Cultivation of a native alga for biomass and 
biofuel accumulation in coal bed methane 
production water
Authors: Logan H. Hodgkiss, J. Nagy, Elliott P. 
Barnhart, Alfred B. Cunningham, & Matthew W. 
Fields
NOTICE: this is the author’s version of a work that was accepted for publication in Algal 
Research. Changes resulting from the publishing process, such as peer review, editing, 
corrections, structural formatting, and other quality control mechanisms may not be reflected in 
this document. Changes may have been made to this work since it was submitted for publication. 
A definitive version was subsequently published in Algal Research, [Vol. 19, November 2016] 
DOI#10.1016/j.algal.2016.07.014.
Hodgskiss LH, Nagy J, Barnhart EP, Cunningham AB, Fields MWF, “Cultivation of a native alga 
for biomass and biofuel accumulation in coal bed methane production water,” Algal Research, 
2016 Nov; 19(1):63-68
Made available through Montana State University’s ScholarWorks 
scholarworks.montana.edu 
Cultivation of a native alga for biomass and biofuel 
accumulation in coal bed methane production water
L.H. Hodgskiss a,b, J. Nagyc, E.P. Barnhartd s 
b,c,e,⁎
a Department of Civil Engineering, Montana State University, Bozeman, MT 59717, Unite
b Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717, United 
c Depa  MT
d U.S. ited
e Energ tate
esu
llen
d 
ro
 fo
as
ed
M 
t g
a s
ro
 production water, and lipid accumulation did not 
production wastewater can be minimally amended and used for the cultivation of a native, 
lipid-accumulating a
Keywo
Powde
basin
Water 
Algae
Bio-oil
Di can 
accu ed pr
carbo igme
furth  emi
utiliz [1]. 
repor has 
expo ironm
prior
nitro
nitro
resou
energ
beco
increasing global population combined with de-
creas
avail
Susta
Rese
indic
and 
the a
quali
strate
nutri
prod
water. Water pro-duced by CBM extraction is typically 
a2+ observed in 
[7]). There-fore, inable Development of Algal Biofuels (National 
arch Council) published a report in 2012 that 
integrated approaches are crucial when developing 
future water, energy, and climate policy [8], particularly 
in regions with changing water demands.ing reserves of fertilizer and the diminishing 
ability of clean water. The Committee on the 
and low concentrations of Mg2+ and C
the majority of the produced water (gen and phosphorus. In addition to carbon, 
gen, and phosphorus, water is an inherent 
rce that has to be considered in the alternative 
y sector. Water and nutrient availability are 
ming an increasing con-cern in light of a rapidly 
stored in lined or unlined im-poundments but can be 
used for agricultural purposes or discharged into streams 
with proper monitoring and treatment [5,6]. However, 
the production water can be problematic due to the high 
concentrations of Na+ and total dissolved solids (TDS), ated the large scale production of 
bio-products would exert a signifi
lready limited availability of nutri
ty water resources. The report s
gies were needed to reuse and recy
ents in future photo-synth
uction facilities [2].ental stresses 
ein), including 
246,601,576 Mcf (Mcf = 1000 cubic feet) of gas and an 
associated 256,305,005 Bbls (8,073,617,107 gal) of  to harvest ([1] and references therer contribute to reduced CO2
ation of atmospheric CO2 
ted algal biofuel research 
sing cultures to a range of envlga.
