全 文 ：第 26卷第 3期
2006年 3月
生 态 学 报
ACTA ECOLOGICA SINICA
Vo1．26．No．3
Mar．．20o6
营养胁迫下球形棕囊藻(Phaeocystis
Scherfe1)的生长行为及溶血活
刘洁生 ，彭喜春 ，杨维东
globosa
性
(1．暨南大学生物工程学系，广州 510632；2．暨南大学食品工程系，广州 510632)
摘要：近年来，我国广东沿海连续出现大面积球形棕囊藻(Phaeocystis globosa Scherfe1)赤潮，产生溶血毒紊等有害物质，给当地的
海洋养殖业造成重大的经济损失。研究不同的生长时期及半连续培养时不同营养盐胁迫下，球形棕囊藻溶血毒素的产生行为。
结果显示，批量培养的球形棕囊藻处于生长平稳期末时，溶血活性最大((21±1)units／L)；半连续培养时，营养盐限制对球形棕囊
藻的生长有明显的抑制作用，其中F 及 N盐限制影响最为明显。同时，营养盐限制也可促进棕囊藻溶血毒素的合成。其中
Fe3 和．Mn2 的限制性时球形棕囊藻溶血活性显著增强。这些结果表明，球形棕囊藻溶血毒素的产生与藻细胞的生长可能受不
同机制的调节，溶血毒素的合成可能是环境胁迫下棕囊藻维持生存的一种策略。
关键词：溶血物质；球形棕囊藻；营养限制
文章编号：1000．0933(2006)03．0780—06 中圈分类号 ：Q178．1 文献标识码：A
Growth and hemolytic activities of Phaeocystis globosa Scherfel at diferent
mutrients condition
LIU Jie-Sheng ，PENG Xi—Chun2，YANG Wei—Dong (1
． D印0 脚眦0， fole ， ，G岫，Igz抽u 510632，China；2．
Departm．~nt ofFood Engineering，Ji mn Unive~ity，Guangzhou 510640，China))．Acts Ecologica Sinica，2O06，26(3)：780—785．
Abstract：In this experiment，the productions of hemolytic substances of Phaeocystis globosa Scherffel at various stages of growth
and under diferent nutrient limited conditions in semi—continuous cultures were studied．The results showed that the hemolytic
activity was highest in stationary phase，but did not decrease as cell entered into senescent phase；the hemolytic activity varied
signifcantly among diferent treatments．Signifcantly higher hemolytic activities were detected in N-and Fe-limited cultures
compared to those under non—limited conditions．However，hemolytic activity of culture under P-limited condition(N：P=150：1)
was the lowest，only 87．5 HU，lower than those under the other conditions．The average hemolytic activities per cell of cultures
under N-limited and Fe-limited conditions were signifcantly higher than those under other nutrient limitation and non-limited
conditions，whereas there was only a few diferences between other three treatments．These suggested that growth and toxicity of
Phaeocystis globosa were regulated by different factors，and that the toxin production might be related to celular physiological
stress，regulated by the availability of nutrients in Phaeocystis globosa．
Key words：Phaeocystis globosa；hemolytic activity；alelopathy
基金项目：国家自然科学基金资助项目(30470321)；国家重点基础研究发展规划973资助项 目(2001CB409710，2001CB409709)；广东省自然科学基
金重点资助项目(021168)
收稿日期：2005．03．10；修订日期：2006．01．05
作者简介：刘沽生(1957一)，男，山东东明人，博士，教授。主要从事功能生物分子与生物安全研究．E-mail：tywd@jnu．edu．al
Foundationitem：The projcotwas supported bythe National Natural Science Foundation ofChina(No．30470321)，the State Key Basic Research and Development
Plan of China(No．2001CB4097l0，No．2001CB409710)，the Key Foundation of Nature Science of Guangdong Province(No．021168)
Received date：2005—03-10；Accepted da le：2006—01 05
Biography：LIU Jie—Sheng，Ph．D，Professor，mainly engaged in functional biological molecule and biological security
． E-mail：tywd@jnu．edu．cn
Acknowledgements We Kre grateful to Me Wu—x．in，who provided practical assistance in the laboratory，to Jiang Tian．jiu，Jian Feng．yi and Fang Ling who either
give 80I good advices，or provided some instruments during this experiment，and to Professor Gin Gee，who correct the English e．xpression of this p印eI
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3期 刘洁生 等：营养胁迫下球形棕囊藻(Phaeocystis globosa scher&1)的生长行为及溶血活性 781
During the period from the autumn of 1997 to the spring of 1998，a massive bloom of Phaeocystis first OCCurred in tlle
coastal waters of East and South China sea，which caused a great impact on the local aquicuhure industry
． Since then，this
harmful algal bloom(HAB)had frequently occured in this tlxea．Phaeocystis had morphological and physiological features
that differed from those previously described for either Phaeocystis globosa Scherfel or Phaeocystis pouehetii
Lagerheim ’ ．Furthermore，the sequence comparison of Phaeocystis 1 8S rDNA clearly showed that it was rernarkablv
similar to several isolates of isolated P．globosa，and hence it was identified as P．globosa rather than P ． poucheti or
another species documented by Chen et al[
．
There were few reports on hemolytic activity of P．globosa in the other waters．He et a1．confirmed that P．globosa
produced hemolytic toxin responsible for the toxicity of fish in the sea and identifed them as a mixture of glycolipids，
which was sim lar to that of Prymnesium parvum 制
． Among the other prymnesiophytes， some species， such as
