1. Microbial Production of
Oils and Fats
Course: Food Microbiology 2014
Lecturer: Dr. Vu Thi Lam An
Group 5: Nguyen Quynh Hai Yen BTFTIU11009
Ho Quang Loc BTFTIU12011

2. Contents
1. Introduction to Single Cell Oils (SCO)
2. SCO production process
3. Applications
4. The significance and safetyness of SCO

3. 1. Introduction to Single Cell Oils.
» What is it?
» Condition for lipid accumulation
» Principles of lipid accumulation

4. • Single Cell Proteins?
• Single Cell Oils (SCO): triglyceride fats generated by
microorganisms.
– Lipid components are similar to those found in plant and animal.
→ Applications: animal feeds, aqua feeds, biodiesel.
What is Single Cell Oils?

5. What is Single Cell Oils?

6. Is it safe for human consumption?
• Microorganism → a human food source from
ancient time.
– Can you name some?

7. Oleaginous microorganism
• Microorganism that gathers >20-25% of their
biomass as oil.
• Many yeast, fungi and several algae are maximum
producers.
– Algae → biodiesel
– Yeast and fungi → edible oil
• They are also capable of producing high levels of
nutritionally important polyunsaturated fatty acids
(PUFAs).

9. Advantages of Oleaginous
microorganism
• Growth on various substrates
→ utilizing by-product = reduce cost
• Ability to synthesize a divert
array of fatty products
→ many useful applications.
• Able to be genetic manipulation
→ selection for highest productivity.

10. Condition for lipid accumulation
• Lipid accumulation is trigger when
– Nutrients in the growth medium (usually Nitrogen
source) is exhausted
– Surplus of Carbon source (usually glucose)
→ Cells stop multiplying.
→ Cells convert Carbon source to Storage oils or fats.

11. Condition for lipid accumulation

12. Stages of Lipid accumulation
in Batch culture
...
Cells reach limit of obesity → stop accumulating.
Lipid accumulation → cells expand
Cells convert C → storage lipid (intracellular)
Exhaustion of N, cells stop divide
Microorganisms grow and multiply until a certain time
Prepare medium: high Carbon, low Nitrogen

13. Biochemistry of Lipid accumulation
Cellular respiration of microorganism
TCA

14. Biochemistry of Lipid accumulation
Acetyl-CoA →(fatty acid biosynthesis)→ triacylglycerols
Enzyme ATP-citrate lyase: Citric acid → acetyl-CoA
(Oleaginous microorganisms only)
Citric acid in mitochondrion → cytosol
TCA is disrupted
[AMP] is diminished;
Isocitrate dehydrogenase in mitochondrion stop working
Exhaustion of N

15. Biochemistry of Lipid accumulation

16. 2. Production of Commercial SCO.

17. Production of commercial oils and fats
from SCO
• Only if cost of production of SCO could compete against
plant oil.
• In fact, production cost of SCO is much higher.
Plant oil = 400-
800$/ton
- 4 tons of sugar (300$ each)
→ 1 ton SCO + biomass
- Cost of extraction and refining
→ food graded oil
- Cost of selling, waste disposal...

18. Production of commercial oils and fats
from SCO
• High production cost
→ Apply for production of specialty oils and fats only.
→ Choose fast growing microorganisms, high oil yield, simple
growth requirements.
→ Focusing on the use of by-products of agriculture and industry
as substrates

19. Production Process
Harvesting
the cells
Cultivation
Drying the
cells
Extraction

20. Production Process

21. Commercial Production Process
• It is important to harvest SCO before Carbon substrate is
completely depleted.
• As the cells will use the lipid that they have accumulated for
their own survival.
• Consequences:
1. Low Oil yield
2. Cells forming lipase → degrade quality of SCO
• Alter flavor, color, functional characteristics.
– How to avoid lipase?
→ Answer: heat denaturation <60ᴼC

22. Commercial Production Process
• SCO usually contain natural antioxidant
→ A clear, bright oil with a minimum of coloring → favorable.
→ Very stable to oxidation
→ Good for consumption
SCO by Cryptococcus curvatus
SCO by Mortierella alpina

23. 3. Applications of Single Cell Oils.
» Cocoa butter equivalent fat
» Polyunsaturated fatty acids (PUFAs)
• DHA
• ARA

24. Cocoa Butter Equivalent Fat
• Cocoa Butter: triacylglycerol, consists of:
- palmitic acid (16:0)
- oleic acid (18:1)
- stearic acid (18:0)

25. Cocoa Butter Equivalent Fat
• Cocoa Butter: very expensive
• SCO = Cocoa Butter Equivalent Fat = cheaper substitute
• However, SCO are low in stearic acid
→ genetic modification is necessary
Cacao Butter Substitutes

26. Cocoa Butter Equivalent Fat
* Henk Smit (Netherlands):
– Using the yeast Cryptococcus curvatus D
– Delete the gene that codes for the Δ9−desaturase, which converts
stearate into oleate
→ Fail, not stable products
* Julian Davies (New Zealand)
– Culturing in O2 deficiency → stop enzyme desaturase from working →
more stearate
→ But uneconomical
* Chance for you!