produce and 
ecursors (e.g., 
nts) that can 
ssions through 
Much of the 
focused upon 
The volume of water used in the energy sector 
represented 15% of global freshwater withdrawals in 
2010 [3], and coal-bed methane (CBM) production 
produces large amounts of water that can negatively 
impact the environment and the economy. According to 
the Wyoming Oil and Gas Conservation Commission 
[4], in 2014 the Powder River Basin produced fferent diatoms and algae 
mulate a variety of value-add
hydrates, fatty acids, and pincrease with additional phosphorus limitation. The presented results show that CBM 
coin-cided with lipid accumulation in CBMrtment of Microbiology and Immunology, Montana State University, Bozeman,
Geological Survey, Wyoming-Montana Water Science Center, Helena, MT, Un
y Research Institute, Montana State University, Bozeman, MT 59717, United S
Coal bed methane (CBM) production has r
Basin of low-quality water in a water-cha
isolated from a CBM production pond, an
indicated the isolate belongs to the Chlo
macro- and micronutrients were evaluated
defined medium. A small level of growth w
and biomass in-creased (2-fold) in amend
highest growth rate was observed in CB
unamended CBM water displayed the lowes
observed in CBM water with nitrate, and 
observed in the defined growth medium. G
rds:
r river 
recyclealgal biofuels 
cant strain on 
ents and high-
uggested that 
cle water and 
etically-driven 
w
b
a
w
m
s,b, A.B. Cunningham a,b,e, M.W. Field
d States
States
 59717, United States
 States
s
lted in thousands of ponds in the Powder River 
ged region. A green alga isolate, PW95, was 
analysis of a partial ribosomal gene sequence 
coccaceae family. Differ-ent combinations of 
r PW95 growth in CBM water compared to a 
 observed in unamended CBM water (0.15 g/l), 
 CBM water or defined growth medium. The 
water amended with both N and P, and the 
rowth rate. The highest lipid content (27%) was 
ignificant level of lipid accumu-lation was not 
wth analysis indicated that nitrate deprivation Recently, the growth of microalgae in low-quality 
ater sources for the purposes of remediation and/or 
iofuel production have been re-ported in the literature, 
nd include dairy wastewater [9] and municipal 
astewater [10]. A primary concern when cultivating 
icroalgae is selecting a species that is a good fit for the 
elected conditions in
different parts of the country (e.g., water, light, nutrients). A recent
study successfully grew a microalgal species in CBM production water
in Australia in which a marine algae species was selected to accommo-
date the high salt content of the water [11]. However, the use of CBM
production water from the Powder River Basin for this purpose has
not been evaluated.
Identifying beneficial uses for low-quality production water from
underground coal seams will become increasingly important as domes-
tic energy production from subsurface reservoirs increases to meet
growing energy demands. An alternative use for CBM production
water could be the growth of microalgae for biodiesel and biomass pro-
duction. Due to photoautotrophic growth, algae utilize solar energy for
growth and CO2 (HCO3−) as the carbon source, but require a low-quality
water source that does not deplete already stressed water supplies. In
addition, CBM production water is inherently high in bicarbonate
(HCO3−) and pH, conditions that foster the utilization of dissolved inor-
ganic carbon by photoautotrophs. Therefore, the low-quality water
needs for algal biofuels and the wastewater streams from CBM produc-
tion present an opportunity to couple water recycling, light-driven CO2
utilization, and production of value added products. We have isolated a
native green alga from a CBM production pond in northeastern Wyo-
ming and evaluated the potential for growth in CBM production water
with the ability to accumulate lipids that can be converted into biodie-
sel. A native species provides the benefit of an organism that is already
adapted to the water and climate conditions of the region, and we have
identified the key nutrients required for the species to grow photoauto-
trophically in CBM production water and accumulate lipids during N-
deprivation.
2. Methods
2.1. Materials
The alga, PW95, was isolated from a CBM production water pond in
the Powder River Basin of northeastern Wyoming (44° 52.613′N 106°
54.700′W). All chemicals were of highest purity available.
2.2. Culture methods
2.2.1. Culture isolation
Cultureswere streaked on Bold's BasalMedium (BBM) agar plates in
a 20 °C incubator with a 14:10 h light:dark cycle (7872 lx, cool white
fluorescent). Subsequent colonies were streaked 3 times for the isola-
tion of a single photoautotroph. DNA sequence analyses (18S and 16S)
suggested a unialgal culture with no detected bacterial sequences, and
heterotrophic plates (R2A) incubated at 20 °C in the dark did not display
bacterial growth. Cultures were routinely checked for heterotrophic
growth.
