Enzyme and Microbial Technology38(2006)
343–350
Significant effect of NH4+on cordycepin production by submerged cultivation of medicinal mushroom Cordyceps militaris
Xian-Bing Mao,Jian-Jiang Zhong∗
石头剪子布
State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,130Meilong Road,Shanghai200237,China
Received14July2004;accepted18October2004
逗比Abstract
Effects of nitrogen sources on cell growth and cordycepin production by submerged cultivation of Cordyceps militaris,a traditional Chine medicinal mushroom,were investigated.In complex medium,peptone was identified as the best nitrogen source for cordycepin biosynthesis.
A study using chemically defined medium indicated that NH4+played an important role in cordycepin biosynthesis.To enhance cordycepin production,fed-batch culture with NH4+feeding was conducted i
n complex medium with peptone.By optimizing the feeding time and feeding amount of NH4+,a maximal cordycepin concentration of420.5±15.1mg/l was obtained,which was70%higher than that in batch cultivation. The fed-batch cultivation process was also demonstrated in a3.5l stirred tank bioreactor and the cordycepin production was incread from 208.8±7.6mg/l in batch culture to346.1±3.6mg/l in fed-batch culture.The information is uful for large-scale efficient production of cordycepin by the mushroom cultures.
©2005Elvier Inc.All rights rerved.
腿的英语单词Keywords:Medicinal mushroom;Submerged cultivation;Cordycepin;Cordyceps militaris;NH4+effect;Fed-batch culture
1.Introduction
In recent years,mushroom has become an attractive source of various physiologically active compounds[1,2].Cordy-ceps militaris,a famous traditional Chine medicinal mush-room,belongs to the class Ascomycetes and DongChongXi-aCao group in Chine herbs[3].It has received extensive attention for medical application due to its various phys-iological activities[3,4].Cordycepin(3 -deoxyadenosine), one of its major bioactive condary metabolites,exhibits antibacterial,antifungal,a
ntitumor/antileukemic,antiviral and immunomodulation activities[5–7].However,it is dif-ficult and time consuming to cultivate the fruiting bodies of C.militaris for obtaining cordycepin[3].Although chemical synthesis of cordycepin is possible,but it is also cumbersome and meets difficult paration[8].Submerged cultivation of C.militaris for efficient cordycepin production is looked at as a promising alternative to fruiting body cultivation and chemical synthesis,becau its culture period is relatively ∗Corresponding author.Tel.:+862164252091;fax:+862164253904.
E-mail address:jjzhong@ecust.edu(J.-J.Zhong).short and its culture process can be optimized to achieve a highly productive process.
In spite of a great need for uful bioactive metabolites production by submerged cultivation of mushrooms,the bio-process development is still far from being thoroughly studied [9–14].Usually,culture medium is important to the yield of any cultivation products and nitrogen source generally plays a significant role,as it is esntial for cell prolifera-tion and metabolite biosynthesis.In submerged cultivation of mushroom Tremella menterica,Wasr et al.demon-strated that types and concentrations of nitrogen sources strongly influenced cell growth and polysaccharide produc-tion and combined u of yeast extract and corn steep liquor enhanced the accumulation of extracellular polysaccharide [13].For the mushroom Paecilomyces sinclairi,Cho et al. showed that mi
xture of peptone–yeast extract led to the high-est mycelial growth,while meat peptone was most favorable to its red pigment production[14].Becau mushrooms are generally cultivated in media containing a complex source of nutrients,it is difficult to evaluate the nutritional effect of individual components in media,due to undefined and some-times variable composition.In order to elucidate the nature of
0141-0229/$–e front matter©2005Elvier Inc.All rights rerved. doi:10.ictec.2004.10.010
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美卡犬
nutritional effect,in this ca,a chemically defined medium must be ud.As far as we know,there have been no reports using chemically defined medium to investigate the effect of a specific nutrient on condary metabolite production by mushroom cultures.
The limited commercialization of mushroom cultivation is mainly attributed to its low productivity[9].A previous work reported a low cordycepin production titer(7.1mg/l) in a laboratory-scale bioreactor in submerged cultivation of Cordyceps sp.[15,16].Therefore,enhancement of cordy-cepin production is critical to overcome the bottleneck for commercial application.As demonstrated in submerged cul-tiv
ation of Ganoderma lucidum,substrate feeding(fed-batch culture)was uful to the efficient production of uful metabolites by mushrooms[10].However,until now,there have been no reports on fed-batch cultivation for cordycepin production.
