Using wastewater from dark fermentative hydrogen production to culture Oleaginous yeast Cryptococcus curvatus to be used as biodiesel feedstock
Zhanyou Chi, Yubin Zheng, Chenlin Li, Bo Hu, Shulin Chen
Department of Biological Systems Engineering, Washington State University, Pullman, WA 99164-6120
BACKGROUND
DHA Producing Alga Strain:
Schizochytrium limacinum SR21
Feedstocks
Oil Crops
Cull Potatoes
Algal Fermentation
Process
Omega-3
Algal cells
before shifting
 Heterotrophic algae growing on glucose and glycerol as
carbon source
Cull Potato
 Underutilized agricultural biomass with low commercial
value
 High lipids content in algae biomass (more than 50%)
 Can provide both carbon and nitrogen sources for
algae’s growth
 Fast growing
 High ratio of DHA to total fatty acids (more than 30%)
Algal biomass
enriched with
lipids
Crude Glycerol
Two-stage Growth of Schizochytrium
Biodiesel Waste Glycerol
Feed additives
to fish
 cell number increasing stage: cell reproduction and rapid cell
number increase with little increase in size and weight of
each cell
 However, it’s good carbon source for algae’s growth
 cell size increasing stage: cells stop reproduction but enlarge
due to lipids accumulation
Biodiesel
Converting them to high value DHA could provide a
great potential market for them
Nutraceuticals and foods
with omega-3
Waste Water
 Involved in the development of infant brains
(Picture from
www.wikipedia.com)
RESULTS
 Supplemental DHA in infant formula is strongly
recommended by WHO
1. Verification of the two-stage growth
 Reduced risk of age-related neurological disorders,
such as Alzheimer’s and dementia
BR cell number
Dry cell wight (g/L)
10
3. Oxygen consumption in the cell increasing stage
OUR
140
BR DCW
120
Cell number (10^6 cells/mL)
12
(Picture from Martek Annual
Report)
100
8
80
6
60
4
40
2
20
SOUR
0.9
0.6
0.5
0.4
0.3
0.2
0.1
Omega-3 Used in Aquaculture
0
0
20
24
28
33
0.07
0.06
0.05
60
feeding
0.04
0.03
0.02
control
0.01
0.00
0.0
0
0
20
40
60
0
tim e (hr)
44
 Aquaculture feed demand increases while ocean fishery
resources decline, using fish meal to support aquaculture
growth becomes non-sustainable
 Organic fish movement requires an omega-3 source that is
not originated from fish meal
 The omega-3 enriched in the algae biomass produced in this
process will be a better source
 The cell number stopped increasing after 24 hours, but the
dry cell weight kept increasing
 The only explanation is that the “body weight” of each cell
was increasing
 Therefore, to improve biomass production:
40
60
 The specific oxygen uptake rate (SOUR) reached its maximum at 8th hr,
and decreased to a very low level after 24th hour
1. produce more cells in the first stage
 Large amount of oxygen was consumed in the cell reproduction stage,
but only a little oxygen was consumed in the lipid accumulation stage
2. grow bigger cells in the second stage
300
high DO DCW
250
low DO cell number
200
high DO cell number
4
150
3
100
cell density (10^6/ml)
low DO DCW
DCW (g/L)
4. Biomass production with shift strategy
Shift time
(hr)
7
Dry cell weight
(g/L)
cell density
(106 cells/ml)
cell body weight
(mg/106 cells)
Control
(no shifting)
21.5
18
24.6
106
0.23
24
25.3
118
0.21
29.3
124
0.24
51
0.42
200
2
Dairy beverages
820
1
50
30
0
0
40
37.9
140
0.27
48
36.2
162
0.22
500
Beverages (non-dairy)
770
Snacks/candy/cookies/crackers
625
Bread
510
Cereal/Breakfast food
465
Yogurt
70
Other
1,500
Total
5,460
(UBS Global Life Sciences Conference, September 27, 2006)
0
8
12
16
20
4
6
8
10
12
14
 To obtain high biomass production with this high cell density culture,
more nutrients and carbon source need to be supplemented in the
shifted culture, to make the algae cells accumulate more lipids inside
cells
6. High cell density culture
Infant Formula
Cheese
30
120
Potential DHA Market in U.S.
5
40
 With the feeding, the cell body weight was enhanced to 0.38 mg/106
cells, 56 g/L algae biomass was obtained
2. High oxygen concentration culture in
6
50
culture time (days)
 Shifting strategy is necessary, in that it produce high cell density at first,
and provide optimal condition for lipids accumulation
the reproduction stage
$ ( millions)
20
tim e (hr)
Time (hr)
 1 million tons of fish oil produced globally per year, 70~80% of
it used in aquaculture as fish meal
5. Enhancing biomass production with fed
batch culture
0.09
0.08
0.8
0.7
mg/10^6 cells/min
 Component of the photoreceptor cells of infant retinas
Optimizing culture conditions of these two stages separately
and using a “shifting strategy” will increase the production
rate
OUR (mg/L/min)
The Health Benefit of DHA (C22:6, -3)
Omega-3 food
Cow Stomach
“Extractor”
Hypothesis
dry cell weight (g/L)
 Difficult to be purified, due to many impurities
24
38
51
time(hr)
 The bottle neck of the algae biomass production
in previous study is low cell density produced in
the cell reproduction stage
 High oxygen concentration in the reproduction
stage produced much more cells
 Algae biomass production could be greatly
enhanced with this high cell density
Dry cell weight (g/L)
 Negative value by-product of biodiesel industry
100
80
60
40
20
0
0
2
4
6
8
Tim e (day)
 Culture with 360× 106cells/ml at initial was conducted, 102.4 g/L algae
biomass was obtained
 DHA Production efficiency was greatly enhanced with this high cell
density culture
 Culture the cell at high oxygen and nitrogen source concentration at high
temperature, then shift the culture to low oxygen and nitrogen
concentration at low temperature
 Biomass production was greatly enhanced with shift strategy
 The cell body weight suppose to be further enhanced, if feed more
nutrients in the culture to the high cell density
 This process is very promising to be industrialized
ACKNOWLEDGMENTS
This research is supported by the Washington State University
IMPACT Center and the Washington State Potato Commission