Journal of Environmental Protec tion, 2013, 4, 70-73
doi:10.4236/jep.2013.41b013 Published Online January 2013 (http://www.SciRP.org/journal/jep)
Studies On Extracting Microcystin-Lr From Microcystis
Aeruginosa By Water Bath*
Fang Li, Wenqing Liu, Nanjing Zhao#, Jingbo Duan, Zhigang Wang, Yujun Zhang, Xue Xiao,
Jing Liu, Gaofang Yin, Chaoyi Shi
Key Laboratory of Environmental Optics and Technology, Anhui Institute of Optics and Fine Mechanics, Chinese Academy of
Sciences, 350 Shu Shan Hu Road, Hefei, Anhui, China.
Received 2013
ABSTRACT
Different temper atures of water bath was used to extract the intracellular microcystin-LR(MC-LR) of M icro cystis a eru-
ginosa. Researching the extraction efficiency under the suitable temperature, so that it could find o ut the best te mpera-
ture a nd ti me for extracting MC-LR from Microcystis aeruginosa cells. Five equal Microcystis aeruginosa was used to
find out the best temperature, extracting at 60˚C, 70˚C, 80˚C, 90˚C and 100˚C for 15 minutes, respectively. Results
sho wed that t he co ntent o f MC-LR extracted with the water under 100˚C wa s the highest. But mean whil e, the type and
the content of impurities was the highest, too. In addition, an o ther six equal Microcystis aeruginosa was extract with the
water under 100˚C for 5min, 10 min, 15 min, 20 min, 25 min and 30 min respectively. It was proved that 20 minutes
was enough for extracting MC-LR from Microcystis aeruginosa, no long time was needed.
Keywords: Boiling Wate r Bath; Extract; Micro cystin-LR
1. Introduction
Cyanobacteria bloom happens more and more frequently
worldwide because of growing pollution, causing many
kind s of mic roc ysti ns(MC s) whic h have dra wn muc h pub-
lic attenti on. M Cs, whic h coul d be detected in 80 percent
water bloom according to the survey, can inhibit the ac-
tivity of protein phosphatase(1, 2Aand3), promote onco-
genesis, or even lead to death.
MCs are ring peptide compounds composition of sev-
en amino acid. They are very stable and release into wa-
ter body after the cellular ruptured, causing serious harm
to the water quality and aquatic. There’re more than 80
different microcystins according to the data has been
already reported. At present, the research of MCs has
bee n focuse s on t he toxicolog y, environmental c hemistry,
preparation of standard toxins used for analysis and the
applications in biochemistry, etc. It can’t obtain MCs
from chemical synthesis. So that extracting MCs from
toxigenic strains efficiently becomes the basis and pre-
requisite of the study of MCs.
At pre se nt t her e ’re many methods to extract MCs from
cellular. ZHANG Ming-ming [1] used 40% methanol
solution as the extract solvent, thawing and refreezing
cells repeatly to extract microcystin-LR(MC-LR). REN
Jing’s results showed that it was efficient to use 75%
methanol as extract solvent, freezing and thawing the
cells as well as ultrasonication. Because cyanobacteria
belongs to prokaryotes, the ce ll wall is mainly mucop ep-
tide, so that both cryogenically freezing and boiling can
destroy cell wall and then MCs released. But freezing
and thawing resulted in low toxin and time consuming.
The research of Jarkko Rapala [3] showed that after the
first freezing and thawing only a small number of cya-
nobacteria colonies had b een dispersed. Sonication for 60
min in the water bath disrupted only the outermost cells
of the colonies. After the second freezing and thawing
and sonication for 15 min a high number of unbroken
cells was still seen. Using methanol to extract MCs, it
must dilute the extract solvent by water or heat and vola-
tile the extract solvent to reduce the concentratio n of the
methanol before solid phase extraction. This increases
the time of solid phase extraction, and the use of organic
regents is bad to the health of the researchers. In addition,
many organics such as phycobiliprotein in cyanobacteria
cells, which could be extracted with MCs, may pollute
the column of the chromatographic. Some reported that
using guard columns to remove the protein, and then
*Foundation item: The national “863” project grants program (2009A-
A063005); Nat u ra l s cienc e fund projects in anhui province (11040606-
M26);
Anhui institute of optics and fine mechanics, director of the
project fund (Y03AG31144).
