Nanjing Forestry University Experience
南京林业大学经验
Gilda Naka
June 6, 2013-July 3, 2013
Gilda Naka
Special Thanks to Dr. Wang Fei, Dr. Liaw Goang, Dr. Zhang Yu，He Qiyang
特别感谢王飞教授、廖广信教授、张瑜副教授、何琦阳同学

The process of producing biofuel through the
transesterfication of triglycerides.
◦ This involves reacting vegetable or animal fats with
short-chain alcohols (typically methanol or
ethanol).

Advantages:
◦ Domestically produced
◦ Biodegradable
◦ Renewable and Clean energy

Industries use chemical methanolysis with
alkalicatalysis
◦ Difficult removal of catalyst
◦ Excessive Energy Requirements

Using lipase is better
◦ Glycerol is by-product (easily recovered)
◦ Simple purification of fatty methyl esters
Two Types of
Lipase
Extracellular

Expensive
(recovery,
purification,
immobilization
process)
Intracellular

Whole-cell
biocatalyst
◦ No purification
necessary
◦ R. oryzae whole
cell with
polyurethane
foaming plastic

Reaction involves two steps:
1) Hydrolyzation（水解反应）

Rate Determining Step
2) Esterification （酯化反应）
 Reaction with ROL:
ROL

Reacts bean oil to glycerol and fatty acids
Maximum methyl ester = 66.6%
Reaction #1: Acyl migration (酰基迁移)

Reaction #2:


◦ Water affects concentration of methyl ester
 Smaller concentration  Slower reaction
 Excess H2O limits Reaction #2
Investigate the optimal amount of water
needed to produce biodiesel via whole cellcatalyzed transesterfication of bean oil

Prepare
Fermentation
Medium
Breed
Cultivation of
R. oryzae
Introduce R.
oryzae to
Medium
Retrieving
Whole-cell
biocatalyst
(foam)
Biodiesel
Production
with Varying
Water Content

Chemical
Mass Percent
Tryptone
2%
MgSO4 · 7H2O
0.05%
NaNO3
0.1%
KH2PO4
0.1%
Bean oil
3%
polyurethane foaming plastic
0.6 g (except control)
pH = 5.5

4 beakers with 80 mL of medium

Medium was autoclaved
◦ 20 minutes at 121° C
Prepare
Fermentation
Medium
Breed
Cultivation of
R. oryzae
Introduce R.
oryzae to
Medium
Retrieving
Whole-cell
biocatalyst
(foam)
Biodiesel
Production
with Varying
Water Content

Ingredients:
◦ D-(+)-Glucose
◦ Potato


2%
20%
50 mL of potato juice and 0.1 g D-(+)-Glucose
Autoclaved for 1 hour then inoculate and place in
incubator at 28°C 180 rpm until mold appears
Agar (2%) was added
to the petri dish as a
storage medium
Prepare
Fermentation
Medium
Breed
Cultivation of
R. oryzae
Introduce R.
oryzae to
Medium
Retrieving
Whole-cell
biocatalyst
(foam)
Biodiesel
Production
with Varying
Water Content

Inoculate ROL (after 10 min UV sanitation of beakers)

Cultivation

60 hours at 28° C at rotation
speed of 180 rpm
Prepare
Fermentation
Medium
Breed
Cultivation of
R. oryzae
Introduce R.
oryzae to
Medium
Retrieving
Whole-cell
biocatalyst
(foam)
Biodiesel
Production
with Varying
Water Content


Wash polyurethane foaming plastic 3-5 times with
water and squeeze
Add 200mL of glutaraldehyde (1%)


Glutaraldehyde contains the lipase in the foam
Covered with gauze and incubated for 1 hour at 25°C,
180 rpm
Add phosphate buffer (diluted 10 times)
 6.5 mL of KH2PO4
 50 mL of Na2HPO4*12H2O
 Put in ice water for 5 min
 Put in watch glass and dry in multi-thermo
incubator for 10 hours at 30°C

Polyurethane Foam with no R. oryzae
Polyurethane Foam with R. oryzae
Prepare
Fermentation
Medium
Breed
Cultivation of
R. oryzae
Introduce R.
oryzae to
Medium
Retrieving
Whole-cell
biocatalyst
(foam)
Biodiesel
Production
with Varying
Water Content

6 beakers





Bean oil (15g)
Methanol (680mL)
25 pieces of R. oryzae foaming plastic (not in control)
Varying percents of water (0%-20%)
In shaker for 12 hours at 37°C

150 μL into 6 test tubes every 12 hours

680 μL methanol added and shake for 12 hours


Methanol added stepwise because poisonous to the mould
Gas Chromatography done on each round of
samples to measure methyl ester content

Use n-hexane as solvent



Sample is diluted 20 times
Nitrogen is the mobile phase
Samples were centrifuged before GC


4 peaks: methyl palmitate, methyl linoleate, methyl
oleate, and methyl linolenate
Methyl heptadecanoate used as interior label
Time (hours)
Water Content
12
24
36
48
60
72
0%
29.20
47.97
44.74
41.28
46.09
35.72
5%
23.45
45.80
65.68
72.29
72.23
71.71
10%
24.42
47.42
55.47
75.01
83.51
82.53
15%
20.12
45.58
54.84
68.37
80.18
65.09
20%
12.74
37.54
47.96
62.14
75.14
69.10
Methyl Ester Content vs. Time
90
80
Water Content
Methyl Ester Content (%)
70
60
50
0%
5%
40
10%
15%
30
20%
20
10
0
12
24
36
Time (hours)

48
60
72
Control had zero peaks throughout

Optimal Water Content was found to be 10%

Most methyl ester produced after 60 hours

One step closer to a most efficient biodiesel
production process

Discovering optimal conditions with cheap
techniques
Push towards cleaner, alternative energy
 Worldwide adaption


Save Mother Earth!

Time stages in which methanol is added


How the amount of catalyst effects biodiesel


Ideal amount of foam for mass transfer
Best type of foaming plastic


Build apparatus
How functional groups play a role in mass transfer
Use Jatropha seed oil

Found in the Guangxi Province
The skills I have gained will help me for years to come!
我所学到的技能在未来很多年都会对我有所帮助！
Special Thanks to Dr. Wang Fei, Dr. Wang Yong, Dr. Moss Elica Dr. Liaw Goang,
Dr. Zhang Yu，He Qiyang
特别感谢王飞教授、廖广信教授、张瑜副教授、何琦阳同学



1) Kazuhiro Ban, Masaru Kaieda, et. “Whole cell
biocatalyst for biodiesel fuel production utilizing
Rhizopus oryzae cells immobilized within
biomass support particles”. Biochemical
Engineering Journal 8 (2001) 39-43
2) Jing Zeng, Wei Du, et. “Study on the effect of
cultivation parameters and pretreatment on
Rhizopus oryzae cell-catalyzed
transesterification of vegetable oils for biodiesel
production”. Journal of Molecular Catalysis B:
Enzymatic 43 (2006) 15-16
3) Yunjun Yan, Li Xu, et. “A synergetic whole-cell
biocatalyst for biodiesel production”. The Royal
Society of Chemistry. 2012.