Chrysochromulina and Prymnesium，were known to produce hemolytic toxins that caused lysis of vertebrate red blood
ce11s[ _
．
Many literatures showed that biosynthesis of HAB toxins were closely related to algal cells growth and their life
cycle¨ 。
． The synthesis of paralytic shelfish poisoning(PSP)in synchronized ceils was confirmed in early G phase and
stop as the cells entered S phase in Alexandrium fundyense ．In the benthic dinoflagellate Prorocentrum lima，
dinophysistoxin一4(DTX,)synthesis was initiated in Gl phase and persisted into S phase，whereas okadaic acid(OA)and
dinophysistoxin-1(DTX1)production occured later during S and G2 phasesn ．These results suggested that toxin
synthesis was coupled to cell cycle events．On the other hand，the latest studies found the dissoluble nitrate and phosphor
clearly fluctuated during the occurrence of P．globosa red tide ．Additionally．the concentrations of Fe and Mn also
varied regularly with the appearance of the red tide ．However，little was known about the changes in hemolytic activity
at diferent stage of growth and production of hemolytic toxins under diferent nutrient conditions in P．globosa．In this
paper，the productions of hemolytic substances of Phaeocystis globosa Scherfel at various stages of growth and under
different nutrient-·limited conditions in semi— continuous cultures were studied to gain more information about toxin
production in P．globosa．
1 Materiais and methods
1．1 Sampling and culture of algae
Phaeocystis globosa Scherfel，strain Shantou 97 was kindly provided by Dr．S．H．Lu at Science and Engineering
Colege of Jinan University in Guangzhou，China．The strain was maintained in our laboratory in artifcial seawater
supplemented with f／2 medium ．In the present study，P．globosa Scherfel was grown in 16 2L—Edenmcyer flasks
each containing 1．5 L f／2 medium．Al media used were prepared by fihration through O．22／~m Milipore filters．Each flask
filled with 1．5L growth medium was inoculated with 50ml exponentially growing P．globosa Scherffel cells at 2．55 x lOs
cels／m1．Cultures were grown at(22±1)℃under Photosynthetic Active Radiation(PAR)of 200 mol‘m。‘s (cool—
white fluorescent tubes)with 12 h light／dark cycle for 15 days．Cels were counted under an inverted microscope and
samples were taken each day．
1．2 Semi—continuous cultures of algae
According to the meth0d developed by Johansson and Granrli 埔 ，after the batch cultures reached high cell densities，
0．8 L of sample was taken each day and replaced with an equal volume of fresh medium．Three N：P supply ratios(of
啪ss)and 1／10 concentration of Fe and Mn of f／2 medium were used everyday in the replacing media(three replicates
of each)(Table 1)．The other nutrients were added to all cuhures at the levels coresponding to medium f／2⋯ ．Vitamins
(B biotin and thiamin)were added folowing the method of SchSne¨ ．
The irL0culated medium was used to estimate the cell density。10 days after semi-continuous cuhures，the ceils were
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782 生 态 学 报 26卷
individually colected for extracting of hemolytic substances．
1．3 Extraction of hemolytic substances
Cultures were centrifuged at 3000 rpm for 10 min．to
harvest ceUs，which were re．suspended in 50 ml of the
mixed solution of mentho1，chloroform and water(13：7：5，
v／v)and agitated vigorously ultrasonically(600M，30min)
at 4℃ ．The chloroform fractions were fuly evaporated to
drynes(25 a【=，0．1 MPa)in a rotatary evaporator，and then
culture was dissolved in 1 IIl 0f 70％ methano1．
1．4 Hemolytic test
Table 1 Inorganic nutrient concentrations(m#L)in the daily added
new med]R in semi-cont~tuous cultures of P．globosa
“
— —
” meting that their concentrations keep the same a8 i"／2 medium
re．dissolved in 70％ methan01． e extract from a 1 L
The hemolytic substances from P．globosa were identified as mixture of glycolipids．and the hemolytic action
resembled that of digitonin，a non-ionic surfactant，80 the hemolytic activity of the extract from P．globosa wfl8 estimated
as a digitonin equivalent． Th e hemolytic test was perform ed according to the method developed by Simonsen and
Moestrup[
． with the following modifications．1 ml of algal extract was added to 4 ml of 0．5％ rabbit blood in isotonic
phosphate bufer(pH 7．2)．After a 30 min incubation at 37℃，the hemolytic activity wfl8 detected spectrophotometrically