In this work,atfirst,the effects of various complex nitro-gen sources on cell growth and cordycepin production were compared.Then,a chemically defined medium was ud to evaluate the effects of different nitrogen compounds.Bad on the experimentalfinding of the positive effect of NH4+on cordycepin production,NH4+feeding in both a shakeflask and a bioreactor was performed to enhance the metabolite production and productivity.As a result,the cordycepin pro-duction titer and productivity obtained were much higher than tho in previous reports.The information obtained is consid-ered uful to the development of a highly efficient process for cordycepin production on a large-scale.The work also provides a uful reference for submerged cultures of other edible and medicinal mushrooms.
2.Materials and methods
2.1.Chemicals
Cordycepin standard,cain acid hydrolysate,cain enzymatic hydrolysate,diethylstilbestrol and sod
思维简史ium ortho-vanadate were purchad from Sigma Chemical Company (USA).Yeast extract was purchad from Oxoid Ltd.(UK). Other reagents,including peptone,were bought from Shang-hai Chemical Reagents Co.(China).
2.2.Maintenance of C.militaris
The strain of C.militaris was purchad from collection bank of Huazhong Agricultural University(Hubei,China). The stock culture was maintained on potato–dextro–agar slants.Slants were incubated at25◦C for7days and then stored at4◦C for subculture[17].
2.3.Effect of complex nitrogen source on cordycepin production
The liquid ed was inoculated with mycelium mat(ca. 5mm2)from a stock slant and grew in a250mlflask con-taining50ml of gluco–salts medium(40g/l gluco,0.5g/l MgSO4·7H2O,0.5g/l K2HPO4·3H2O and0.5g/l KH2PO4) and10g/l yeast extract at25◦C on a rotary shaker at110rpm for5days.
Effect of complex nitrogen sources on the mushroom cul-ture was studied by using various complex nitrogen sources, ast extract,peptone,cain acid hydrolysate,cain enzymatic hydrolysate an
d combination of yeast extract and peptone at1:1(w/w).The culture medium consisted of gluco–salts medium and10g/l of a nitrogen source to be investigated.The cultivation was inoculated with5ml liq-uid ed with ca.1.2g cells by dry cell weight(DW)per liter and then conducted in a250ml shakeflask containing 50ml medium on a rotary shaker(25◦C,110rpm)in the dark.Nitrogen source was autoclaved parately and added into culture medium before inoculation.
2.4.Effect of nitrogen compounds in chemically defined medium
The1st stage liquid ed was inoculated with mycelium mat(ca.5mm2)from a stock slant and grew in a250mlflask containing50ml of gluco–salts medium and10g/l yeast extract at25◦C on a rotary shaker at110rpm for5days.The 2nd stage liquid ed was inoculated with the1st stage culture (10%,v/v)and then incubated in a250mlflask containing 50ml of gluco–salts medium and10g/l peptone on a rotary shaker for7days.
Experiments were performed in a250ml shakeflask containing50ml medium with inoculation of about2.7g DW/l from the2nd stage liquid eds.The inoculation cells were apticallyfiltrated and washed three times with distilled water before being transferred into a culture flask.The cultivation medium was compod of10g/l glu-co,0.5g/l MgSO4·7H2O,0.5g/l K2HPO4·3H2O,0.5g/l KH2PO4,
0.1g/l CaCl2,0.1g/l FeSO4·7H2O and40mM of a nitrogen compound to be investigated.Nitrogen com-pounds investigated were20kinds of amino leucine,tyrosine,asparagine,proline,valine,isoleucine, phenylanaine,tryptophan,aspartic acid,lysine,threonine, arginine,glutamic acid,glutamine,histidine,methionine, glycine,alanine,rine and cysteine)and ammonium sul-fate(inorganic nitrogen source),while peptone(com-plex nitrogen source)was taken as a control.Nitrogen sources were autoclaved parately before addition to culture medium,except that glutamine and asparagine werefilter-sterilized.
