of the river sediment
community to dilbit, whereas the present study used a community that
had not previously been exposed to dilbit. While some regions may
have previous or continuous exposure to oil over time, priming a re-
sponse to a large spill, other areas may be more pristine. Thus, lower
degradation rates are likely to occur following a dilbit spill in marine
environments that have not been previously exposed to oil.
The short time frame of this experiment does not allow us to de-
termine if all of the alkanes would have been degraded given sufficient
time. It is also not clear if the advantage of additional nitrogen and
phosphorous would have accelerated the removal rates over the long
term. There are some indications that this would not have occurred.
Looking at the microbial community itself, by t5 there was no differ-
ence in the community composition among treatments (Fig. 5) and
while growth in the nitrate + phosphate and ammonium + phosphate
treatments had decreased and was showing declining cell numbers,
growth was still occurring in the other treatments (Fig. 1). Together,
these patterns suggest that a hydrocarbon degrading community de-
veloped in all treatments with dilbit regardless of inorganic nutrient
concentrations. Additionally, looking at the chemical data, there was no
significant difference in TPH concentrations among treatments at t5.
While alkane degradation rates were determined for two of the treat-
ments, PAHs and alkylated PAHs did not change. Together, these three
groups of compounds make up approximately 45% of AWB (King et al.,
2014), with the rest composed of resins and asphaltenes, which are
even more resistant to biodegradation. Thus, it is not surprising that
TPH showed little change over the experiment. While TPH did not
change, the composition of the oil as measured by GC-FID did show a
clear change over time, but no clear difference was observed among
treatments. This suggests that weathering of the oil was occurring in all
of the treatments. However, we cannot discount that some of the
weathering was abiotic, which should not be impacted by inorganic
nutrients.
Although the experiment was carried out at low temperatures, the
microbial community structure changed rapidly, with the largest shifts
between t0 and t1 as well as t1 and t2. After t2 shifts in community
structure were smaller. The addition of phosphate, either alone or with
a nitrogen source appeared to increase the rate at which the community
387
Marine Pollution Bulletin 139 (2019) 381–389
changed. At t1 there is a clear difference between the treatments
amended with phosphate and those that were not (Fig. 5). The differ-
ence in these two clusters is mainly due to larger increases in the re-
lative abundance of Glaciecola spp., Colwelliaceae and Rhodobacter-
iaceae with additional phosphate (Fig. 5). While all three taxa increased
in the treatments without excess phosphate relative to t0, the increases
were smaller. Glaciecola spp. have previously been observed to increase
in North Atlantic waters in response to oil additions (Tremblay et al.,
2017) and has been documented in hydrocarbon contaminated cold
waters, although in a mesocosm experiment Glaciecola spp. was more
abundant in the control treatments (Chronopoulou et al., 2015). In the
present experiment, Glaciecola spp. increased with oil at t1, and was one
of the most abundant taxon at t5, with average relative abundances of
22.9 and 30.3% in dilbit treatment without and with additional phos-
phate, respectively. In the controls at t5, Glaciecola spp. was the second
most abundant component of the community, but only reached average
abundances of 18.8%.
At t5, the control flasks were dominated by organisms that are as-
sociated with hydrocarbon degradation, including Rhodobacteraceae,
Oleispira spp. and Colwelliaceae (Jimenez et al., 2007; Ortmann and Lu,
2015; Ribicic et al., 2018; Yakimov et al., 2003), although not all
Rhodobacteraceae or Colwelliaceae degrade oil and are common
members of coastal microbial communities (Bowman, 2014; Elifantz
et al., 2013; Ortmann and Ortell, 2014). Sediminicola spp. were also
abundant in the controls at t5, but were also present in the dilbit treated
samples, although they averaged only 2.1% vs. 5.3% in the controls.
Sediminicola spp. (identified as NS3a marine group of the Flavobacter-
iaceae in the Silva database (Yilmaz et al., 2014)), are commonly as-
sociated with blooms and likely capable of degrading complex organic
matter (Kirchman, 2002; Yang et al., 2015). This group may have been
responsible for the increase in ammonium in many of the treatments,
due to remineralization of organic matter present in the incubations.
Along with Rhodobacteraceae, other taxa associated with hydro-
carbon degradation were abundant in the oil exposed flasks at t5.
Octadecabacter spp., within the family Rhodobacteraceae, has pre-
viously been associated with oil contamination under cold conditions
(Brakstad et al., 2008). This organism increased by t1 in the presence of
oil under all treatments relative to t0, but decreased back down to
starting abundances by t5. Other taxa included Gammaproteobacteria
identified as HTCC2207 (SAR92) and HTCC2188 (OM182), which in-
creased in the presence of oil with larger increases in treatments with
additional phosphate (Fig. 5). By t5 HTCC2188 increased 1.3× with
dilbit, and 1.8× with dilbit and additional phosphate. HTCC2207
was < 0.5% of the community at t0, but increased to 0.9 and 1.2% with
dilbit and with dilbit and phosphate, respectively. A draft genome of
HTCC2207 (Alteromonadales, Gammaproteobacteria) includes anno-
tations for a putative alkane monooxygenase gene (Stingl et al., 2007),
suggesting these organisms can degrade alkanes (Rojo, 2009).
HTCC2188 has been detected previously in coastal waters (Liu et al.,
2016), but why it responded to dilbit is unclear. This organism is
identified as either belonging to the OM182 of the Altermonadales
(Greengenes, McDonald et al., 2012) or Pseudohongiella spp. in the
Oceanospirillales (SILVA, Yilmaz et al., 2014). Both of these Gamma-
proteobacteria families are associated with hydrocarbon degradation
(e.g. Kostka et al., 2011; Ortmann and Lu, 2015; Redmond and
Valentine, 2012), so it is possible that HTCC2188 may share this ability.
The addition of dilbit to a natural microbial community that had not
previously been exposed to oiling resulted in a rapid shift in the mi-
crobial community. Bacteria commonly associated with both cold water
and hydrocarbons increased, with faster increases in treatments
amended with phosphate, with or without inorganic nitrogen. Cell
abundances increased fastest in the ammonium + phosphate and ni-
trate + phosphate treatments, in which alkane degradation rates could
be calculated. Although the rates measured here were lower than ob-
served for a freshwater microbial community enriched on dilbit
(Deshpande et al., 2018), they were higher than rates measured for a
A.C. Ortmann et al.
coastal community in the absence of additional inorganic nutrients
(King et al., 2014). The results observed here suggest that there may be
an initial benefit to increasing inorganic nutrients to stimulate biode-
gradation of dilbit, but in the short time frame of this experiment, the
advantage was minimal and disappeared by t5. Under natural condi-
tions, where water is constantly moving and resupplying nutrients, it is
unlikely that a coastal community at low temperature would become
nutrient limited; hence, there would be no advantage to adding in-
organic nutrients in an attempt to increase the rate of dilbit biode-
gradation. Increases in inorganic nutrients during this experiment
suggest that organic sources of nutrients may be used by microbial
communities during the biodegradation of hydrocarbons.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.marpolbul.2019.01.012.
Acknowledgements
Thanks to S. Ryan and B. Robertson for technical and logistical as-
sistance. C. Anstey processed inorganic nutrient samples. This research
was supported through the Oceans Protection Plan program of the
Government of Canada.
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