2.2.2. Culture conditions
Liquid BBMwas used routinely for culturemaintenance and biomass
production at 20 °C. Flasks were placed on a shaker rotating at 125 rpm
to encourage mass transfer of CO2. Flask experiments were inoculated
from a stock culture of PW95 grown in liquid BBM. Each inoculum
was centrifuged (1750 ×g for 5 min) and washed twice with sterile
CBMwater or BBMdepending on the intended use. CBMwaterwas sup-
plied from well FG-09 (45° 26′ 5.8914″N 106° 23′ 31.416″W) in the
Powder River Basin, and this water was used for all experiments. CBM
production water was filter sterilized using a bottle top 0.2 μm, polye-
thersulfone (PES), Nalgene Rapid-Flow vacuum filter (Thermo Scientif-
ic) in order to better understand the direct responses of the algal isolate
in CBMproductionwater. Growth experiments at 20 °Cwere performed
with biological duplicates in 500 mL Erlenmeyer flasks containing
250 mL of sterile CBM water or Bold's Basal Medium (BBM; 0.5 mM ni-
trate, 0.05mMphosphate). Five different nutrient conditionswere eval-
uated using CBMwater: CBMwater without additions; CBMwater with0.5 mM nitrate; CBM water with 0.5 mM nitrate and 0.05 mM phos-
phate; CBM water with 0.5 mM nitrate, 0.05 mM phosphate, and
0.3 mM magnesium sulfate; and CBM water with 0.5 mM nitrate,
0.05 mM phosphate, 0.3 mM magnesium sulfate, and micronutrients
(i.e., trace vitamins andminerals) used in BBM [12]. Allflaskswere inoc-
ulated with 15 mL of inoculum from the same stock culture.
2.3. Analysis methods
2.3.1. Determination of biomass dry weight
Nitrocellulose or acetate cellulose 0.2 μm filters (25 mm diameter)
were used to periodically determine the dry weight biomass of the cul-
tures. Culture samples were vacuum filtered and rinsed with water to
remove excess salts before air drying in an oven set at approximately
90 °C for 48 h. Filters were weighed before and after use to determine
dry weight biomass.
2.3.2. Determination of chlorophyll
Culture samples (1 mL) were centrifuged (16,162 ×g for 15 min) in
microcentrifuge tubes and 950 μL of supernatant was frozen at−80 °C
for water chemistry analysis. The cell pellet was submerged in 1 mL of
100% methanol, disrupted by sonication and vortexing, and stored at
4 °C for at least 24 h in a covered box. After 24 h, the samples were cen-
trifuged (16,162 ×g for 5 min). The supernatant was extracted and an-
alyzed in disposable plastic cuvettes using a ShimadzuUV-1700 UV–VIS
spectrophotometer at wavelengths of 665.2 nm, 652 nm, and 632 nm.
Chlorophyll concentrations in μg/mL were estimated as previously de-
scribed by Ritchie [13].
2.3.3. Determination of lipid content
Lipid accumulation was tracked using a Nile Red staining method
[14]. Culture samples (1 mL) were stained with Nile Red stock solution
(4 μL of 0.25 mg Nile Red/mL of acetone). Samples were stored in the
dark for 5 min. The set time of 5 min was determined by performing a
time assay with PW95 to determine the optimum time to measure
Nile Red fluorescence as described by Chen et al. [15]. After 5 min,
200 μL duplicates were pipeted into a 96 well plate and fluorescence
was read using a Synergy H1 hybrid flourometer/spectrophotometer
reader. Gen5 microplate reader software was used to evaluate the fluo-
rescence at an excitation of 480 nm and emission of 580 nm. Samples
were diluted 2, 4, or 10 times as necessary to attempt to keep Nile Red
measurements within a linear range [15].
2.3.4. Determination of anion concentrations
Nitrate and phosphate concentrations were determined with a
DIONEX ICS-1100 and Chromeleon Chromatography Management Sys-
tem with an ASRS 4 mm suppressor. A 4.5 mM sodium carbonate and
1.4 mM sodium bicarbonate eluent mix was used. Values for pH were
measured using a standard bench top Oakton pH 11 Series meter.
2.3.5. Determination of FAME content via GC-MS
Cultures were harvested once a Nile Red peak was observed and
processed as described previously [16]. Flasks were transferred to
50 mL sterile Falcon tubes and centrifuged (4816 ×g for 10 min). The
supernatant was decanted and the biomass was flash frozen at−80 °
C until the sample was lyophilized. After lyophilization, biomass was
weighed in glass tubes, and submerged in 1 mL of toluene and 2 mL of
sodium methoxide. The samples were vortexed and placed in a 90 °C
oven for 30 min (samples were vortexed every 10 min). After 30 min,
2 mL of 14% boron trifluoride in methanol were added to each sample
and vortexed. Samples were again incubated for 30 min at 90 °C (sam-
pleswere vortexed every 10min).When finished, 0.8mL of hexane and
0.8mL of 15%NaClwere added to each sample. Sampleswere incubated
for 10 min and centrifuged (2500 ×g for 2 min). The top layer in the
tube (containing the lipids) was extracted and analyzed on an Agilent
Technologies 6890 N Network GC system to determine lipid concentra-
tion and composition.