2.5.Pul feeding in shakeflasks
Medium components and inoculation density were the same as tho in Section2.3and peptone was ud here as the nitrogen source.To investigate feeding time,a single feeding of20mM ammonium sulfate(40mM of NH4+)to medium was made at days3,5,7or9.To study the effect of feed-ing amount5,10,20and40mM of ammonium sulfate was,
X.-B.Mao,J.-J.Zhong/Enzyme and Microbial Technology38(2006)343–350345
respectively,fed to medium at day7,when residual NH4+ was about4mM.
2.6.Batch and fed-batch culture in bioreactors
The bioreactor ud was a3.5l(working volume)stirred tank bioreactor with two six-bladed turbine impellers(6.5cm
i.d.).The vertical distance between two turbine impellers was
7.0cm and the lower one was5.0cm above the reactor bot-tom.The reactor was aerated through a ring sparger with a pore size of0.8mm,which was positioned below the impeller and2.5cm above the reactor bottom.Medium components and inoculation density in bioreactor were the same as in shakeflasks(Section2.5).Cultivation was conducted at25◦C in the dark.Rotation speed was150rpm throughout the cul-tivation.Dissolved oxygen(DO)in the culture medium was monitored by a Mettler Toledo DO Probe(Switzerland).DO remained around50–60%of the liquid with air saturation withinfirst4days and then controlled at about30%of air saturation until the end of cultivation by adjusting airflow rate(only air aeration).The culture pH value was monitored throughout cultivation without control.Fed-batch culture was performed by adding20mM of ammonium sulfate at differ-ent cultivation time.
2.7.Analys
2.7.1.Dry cell weight,pH,residual sugar and NH4+
For sampling,three identicalflasks were taken each time. In bioreactor cultivations,about40ml of cell culture was taken once from each reactor.DW was estimated byfil-tering a culture broth through pre-weighedfilter paper and washed with a large amount of distilled water and dried at 60◦C for sufficient time until a constant weight was obtained. After sampling,one part of culture supernatant was ud for pH measurement by a pH meter and another part was stored at−20◦C,which was later thawed for analys of residual sugar,NH4+and cordycepin concentrations.Resid-ual sugar concentration was assayed by phenol–sulfuric acid method[18].Residual NH4+concentration was assayed by phenol–hypochloride method[19].
2.7.2.Cordycepin concentration
The cordycepin concentration(extracellular product)was analyzed by HPLC[8,16,17].A column Kromasil C18(4.6mm×250mm,5m particle sizes)of Eliter Company (Liaonin,China)was ud.The mobile pha was a mixture of methanol/water with0.01M of potassium dihydrogenphos-phate,wasfiltered with membrane of0.45m aperture sizes and was driven by double pump(model:Waters150,Milli-pore,USA).Elution was performed at aflow rate of1ml/min with column temperature at30◦C.The UV wavelength of 254nm was monitored by a tunable absorbance detector (model:Waters486,Millipore,USA).Samples from Eppen-dorf tubes were thawed and then centrifuged
at15,000×g for15min.The sample supernatant was ud for assay of cordycepin concentration[17].
3.Results
3.1.Comparison of various complex nitrogen sources
Table1shows effects of various complex nitrogen sources, ast extract,peptone,cain enzymatic hydrolysate, cain acid hydrolysate and combination of yeast extract and peptone on cell growth and cordycepin production in sub-merged cultivations of C.militaris.All the complex nitrogen sources well supported cell growth,but they had very differ-ent effects on cordycepin production.Although yeast extract was the best for cell growth,peptone was most beneficial to cordycepin biosynthesis.By using peptone,the cordy-cepin production and productivity reached245.8±3.8and 14.5±0.2mg d−1,respectively.Combination of yeast extract and peptone did not improve the cordycepin production and productivity compared with peptone alone(Table1).
3.2.Evaluation of nitrogen compounds using chemically defined medium
Peptone contained20kinds of amino acids and NH4+ [20,21].In addition,peptone itlf might be decompod by extracellular proteas into amino acids,which could also be consumed by mushroo
m cells[21,22].To understand the favorable effect of peptone on the cordycepin production by C.militaris,which was found in the above experiments,there-fore,it is logical for us to investigate the effects of individual amino acids as well as NH4+.After our preliminary exper-iments,a chemically defined medium mentioned in Section 2.4was ud here for the investigation.