by measuring the absorbance at 540 nm(10 mm cuvette)．Tests were performed in triplicate，and 70％ methanol was used
a8 contro1．
Rabbit blood was drawn out ofwhite rabbits obtained from the animal laboratory of Jinan University and used within 3
days．The blood was centrifuged(1000 r／mln，lOrnin)，and the serum and upper layer of white blood cels(bufy coat)
was removed．Erythrocytes were washed 3 times with phosphate-bufered saline(PBS；pH 7．2)and brought to final
concentration(v／v)of 0．5％ in isotonic citrate bufer(NaC1 73 mmol／L，sodium citrate 42 mmol／L，gluc0se 114
mmol／L)．
2 Results
2．1 Cells growth
Cell growth of Phaeocystis glosbosa in batch culture was demonstrated in Fig．3．After three days’culture，algal
cells entered its exponential phase，and then the cels abruptly increased from 2．92 x 105 cels／ml to 1
．63 x 10 cells／ml，
lasted ab out 8 days after that the culture got into its stationary phase of growth，then in about 12 days，the growth went to
dec】ine．
耋
2500
2000
l500
l000
500
O
0 2 4 6 8 10 12 14 16
Culture days(d)
25
l5
10
5
0
0 2 4 6 8 10 12 14 16
Culturedays(d)
Fig-1 Cell growth of Phaeocystis glosbosa Scherfel in batch edtum Fig
．2 Hemolytic activity of Phaeocystis glosbosa Seherfel in batch culture
2．2 Hemolytic activity during the course of its growth stage of P ． globosa cells
Figure 2 presented the changes in hemolytic activity from algal cells at various stages of growth
． Litle or no hemolytic
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3期 刘沽生 等：营养胁迫下球形棕囊藻(Phaeocystls globosa Sche- 1)的生长行为及溶血活性 783
activity was detected within 4 days after the inoculation when the nutrient was suficient in cultures
． Then。hemolytic
activity increased apparently from day 4 to 10．After day10，the augment of hemolytic activity became slower and the
maximum of hemolytic activity reached 21±1 units／L in day 12．
2．3 Cell density and hemolytic activity of P．globosa cells at the different nutrition conditions
The growth curves of P．globosa under diferent nutrient conditions in semi continuous cultures were obser、red flmm
the beginning of culture．Th e new added nutrients were not enough to support the cells growth at 1
．
94×106·ml一‘that was
reached in the non—limited batch cultures(Fig．1)．As a result，al cultures experienced a rapid drop in call density
during the following ten days．Th e cels densities difered signifcantly among the diferent treatment．Th e cel density
under Fe3 ．1im ted condition was the least in al the treatments
． Th e cell densities of cultures under N．1im ted condition
were significantly lower than those under P-limited and non—limited condition．Th ese results suggested that Fe limi tation
had the most significant efect on cell density，and that nitrogen limi tation had more influence on cel density than
phosphords limitation．
Th e hemolytic activity varied sigYlifcanfly among 2000
different treatments． In N． and Fe” ．1imited cultures．
sign ifcantly hisher hemolytic activities were detected
compared to those under non．1imited condition，P-and Mn2 ．
1imited conditions． However， hemolytic activity of culture
under P．1imted condition(N：P=150：1)was the lowest，
only 87．5 HU／L，lower than that under the other conditions
(Fig．4)．Their average hemolytic activities per cel of
cultures under N．1imi ted and Fe．1imited conditions were also
higher than those under other and non—lim ted conditions，
whereas the activity in Fe—limited culture was higher than that
in N—limted culture (Fig．4)．
1500
1000
10 12 l4 l6 l8
Culture days(d)
Fig．3 Cell densities of Phacocystis globosa Scherfel in semi-continuous
cultures under diferent nutritional conditions．
黾 2．5
2．0
1．5
1．0
0．5
0
Diferem nutrition
Fig．4 (I)Hemolytic activity of Phaeocystis globosa cels and(Ⅱ)Hemolytic activity per P．globosa cel in semi‘continuous cultures under diferent
nuIrient c0nditi0n8．N0te8：1．N：P=1．5：1；2．N：P兰15：1；3．N：P=150：1；4．1／lO[Fe3 ]；5．I／lO[Mn2 ]+[F ]and[M玎2 ]refer to the
∞ncentration of Fe3 and Mn2 in t／2 medium．
3 Discussion
Changes of hemolytic activities were presented at various growth stages of P．globosa cells．Th e hemolytic activity
was highest in stationary phase．but did not decrease as cels entered into senescent phase(Fig．2)，suggesting that
growth and toxicity of P ．globosa were regulated by diferent factors，and that production of hemolytic substances in P·