2.3.6. Determination of SSU rRNA sequence
Cells for DNA extraction were taken through three freeze-thaw cy-
cleswith liquid nitrogen, and cellswere lysedwith sterile sand in amor-
tar and pestle (3 times). A FastDNA Spin Kit for Soil (MP Biomedicals,
Santa Ana, CA) was used to extract the algal DNA. Quality of the extract-
ed DNAwas checked by agarose gel electrophoresis and DNA yield was
quantified using a Nanodrop UV–Vis spectrophotometer and a Qubit
(1.0) Fluorometer. The 18S rRNA gene of the isolate was amplified
using PCR and analyzed with Sanger sequencing through Functional
Microalgae Culture Center, and Neospongiococcum H6904 (UTEX
2284) was previously classified as Chlorochytrium [21]. The results indi-
cate that isolate PW95 is a green alga that can be classified as a
Chlorophyceae. However, it should be noted that despite the high degree
of sequence homology between the SSU rRNA gene sequences for these
isolates, the extent of phenotypic and/or genotypic similarity is not
known.
3.3. Growth of isolate PW95 in sterile CBM water and BBM
The growth of PW95 was compared between unamended CBM
water, amended CBM water (N, N + P, N + P + MgSO4,
N + P + MgSO4 + micronutrients) and a defined growth medium
(BBM). The lowest biomass was in unamended CBM water, although
some growth was observed (most likely due to carry-over nutrient
and/or stored polymer) with a subsequent decline in biomass (Fig. 1).
The fastest initial growth ratewas observedwith theN+P amendment
that was approximately 2-fold the observed growth rate in unamended
CBM water. The other conditions displayed initial growth rates that
ranged from 0.03/h to 0.04/h, including the defined BBM, and these re-
sults indicated that the isolate could grow faster in amended CBM (i.e.,
N+P)water than BBM (Fig. 1). The biomassmaxima for the tested con-
ditions were 1.7-fold to 2-fold higher than unamended CBMwater, and
the addition of P increased the growth rate but not biomassmaxima. In-
terestingly, the CBM water amended with all additions did not show
higher biomass compared to the other amendments that had similar
maximum biomass (approximately 0.3 g/l) at the tested N concentra-Biosciences (Madison, WI). The 18S rRNA gene was PCR amplified
(NS1, 519R, 576F, and 1148R) using the following parameters: initial
2-steps of 80 °C for 1.5 min, 94 °C for 2.0 min, and then 40 cycles of
94 °C for 0.5 min, 52 °C for 1.0 min, and 72 °C for 1.25 min with a final
extension of 72 °C for 7.0 min. The nucleotide sequences were com-
pared to the National Center for Biotechnology Information (NCBI)
BLASTn database to determine the phylogenetic relationship of the iso-
late to previously observed sequences.
3. Results and discussion
3.1. Water chemistry of CBM production water
Comparison of geochemical results from the selected CBM produc-
tion water and BBM indicate some nutrients were similar but the con-
centrations varied (Table 1). The tested CBM water was typically
pH 8.1 to 8.5, and was bicarbonate dominant with a typical alkalinity
of 23 meq/kg. The tested CBM water had a substantial amount of man-
ganese and iron (3.6 × 10−4 and 3.58 × 10−4 mM, respectively) when
compared to standardized BBM. Typically, iron availability can be a
point of concern for algal growth and supplementation of iron will in-
crease biomass productivity [17]. Lack of iron does not appear to be a
growth-limiting factor when using CBM water as a growth medium.
However, magnesium and sulfate, two key nutrients in algal physiology
[18,19], were 5-fold and 4-fold lower, respectively, when compared to
standard BBM. From the water chemistry, a SAR (sodium adsorption
ratio) value of 64.3was calculated; therefore, the tested CBMwater like-
ly represents production water that could be harmful to soil health if
used for irrigation purposes [20]. The followingwere unique to the test-
ed CBM production water and not included for BBM: Ba2+, Sr2+, Si, F−,
and Br−. In addition, the absence of nitrate and phosphate in CBMwater
was a major difference.
3.2. Isolate PW95 identity
Based upon the small subunit (SSU) rRNA sequence (600 nt),
PW95 is predicted to be a member of the Chlorococcaceae or
Chlamydomonadaceae family, and has 100% sequence identity with
Chlamydomonadaceae KMMCC206 and 100% sequence identity with
Neospongiococcum H6904 [21]. Chlamydomonadaceae KMMCC 206 was
isolated from fresh water in Korea and is part of the Korean Marine
Table 1
Nutrients observed in CBM water and BBM.