Table1
Effects of complex nitrogen sources on cell growth and cordycepin production in submerged cultivation of C.militaris
Parameters Complex nitrogen sources
YE a Pep CEH CAH Pep+YE
Maximal DW(g/l)21.27±0.42(day5)b16.12±0.51(day13)18.53±0.26(day9)17.33±0.35(day9)19.84±0.65(day9) Average growth rate(d−1) 3.20±0.070.92±0.03 1.54±0.02 1.43±0.03 1.65±0.06 Maximal cordycepin production(mg/l)10.8±0.8(day11)b245.8±3.8(day17)140.0±4.5(day15)18.4±0.5(day15)97.5±1.6(day15) Cordycepin productivity(mg d−1) 1.0±0.114.5±0.29.3±0.3 1.2±0.0 6.5±0.1
a Pep,peptone;YE,yeast extract;CEH,cain enzymatic hydrolysate;CAH,cain acid hydrolysate.
b Cultivation time when a maximal DW or maximal cordycepin production titer was reached.
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Table2
Effects of nitrogen compounds on cell growth and cordycepin production by C.militaris in chemically defined medium a
Nitrogen sources DW(g/l)Cordycepin production(mg/l)
Day3Day5Day7Day3Day5Day7 Peptone 6.02±0.717.54±0.537.08±0.1239.0±3.8100.2±3.8135.2±1.1 Alanine 5.52±0.117.89±0.217.33±0.1834.1±4.164.8±3.280.5±2.6 Arginine 4.57±0.04 5.30±0.82 5.36±0.4517.5±2.618.4±5.346.1±5.9 Asparagine 5.85±0.10 6.64±0.23 6.69±0.479.2±0.39.1±1.417.7±1.0 Aspartic acid 5.28±0.93 5.36±0.11 6.84±0.4517.7±2.830.2±1.740.2±2.0 Cysteine 5.76±0.23 5.82±0.11 6.11±0.2483.8±2.3130.7±6.8144.0±1.8 Glutamic acid 5.55±0.38 5.95±0.41 6.9±0.5929.2±0.942.8±现代管理理论
1.847.4±1.9 Glutamine 5.36±0.23 6.56±0.31 6.94±0.0331.5±1.235.1±3.049.3±4.8 Glycine 5.65±0.30 6.91±0.16 6.70±0.4840.9±2.943.8±13.265.3±6.2 Histidine 4.95±0.21 6.31±0.33 6.65±0.1020.9±0.535.7±3.055.0±5.2 Isoleucine 5.90±0.32 6.22±0.427.13±0.5218.0±1.221.5±1.428.7±0.9 Leucine 5.17±0.65 5.47±0.18 6.45±0.2713.4±0.314.6±0.810.4±0.2 Lysine7.78±0.657.63±0.388.18±0.2518.1±2.726.0±1.145.6±4.7 Methionine 3.91±0.21 4.15±0.16 3.68±0.0921.4±0.726.4±2.561.3±9.9 (NH4)2SO4 5.24±0.23 5.93±0.13 5.80±0.0855.3±1.8146.0±15.4205.2±9.6 Phenylalanine 5.87±0.10 6.99±0.527.96±0.3113.7±1.026.1±0.431.3±2.0 Proline 4.86±0.03 6.07±0.01 6.36±0.177.9±0.216.2±0.318.4±0.7 Serine 5.32±0.347.18±0.147.46±0.1452.9±6.874.3±2.699.6±1.7 Threonine7.90±0.26 6.66±0.17 6.22±0.1436.8±1.751.2±0.945.7±1.4 Tryptophan 6.12±0.337.24±0.187.56±0.328.7±0.427.9±1.434.9±1.6 Tyrosine10.88±0.3411.15±0.0111.97±0.0414.4±2.510.0±0.616.3±2.8 Valine 4.93±0.01 5.68±0.34 6.57±0.0114.8±1.019.1±1.920.6±1.3 Days3,5and7denote the cultivation time.
a Maximal cordycepin production was reached at day7in preliminary experiment using ammonium sulfate as nitrogen source.