#Corresponding author.

Studies On Extracting Microcystin-Lr From Microcystis Aeruginosa By Water Bath
71
elute MCs. So me adjusted the PH value to 4 with diluted
solution of sulfuric acid, so that the protein was denatu-
ration and precipitated and the column was defended
from pollution. All these methods was time consuming
and increased the cost. Metcalf et al. [5] first extracted
MCs with boilin g water, usin g deionized water as extrac-
tion so l vent. T he re sult sho wed that one mi nute was ver y
good for 150 μL extraction solvent. The use of boiling
water based upon the heat resisitance of MCs. MCs
would not lose activity after heating in the water of
100˚C for 30 min, and was hard to resolve under 300˚C
[6]. This method avoided the use of metha nol during the
extraction process which was unsafe to the analyst. In
addition, it lowered the cost, and shortened the time of
extraction. LEI La-mei et al [7] also studied extracting
MCs with boiling water, and contrasted with the tradi-
tional method using methanol, found that the relative
error was between 0.2% and 16.59%. 12 min of heating
time was enough in their study and deionized water was
better than disti lle d water for the extra c tion of MC s.
In conclusion, the use of boiling water to extract MCs
from cyanobacteria cells has a good application prospect.
But at present this method is not very mature as there are
not many related reports. This study systematically re-
searched extracting MC-LR within the cells o f Microcys-
tis aeruginosa, obtaining the best temperature and time.
And good results had been achieved with the recovery of
89.3%. A great quantity of organic solvent was avoid
during the extraction process. The entire analysis could
be finishe d i n one day.
2. Material and Method
2.1. Instruments
The main instruments needed in this experiment were as
follows: Visiprep SPE Vacuum Manifold-DL, 24-port
model; Supelclean ENVI-18 cartridges(500 mg/3 mL);
Agilent 120 0 HPLC (Eclipse XDS-C18 chromato grap hic
column); HP400G incubator(Wuhan Ruihua Instrument
& Equipment Co.Ltd); Molecular ultra-pure water ma-
chine; sterilizer; The Labnet 6Liter Water Bath; H-1650
Ultracentrifuge; NMT-2800 Pressure Blowing Concen-
trator.
2.2. Reagent
The main reagents are as follows: methanol (chromato-
graphic grade), trifluoroacetic acid (TFA, chromato-
graphic grade); deionized water; phosphoric acid; 0.05
mol/L KH2PO4 (chromatographic grade); Microcystis
aeru ginosa(FACHB-905) was purchased from institute
of hydrobiology, Chinese academy of sciences and cul-
tured in BG-11 medium.
2.3. Extraction of MC-LR
2.3.1. The Experiment of Selecting the Best
Temperature
Added 10 equal parts of Microcystis aeruginosa which
were in logarithmic growth phase into 10 Erlenmeyer
flasks, centrifuging and removing the supernatant. The
cells were transferred into other 10 Erlenmeyer flasks
with deionized water. Then put them into water bath at
60˚C, 70˚C, 80˚C, 90˚C and 100˚C, respectively. Two
parallel samples were managed for each temperature.
After 15 minutes took them out and centrifuged, the su-
pernatant was transferred into 10 clean Erlenmeyer flasks,
respectively. Washing the residue with deionized water
and centrifuged again, combining the supernatant. The
supernatant were loaded into SPE cartriege, rinsed with
10 mL deionized water and 20% methanol, and then the
MC-LR was eluted by 90% methanol which containing
0.1% TFA). The eluents were dried under nitrogen in
40˚C water bath, the rest substances was dissolved in 0.5
mL methanol(two times) and transferred into autosamp-
ler vial, dried again with strea m of nitrogen, d iluted wit h
50% methanol to 200μL, and detected by HPLC.
2.3.2. The Extracting Time under 100˚C
Added 12 equal parts of Microcystis aeruginosa which
were in logarithmic growth phase into 12 Erlenmeyer
flasks. The isolation of cells was the same as the above.