Medium Nutrient (mM)
Ca2+ Mg2+ Na+ K+ B
CBM water 0.10 0.06 25.65 0.12 0.03
BBM 0.17 0.30 1.10 0.06 0.18
Supplemented CBM water 0.17 0.36 26.75 0.19 0.18
Mn2+ Fe Cl− SO42−
CBM water 3.6E-04 3.58E-04 1.79 0.09
BBM 7.28E-06 1.79E-05 0.94 0.38
Supplemented CBM water 3.72E-04 3.76E-04 2.13 0.41Fig. 1. Dry weight biomass of isolate PW95 grown in CBM water ( ), CBM water with
0.5 mM NO3− ( ), CBM water with 0.5 mM NO3− and 0.05 mM PO43− ( ), CBM water
with 0.5 mM NO3−, 0.05, mM PO43−, and 0.3 mM MgSO4 ( ), CBM water with 0.5 mM
NO3−, 0.05 mM PO43−, 0.3 mM MgSO4, and micronutrients ( ), and BBM with 0.5 mM
NO3− and 0.05 mM PO43− ( ). Arrows mark when nitrate was depleted. Error barstions of 0.5 mM (Fig. 1). Even though phosphate was not added to the
amendment with only CBM water and nitrate, it is possible that the
microalgae in the inoculum had stores of internal phosphate that
could be utilized once transferred to amedium lacking the nutrient [22].
All culture conditions displayed an increase in pH that ranged from
9.5 to 10.75, and the highest pH was observed with the three most
amended culture conditions (Fig. 2). The nitrate-only amendment and
BBM culture reached similar pH levels (10.3) before declining photo-
synthesis and subsequent pH decline.represent one standard deviation of the combined biological and technical replicates.
was delayed but sustained for a longer period of time compared to the
other two conditions (N+ P or N+ P+MgSO4). Altered lipid accumu-
lation (rate, duration, and peak) has been reported for both green algae
and diatoms in response to varying combinations of N and P limitation
[23–25]. The results indicated that algal biomass and lipid could be ac-
cumulated in nitrate-amended CBM production water.
In each condition that accumulated lipids, a strong correlation was
observed between nutrient depletion and the onset of lipid accumula-
tion (Fig. 4). This correlation has been previously documented for
green algae and diatoms [26,27]. Theoretically, a lack of nitrogen causes
an inability to produce new biomass, therefore, halting cell division and
growth. This is supported by transcriptomic studies that have shown a
depletion of nitrogen causes an overexpression of lipid biosynthesis
and pentose phosphate pathway genes along with reduced expression
of fatty acid degradation genes [27,28].
A higher pH was observed for each condition that accumulated a
substantial amount of lipids (Fig. 2). pH stress has been observed to co-
incide with lipid accumulation in other microalgae [24,29]; however,
pH may also be a consequence of increased photosynthesis (and there-
by increased photosynthate). It can be difficult to determine whether
the lipid accumulation was a result of high pH, nitrate depletion, or a
combination of the two conditions. In this study, nitrate depletion was
possibly the cause of lipid accumulation because the N-deprived state
was observed before the spike in Nile Red fluorescence, whereas the
highest pH values were observed in tandem with the Nile Red peaks.