globosa may be enhanced when the cells were grown in nutrient deficient condition-
Nitrogen limitation had a signifcant promotion on the production of hemolytic substances in P．globosa contrasted
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784 生 态 学 报 26卷
with s0me relevant rep0ns that P．starvation had more，which was in agreement with previous studies showing that N_
limitati0n could pr0mote toxicity of P．parvum[17]
．
However，it was interesting to note that Fe limitation also stimulated
the producti0n of hemolytic substances in P． 060船 ．According to 0ur knowledge，it was the first time that Fe‘lim
，
itation
had been reported．
，IIli8 studv had showed that there was a close relationship between environmental conditions and toxicity of P．
glob0s口．High toxicity had been observed with the increase in cell density and loss in nutrients，indicating that the growth
and toxicity of P．globosa were regulated by diferent factors．It could be predicted that the growth and toxin production of
P．globosa mi ght have diferent optimal requirements，and that toxicity of P ．globosa was fully exhibited only when the
growth was limited．
The relationship between the environmental conditions and phytoplankton toxicity was still cryptic although it had been
studied in the last years．The influence of lim ted conditions of some crucial nutrients on toxicity had been presented for
many phytoplanktons that produced distinct toxins in chemical structure，but the increase in toxicity under certain nutrient
limited condition had been observed extensively．Several studies showed that algal toxins had a negative infl uence on both
herbivorous zooplankton and other algae[2o，
． Th e negative influence of toxins，in fact，had a function of competing
organisms and grazers，so called“allelopathy”，and would certainly be of value for phytoplankton．During the break of the
HAB of Phaeocystis globosa，cell density of P．globosa swihly increased in a short time an d the available nutrients in
environment were rapidly exhausted．Hence，it could be speculated that production of hemolytic substance in P．globosa
gave the alga an opportunity to gain sufficient nutrient for its growth by inhibition on other aquatic or algae in the same
water．Th e closely related P．poucheti was reported recently to be poor competitor for N at both light levels㈨ ．Gran61i
and Johansson also reported the increase in the production of allelopathic substances by Prymnesium parvum cells grown
under N+deficient condition ．It was reasonable to speculate that the lower ability in competing for N results in the
sensitivity to N—lim ted condition for P．globosa due to the similarity in morphological and overlapping ranges between P．
pouchetii and P． globosa． In fact， both species were commonly referred to as P．globosa until recent genetic
analysis 驯
． Th us，from an ecological point of view，an increased toxin production when nutrients were limited would be a
great advantage，giving the algae an opportunity to proliferate where they would otherwise be incapable of competing with
competitively superior species of algae．However，the influences of hemolytic toxins pmduced by P．g2060s口on growth of
other algae had not been studied in this paper，which deserved to be addressed in the further studies．
4 Conclusions
Th e toxicity of P．globosa was closely related with algal cell growth stages．The hemolytic activity of toxic substances
reached the maximum at the end of stationary phase，but lower at early exponential phase．Moreover，the hemolytic
activity of P．globosa was also related with diferent nutrient conditions．Signifcantly higher hemolytic activities were
detected under nitrogen(N)and iron(Fe)-limited conditions compared to those under non，limited condition as well as P-
and Mn·limited conditions in semi·continuous culture． Th e increase in hemolytic activity was probably related to
physiological stress from nutrition limitation，which may explain the diference in hemolytic activity in P．globosa at
different coastal areas．According to the results，we considered that an increased toxin production in nutrients starvation
would give the algae an opportunity to proliferate under specific conditions that limited cel growth．
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3期 刘洁生 等：营养胁迫下球形棕囊藻(尸^ globosa Scher雎1)的生长行为及溶血活性 785
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