27. Polyunsaturated fatty acids
• Omega-3 and omega-6: cannot synthesize by
human body.
– Omega-6: from plant food.
– Omega-3 (EPA, DHA):
• Are long chain PUFAs
• Most plants are unable to synthesize
• Must obtain from animal food.

28. Dietetic significant of PUFAs
• Long chain PUFAs:
– Key components of cell
membrane
– Inflammation response
• Arachidonic acid (n-6)
and DHA (n-3):
essential for brain and
neural development
• In general, our diet
contains too much n-6
and too little n-3

29. SCO as source of Omega-3
• Animal source of Omega-3:
– Oily fish → distinct fishy taste
– Animal organs (esp. liver)
→ Unfavorable + shortage of supply + extensive purification
→ Costly
→ Why not SCO?

30. • In this presentation, we will also briefly introduce to
you:
– Arachidonic Acid (ARA) production process.
– Docosahexaenoic acid (DHA) production process.

31. Arachidonic Acid (ARA)
Processes for the production of various PUFAs
Arachidonic Acid is a long chain
polyunsaturated fatty acid with twenty carbon
atoms and four double bonds. Its systematic
name is (all-cis)- 5,8,11,14-eicosatetraenoic
acid(ETA)

32. Arachidonic Acid (ARA)
Processes for the production of various PUFAs
Biosynthetic pathway of polyunsaturated fatty
acids in Mortierella alpina
ARA is synthesized through
many enzymatic steps of fatty acid synthesis,
elongation of fatty acid and desaturation of
fatty acid

33. Microorganism ARA
yield/cultivation
period
Scale
Submerged culture
Mortierella alpina 1S-4 13 g/L/10d 10-kL fermentor
M. alpina ATCC 32222 11 g/L/11d 250-mL flask
M. alpina ATCC 32221 11 g/L/16d 500-L fermentor
M. alpina UW-1 5.5 g/L/6d 20-L fermentor
M. alpina LPM 301 4.5 g/L/8d 30-L fermentor
Mortierella alliacea YN-15 7.1 g/L/6d 50-L fermentor
Mortierella schmuckeri S12 2.3 g/L/3d 14-L fermentor
Mortierella sp. S-17 0.96 g/L/7d 1-L flask
Mortierella elongata SC-208 0.49 g/L/5d 250-mL flask
Pythium irregulae ATCC
10951
3.1 g/L/8d 250-mL flask
Solid-state culture
M. alpina IFO 8568 13 g/kg-medium/20d
M. alpina CCF 185 36 g/kg-medium/21d
Arachidonic Acid (ARA)
The fatty acid
composition
of 50 Mortierella
subgenus isolates
was analyzed Amano
et al
They found that ARA
composition of alpina
was higher than
those of any

34. Processes for the production of various PUFAs
Docosahexaenoic acid (DHA)
Docosahexaenoic acid (DHA,
22:6) is a particularly important
ω ‐3
PUFA, with a 22‐carbon chain
and six double bonds.

35. Processes for the production of various PUFAs
Docosahexaenoic acid (DHA)

36. Microbial PUFAs on market
ARA for muscle
stimulation
ARA-rich SCO and DHA-
rich SCO in infant
formulae in many
countries of Europe,
Australia, and the Far East

37. Advantages and Disadvantages of SCO
ADVANTAGES:
1. Simple fatty acid profile → easy for extraction and
purification.
2. Both quality and quantity of the product can be guaranteed.
3. SCO is not affected by geographical or environmetal factors
(e.g pollution etc.)
4. Rapid production: increase cells in a short time and
independence from climatic conditions.
5. SCO act as a novel source of polyunsaturated fatty acids,
such as DHA and AA for nutritional supplementation.

38. Advantages and Disadvantages of SCO
DISADVANTAGES:
1. Limited production capacity.
2. Consumer acceptance.
3. DHA from fish oil are linked to growth retardation in infant
→ also DHA of SCO

39. Understanding 2 routes for safety
evaluation of a food ingredient
1. Petition for FDA approval as a food additive.
– FDA will review before products are available on the market.
– FDA is responsible for their decision.
2. Manufacturer can determine that a substance is GRAS if
there is scientific consensus among qualified experts
about its safety under the intended condition of use.
→ SCO is only FDA approval.

40. Safety of SCO
• SCO is only FDA approval. Why?
→ For some years, high level of SCO-ARA in the human diet was
thought to induce potential adverse effects on blood
clotting.
• However, later intensive and prolong studies demonstrate
the lack of evidence of toxicity in volunteers.
• Recently, microbial oils have been shown to be no more toxic
than oils from traditional sources.
• More researches are needed!

41. Summary
• SCO are now used to produce high value oils rich in
arachidonic acid (ARA) and docosahexaenoic acid
(DHA).
• These oils are used in infant nutrition and also as
nutriceuticals and food additives appropriate for adults.
• Potential therapeutic applications are very promising.
• Production of SCO:
– SCO can be produced in large scale.
– To ensure stabilization of SCO, must harvest the cells before
substrates are totally depleted.
– SCO extraction is much the same as used for plant seeds.

42. References
• James, P.W., Colin, R. Microbial production of fats and oils. Food Biotechnology.
2006, pp. 443-472
• http://www.ukessays.com/essays/education/microbial-production-of-fats-and-
oils.php