Fig. 2. pH of CBMW when grown in CBM water ( ), CBM water with 0.5 mM NO3− ( ),
CBM water with 0.5 mM NO3− and 0.05 mM PO43− ( ), CBM water with 0.5 mM
NO3−0.05, mM PO43−, and 0.3 mM MgSO4 ( ), CBM water with 0.5 mM NO3−, 0.05 mM
PO43−, 0.3 mM MgSO4, and micronutrients ( ), and BBM with 0.5 mM NO3− and
0.05 mM PO43− ( ). Arrows mark when nitrate was depleted. Error bars represent one
standard deviation of the combined biological and technical replicates.Lipid accumulation was not observed in cultures grown in BBM, un-
amended CBM water, nor CBM water amended with all additions,
whereas CBM water with added N, added N + P, and added
N + P + MgSO4 did display increased lipid accumulation based upon
Nile Red fluorescence (Fig. 3). Of these conditions, each that had phos-
phate reached a lipid accumulation peak more quickly than the condi-
tion without added phosphate. The rate of lipid accumulation was
slightly lower for the N-only amendment (1.5-fold), and the increase
Fig. 3. Lipid production of CBMWwhen grown in CBMwater ( ), CBMwater with 0.5mM
NO3− ( ), CBMwater with 0.5 mMNO3− and 0.05 mM PO43− ( ), CBMwater with 0.5mM
NO3−0.05, mM PO43−, and 0.3 mM MgSO4 ( ), CBM water with 0.5 mM NO3−, 0.05 mM
PO43−, 0.3 mM MgSO4, and micronutrients ( ), and BBM with 0.5 mM NO3− and
0.05 mM PO43− ( ). Arrows mark when nitrate was depleted. Error bars represent one
standard deviation of the combined biological and technical replicates.Fig. 4. (A) Lipid accumulation (■) and nitrate depletion (□) in CBM water with 0.5 mM
NO3− and (B) lipid accumulation (■), nitrate depletion (□), and phosphate depletion (Δ)
in CBM water with 0.5 mM NO3− and 0.05 mM PO43−. Phosphate concentration is
multiplied by 10 for scaling purposes. Error bars represent minimum and maximum of
technical duplicates.
However, further work is needed to discern the mechanism(s) of lipid
accumulation in the described isolate in terms of photosynthetic rates
at different light intensities associated with changes in nitrate levels
and pH.
A lack of lipid accumulation in CBM water without added nutrients
can likely be attributed to the poor growth from an absence of nitrate
and phosphate. In the conditions with more replete nutrients (N, P,
MgSO4, and micronutrients; BBM) in which lipid accumulation was
not observed, nitrate and phosphate were confirmed to be depleted at
the same time as the other growth conditions that did accumulate
lipids. A possible explanation could be the presence of micronutrients
in these conditions that decouples nutrient deprivation and lipid accu-
mulation. A recent study evaluated the proteome of Chlamydomonas
reinhardtii and observed that vesicular proteins involved in lipolysis
were negatively affected when Zn, Cu, Mn, and Fe were lacking [30].
Perhaps the presence ofmicronutrients allowed these enzymes that de-
grade lipids to stay functional and therefore prevent a lipid accumula-
tion event. The presence of micronutrients could also override the
signaling associated with short-term deprivation of macronutrients
and/or interactions with potential heterotrophs under mixed culture
conditions, but further work is needed to discern cellular responses in
low-quality water amended with different nutrients, particularly for
algal species that are indigenous to terrestrial environments.
For all cultures, except CBM with only nitrate, chlorophyll was ob-
served to decrease when nitrate was depleted (Fig. 5). This response
to nitrate depletion has been previously documented [31]. A possible
explanation for the decline in chlorophyll could be the damaging effects
of reactive oxygen species formed from oxidative stress during photo-
synthesis. A recent study showed an increase in antioxidant enzymes
It is interesting to note that under the condition of nitrate-only in
CBM water, chlorophyll steadily increased and was maintained longer
compared to the other conditions, and this coincided with biomass as
well. These results suggested that a nitrate-only condition more analo-
gous to environmental conditions for the isolate favored biomass and
lipid accumulation.
3.4. Lipid analysis
Gas chromatography-mass spectrometry (GC-MS) analysis of
the extracted lipids of trans-esterified samples revealed a total FAME
content of 27%, 20%, and 25% weight of fatty acid/weight of biomass
(w/w) for the conditions with added N, added N and P, and added N,
P, and MgSO4, respectively (Fig. 6). These values correspond to previ-
ously reported values in the literature for other algae and diatoms [33,
34]. All three lipid contents match very closely to values observed in
Dunalliella tertiolecta (20% and 24% lipid content) when grown in CBM
production water from Australia [11]. A primary difference between
the two studies was the need to add a significant amount of salt to the
coal seam gas water for optimum D. tertiolecta growth as the organism
is a marine species. Our results highlight the advantage to using PW95
for growth in the tested CBM water, as the alga is indigenous to the re-after nitrogen depletion indicating that themicroalgae were experienc-
ing oxidative stress [32]. Concurrently, microalgae have been observed
to degrade proteins and RNA aswell as down regulate protein synthesis
to adapt to a lack of nitrogen in the environment [28]. The combination
of these two responses may be a result of recycling nitrogen contained
in chlorophyll that is balanced with the need for some photosystems
to be maintained for the harvesting of light energy.