The flasks were put into 100˚C for 5min, 10min, 15min,
20 min, 25 min and 30 min, respectively. Two parallel
samples for each gradient. The crude extract was centri-
fuged and loaded into SPE as above.
2.4. HPLC Conditions
Column temperature at 40˚C, using methanol and the
buffer solution of phosphoric acid (PH = 3) at 57:43(V/V)
as the mobile phases, velocity of flow was 1mL/min,
wavel ength of the U V-vis detectors was 380 nm.
3. Results and Discussion
The contents of MC-LR extracted under different tem-
perature were showed in Figure 1. The algae cells were
extracted 15 minutes under each temperature. As it
turned out, t he higher the tem peratur e, the muc h MC-LR
was extracted. In the range of 70˚C to 100˚C, the co ntent
of MC-LR and the temperature had a good linear rela-
tionship. The data showed that every 10˚C degree tem-
perature rise would increase the content of MC-LR about
15 μg/L. Thus it could be seen that the appropriate tem-
perature for extracting MC-LR from Microcystis aerugi-
nosa was 100˚C. But the disadvantage was that the pig-
ments and other impurities in the algae cells would ex-
tracted out simultaneously. These impurities would in-
terfere with the detection of MC-LR. Research about
eliminating influe nce from these impur ities is c ontinuing.

Studies On Extracting Microcystin-Lr From Microcystis Aeruginosa By Water Bath
72
The results of extracting MC-LR for six different
times in 100˚C water bath was shown in Figure 2. It
demonstrated that the content of MC-LR was the highest
when extracted for 20 min. For 5 minutes, it was too
short that a large amount of cells were still integrity, so
the MC-LR inside the cells couldn’t be released. If the
time was too long, part of the MC-LR was degraded un-
der the high temperat ure, resulting in the reducing of the
content of MC-LR.
4. Conclusion
Tests results showed that there were some advantages to
extract MC-LR from Microcystis aeruginosa cells by
water bath, listing as follows: firstly, it was quick, con-
venient and reducing the costs. The whole procedure
could be finished in one day. Secondly, the denaturation
of protein under high temperature made it no need to
adjust PH value with sulfuric acid. Thirdly, this method
avoided the use of methanol during the extracting proc-
60 70 80 90100
70
80
90
100
110
120
The content of MC-LR μg/L
Th e extracting tem perature /℃
69. 22
73.71
89. 31
103.97
117.80
Figure 1. Results of extracting MC-LR under different
temperature.
510 15 20 25 30
240
260
280
300
320
340
The content of MC-LR μg/L
e x tra ctin g time (min)
249. 23
321. 26
327.72 328. 16
311. 76
302. 31
Figure 2. The time of extracting MC-LR from Microcystis
aeruginosa under 100˚C.
ess and therefore reduced injuries to researchers. This
experiment demonstrated it was valid to extract MC-LR
from the cells of Microcystis aeruginosa under 100℃
for 20 minutes. The recovery was over 89.3%. Even so,
there are some problems, namely meanwhile the pig-
ments and other i mpurities would b e extrac ted o ut, which
could interfere with the determination of MC-LR. Expe-
riments had been designed, attempting to remove the
impurities by gradient rinsing the column with different
levels of methanol, namely water, 10%, 20%, 30%, and
40%methanol. Resulting that methanol below the con-
centration of 30% couldn’t remove the pigments. But
40% methanol would elute a large part of MC-LR. So it
was obviously unsatisfactory to remove the impurities
with different concentrations of methanol. In addition,
the time of extracting MC-LR might relate to the con-
sumption of the deionzed water for suspending the algae
cells and the density of the cells. This had to be studied
further and co mprehensively. At the moment, there were
not many data for e xtra c ting M Cs with boili ng water bath.
As this method had a promising prospect it should be
continually studied and improved.
5. Acknowledgements
We should like to express our sincere thanks to the edi-
tors and the reviewers for their useful comments. This
study is supported by the funds as follows: National
“863” Program of China(2009AA063005), Anhui Pro-
vincial Natural Science Foundation(11040606M26),
Foundation of Director of Anhui Institute of Optics and
Fine Mechanics (Y03AG31144), and this is gratefully
acknowledged.
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