S u r v e y a n d C h a ra c t e r i z a t i o n o f A l g a l S t ra i n s A d a p t e d t o
G r o w t h o n Wa s t e Wa t e r a s B i o f u e l Fe e d s t o c k s
Sage R. Hiibel, Brian P. Kelly, John C. Cushman
Department of Biochemistry and Molecular Biology, MS200 - University of Nevada, Reno 89557
Project Objectives
Microalgae Isolates
• Create a culture collection of indigenous microalgae
for biofuel research in Nevada.
• Investigate the value of waste water as a nutrient
source for growth of lipid-rich algal species.
• High level of diversity observed among isolates
from different sites and within the same sampling
location (Fig. 2 and Table 1).
• Species identification difficult from morphology.
Growth on Waste Water
Methods
Sampling Locations
o  Truckee Meadows Water Reclamation Facility (TMWRF) in
Reno (Fig. 1).
o  Moody Lane Waste Water Treatment Plant in Fallon.
• Steamboat Creek up and downstream of TMWRF.
• Agricultural areas near
Fallon, NV.
• UNR Greenhouse Facility.
8
9
Project Sponsor
US Department of Energy
NSHE’s Nevada Renewable
Energy Consortium
7
5
6
3
4
1
2
11
12
Figure 1. Aerial view of TMWRF and
Steamboat Creek with sampling
locations indicated. Descriptions of
each location are given in Table 1.
TMWRF 10 - #1
TMWRF 1 - #3
TMWRF 11 - #1
TMWRF 3 - #1
TMWRF 11 - #2
TMWRF 11 - #3
• Several strains grow well with elevated centrate
concentrations (Fig. 4).
• Longer lag times observed with increasing centrate
concentrations (Fig. 5).
Fallon 2 - #1
Fallon 2 - Green
Fallon 4 - #2
Fallon 5 - #1
Fallon 2 - Gold
Fallon 4 - #1
UNR GH - #A1
Figure 2. Micrographs of the various environmental isolates at 60X magnification;
scale bar represents 25 µm.
Table 1. Descriptions of sampling locations for the indigenous Nevada microalgae
collection. The number of potential isolates and their putative identifications for
each location are also listed.
TMWRF 2
Description
Primary sedimentation tank
20
10
0
Figure 4. Maximum centrate concentrations (% v/v) demonstrating growth with
fresh, brackish, and salt water microalgae strains. Brackish and salt water strains
required 0.6M and 1.2M NaCl, respectively, for growth.
0.30
0.25
-
TMWRF 3
Aeration tank 2C
2
TMWRF 4
Phosphorus stripping tank
-
TMWRF 5
Phosphorus stripping tank
1
TMWRF 6
Phosphorus stripping tank
-
TMWRF 7
Secondary sedimentation tank
-
TMWRF 8
Secondary sedimentation tank
-
TMWRF 9
Post-aeration tank
-
TMWRF 10
Backwash settling tank
1
Class: Chlorophyceae
TMWRF 11
Steamboat Creek - outfall
4
Class: Chlorophyceae
TMWRF 12
Steamboat Creek - upstream
-
0.20
Class: Chlorophyceae
Division: Cyanobacteria; Order: Oscillatoriales;
Genus: Geitlerinema, Leptolyngbya, Microchaete,
Stichococcus, or Pseudanabaena
10% Centrate
25% Centrate
40% Centrate
0.15
0.10
0.05
0.00
0
Agricultural pond
-
Fallon 2
Moody Lane Waste Water Treatment
Plant - fallow aeration tank
3
Fallon 3
Moody Lane Waste Water Treatment
Plant - evaporative holding pond
-
Fallon 4
Dairy milking parlor washout pond
2
Class: Chlorophyceae
Fallon 5
Irrigation drainage ditch
1
Division: Cyanobacteria; Order: Oscillatoriales;
Genus: Geitlerinema, Leptolyngbya, Microchaete,
Stichococcus, or Pseudanabaena
1
Bristol
-0.05
Fallon 1
Recurring open pond contaminant
30
Potential
Isolates Putative Isolate ID
3
Class: Chlorophyceae
Water activated sludge secondary
sedimentation tank
UNR GH
40
■  Neochloris – fresh water
■  Nannochloropsis – brackish water
■  Dunaliella – salt water
UTEX 138
UTEX 776
UTEX 777
UTEX 1185
UTEX 1249
UTEX 1707
UTEX 1981
UTEX B778
UTEX B947
CCAP 213/4
CCAP 254/1
CCAP 211/78
CCAP 849/1
CCAP 849/2
CCAP 849/3
CCAP 849/4
CCAP 849/5
CCAP 849/6
CCAP 849/7
CCAP 849/8
CCAP 849/9
CCAP 849/10
UTEX 199
UTEX 200
UTEX 999
UTEX 1000
UTEX 1644
UTEX 2192
UTEX 2358
UTCC 197
UTCC 420
UTCC 457
CCAP 19/6B
CCAP 19/18
CCAP 19/24
CCAP 19/27
SAG 19.5
SAG 41.89
SAG 42.89
SAG 43.89
TMWRF 11 - #4
Figure 3. Example of the 96-well plate
screening of Dunaliella species; photo taken
after 8 days of growth. Percentages in rows
indicate the centrate concentrations (% v/v).
Columns were inoculated with:
a,l - sterile water; b – 2X ASW; c - UTEX 199;
d - UTEX 200; e - UTEX 999; f - UTEX 1000;
g - UTEX 1644; h - UTEX 2192; i - UTEX 2358;
j - UTCC 197; k - UTCC 420.
TMWR 3 - #2
50
Sampling
Location
TMWRF 1
• Waste Water Treatment Facilities
10
TMWRF 5 - Green
TMWRF 1 - #2
A600
Biofuel Potential Screening
• 96-well plate format.
• Maximum centrate concentration, growth rate, and
biomass production.
TMWRF 1 - #1
a b c d e f g h i j k l
2X ASW
14%
20%
30%
35%
40%
50%
DI H2O
% Centrate
Culture Collection Development
• Aqueous samples collected from waste water
treatment facilities and agricultural areas.
• Samples spread on agar plates with maintenance
media and 5% centrate from waste water
treatment facility.
• Individual colonies re-streaked on agar plates two
additional times to isolate single species.
• Single colonies cultured in liquid maintenance
media with antibiotics to remove bacterial
contamination.
• Axenic cultures screened for biofuel potential.
• 96-well format facilitates high-throughput screening
of multiple species and concentrations (Fig. 3).
Division: Cyanobacteria; Order: Oscillatoriale;s
Genus: Geitlerinema, Leptolyngbya, Microchaete,
Stichococcus, or Pseudanabaena
Scenedesmus dimorphus (Turpin) Kützing
10
20
30
Days
40
50
60
Figure 5. Growth of Neochloris pseudostigmata (UTEX 1249) on maintenance
medium, 10, 25, and 40% (v/v) centrate concentrations.
Conclusions
• Waste water offers great potential as a nutrient and
water source for microalgae cultivation.
o  Dose-dependent growth inhibition observed in centrate.
• Wide diversity of indigenous microalgae preacclimated to waste water environments exists.