Fig. 5. Total chlorophyll (a, b, and c) of CBMW grown in CBM water ( ), CBM water with
0.5 mMNO3− ( ), CBMwater with 0.5 mMNO3− and 0.05 mM PO43− ( ), CBMwater with
0.5 mM NO3−, 0.05 mM PO43−, and 0.3 mM MgSO4 ( ), CBM water with 0.5 mM NO3−,
0.05 mM PO43−, 0.3 mM MgSO4, and micronutrients ( ), and BBM with 0.5 mM NO3−
and 0.05 mM PO43− ( ). Shaded area shows time frame when nitrate was depleted.
Error bars represent one standard deviation of biological and technical duplicates.gion. Although primary nutrients such as nitrogen and phosphorus are
needed, other amendments to the water are not needed for PW95
growth because low-quality CBM water is a natural habitat.
The predominant fatty acids in each casewere palmitic acid (C16:0),
oleic acid (C18:1 (ω-9)), linoleic acid (C18:2 (ω-9,12)), andα-linolenic
acid (18:3 (ω-9,12,15)). These fatty acids match those predominantly
observed in other plant-based sources of biodiesel such as canola and
palm oil [35], implying that they could be effectively utilized as a biodie-
sel source. When compared to other algal species, PW95 is lacking in
palmitic acid accumulation and contains a higher amount of C:18 unsat-
urated fatty acids, especially α-linolenic acid [34]. Interestingly,
linolenic acids can be a value added product because of use as a dietary
supplement as an omega-3 fatty acid. The decrease in lipid content ob-
served in the conditionswith phosphate could be a result of a non-opti-
mal phosphate concentration. Varying the phosphorus concentration
has been observed to have an effect on the amount of lipids produced
by microalgae and diatoms [25,36].
However,more researchneeds to be done to characterize the species
in terms of maximal biomass and lipid accumulation. The isolate PW95
can grow on ammonium as well as nitrate (Hodgskiss and Fields, un-
published data), and the ability to assimilate other nitrogen sources
Fig. 6. Lipid composition of PW95 grown in CBM water with 0.5 mM NO3− (white), CBM
water with 0.5 mM NO3− and 0.05 mM PO43− (light grey) and CBM water with 0.5 mM
NO3−, 0.05 mM PO43−, and 0.3 mM MgSO4 (dark grey). Lipid content is % w/w of fatty
acid to biomass. Error bars represent one standard deviation among technical and
biological duplicates.
could prove beneficial when determining how to best supplement CBM
water in the Powder River Basin, particularly low-cost nitrogenous
waste products. Concurrently, moving from laboratory scale to pilot
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Technol. 35 (2014) 209–219.
[11] V. Aravinthan, D. Harrington, Coal seam gas water as a medium to grow Dunalliella
tertiolectamicroalgae for lipid extraction, Desalin. Water Treat. 52 (2014) 947–958.and non-sterile CBM water are planned for future work to assess the
cellular responses and lipid accumulation of this novel environmental
isolate in low-quality CBM production water.
4. Conclusion
In summary, a green algae species, PW95, was isolated from a coal
bed methane production pond in northeastern Wyoming. Preliminary
identification based upon SSU rRNA gene sequences suggested isolate
PW95 belongs to the Chlorococcaceae family. The native isolate accumu-
lates lipid after nitrate depletion in CBM production water amended
with nitrate. The highest lipid content achieved was 27% (w/w) when
only nitrate was added to CBM water. The addition of other nutrients
to CBM production water resulted in similar biomass maxima but
lower lipid contents. The predominant fatty acids were C16:0, C18:1
(ω-9), C18:2 (ω-9,12), and 18:3 (ω-9,12,15).
Acknowledgments
The authors would like to thank the CBM research group and the
Algal Research group at Montana State University for helpful discus-
sions, and Drs. D. Ritter and J. McIntosh at the University of Arizona De-
partment of Hydrology andWater Resources for water geochemistry of
FG-09 CBM production water. The authors would also like to thank
Bradley Ramsay for help in the field as well as Jacob Valenzuela, Erika
Whitney, andWhitney Grimsrud for laboratory assistance. The present-
edworkwas supported byMBRCT #14–16 from the State ofMontana as
well as DE-FE0024068 from the Department of Energy, Office of Fossil
Energy and the U.S. Geological Survey Energy Resources Program
(USGS-ERP). Any use of trade, firm, or product names is for descriptive
purposes only and does not imply endorsement by the U.S.
Government.
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