加热坐垫Table2shows that different nitrogen compounds had dif-ferent effects on cell growth.Tyrosine,lysine,p
henylalanine and threonine were beneficial to cell growth and a maximal DW of11.97±0.04g/l was reached at day7by using tyro-sine.It was obrved that peptone with the same amount of molar nitrogen for cell growth was inferior to the amino acids.The types of nitrogen compounds also significantly affected cordycepin biosynthesis.Ammonium sulfate was the best for cordycepin production,and in this ca,its maxi-mal concentration of205.2±9.6mg/l was obtained at day 7.Cysteine,rine,alanine and glycine were also beneficial to cordycepin production.However,peptone was inferior to ammonium sulfate and cysteine for the metabolite produc-tion(Table2).Compared with that of ammonium sulfate,a similar titer of cordycepin production was achieved by using ammonium chloride(data not shown).
3.3.Effect of NH4+concentration in chemically defined medium
Fig.1shows effects of different concentrations of NH4+on cell growth and cordycepin production in chemically defined medium.The maximal DW incread rapidly with increa of NH4+concentration from0to10mM,but further increa of NH4+level up to160mM led to its slight decrea.The cordy-cepin production titer was incread with increa of NH4+ concentration within a range of0–40mM.The maximal cordycepin production reached209.6±9.2mg/l at40mM of NH4+on day7.From Fig.1,NH4+was considered as the limiting nutrient for cordycepin biosynthesis when its initial concen
tration was below20mM.
3.4.Fed-batch cultures with NH4+in shakeflasks
From the above results,it was understood that NH4+was very important to enhance the cordycepin production by C. militaris,but its relatively high concentration had a bit detri-mental effect on cell growth.Additional experiments indi-cated that combination of NH4+with amino acids could not improve the cordycepin production(data not shown).
More-Fig.1.Effects of various initial NH4+concentrations on cell growth and cordycepin production by C.militaris in chemically defined medium.Sym-bols:cell growth by maximal DW( );maximal cordycepin production ( );residual NH4+concentrations when maximal cordycepin production was obtained( ).
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347
Fig.2.Time cours of cell growth(A)(solid line),pH value(A)(dashed line),residual sugar(B),residual NH4+(C)and cordycepin production(D)in fed-batch cultures of C.militaris with feeding of40mM NH4+at different cultivation time.Symbols:control( );NH4+feeding at day3( );day5( );day 7( );day9( ).
over,it was obrved that the maximal cordycepin production titer in chemically defined medium(Table1)was lower than that in complex medium(Table2).In order to further enhance cordycepin production,therefore,fed-batch cultivation with NH4+was applied to cultivation in complex medium.
The optimum time and amount for NH4+feeding was important.Fig.2shows the effect of feeding of40mM NH4+ at days3,5,7and9in fed-batch culture.Addition of NH4+ showed a slightly inhibitory effect on cell growth compared to control(Fig.2A).In control cultures,the medium pH rapidly decread from4.8to3.0and then almost remained con-stant;in contrast,the pH all went on decreasing to2.2or so at late stage in fed-batch cultures.Ammonium addition also did not obviously affect gluco consumption(Fig.2B), but it showed a significant effect on cordycepin production after7days.Although NH4+feeding at different feeding time showed a similar stimulatory effect,its add
ition on day7was the best to cordycepin production,which reached 420.4±15.1mg/l at day17and was about70%higher than the control(Fig.2D).
Fig.2C shows the dynamic changes of residual NH4+in medium.As obrved,peptone had18.8mM NH4+,which may be the reason why peptone was the best complex nitrogen source for cordycepin production.But,it was also obrved that a relatively higher concentration of peptone would inhibit cordycepin formation[unpublished result].In cultures of control,residual NH4+decread to4.0±0.1mM at day7 and was completely consumed at day9.When NH4+was fed to medium at day7,the highest cordycepin production titer was achieved.The results suggest that a single feeding of NH4+should be done when residual NH4+concentration decread to about4mM.
Fig.3shows effects of feeding amount of NH4+,i.e.10,20, 40and80mM,on cell growth and cordycepin production.A low feeding concentration(10mM)did not affect cell growth compared to the control,however,a relatively higher feed-ing concentration showed a slightly inhibitory effect.It was obrved that all feeding concentrations of NH4+prented significantly stimulatory effects on cordycepin production. Relatively high concentrations of NH4+(40and80mM) showed the best effects on cordycepin production and a high production titer of415.1±4.4mg/l was reached.Concern-ing NH4+consumption,the lowest concentration of residual NH4+was18.5±1.2and61.1±2.3mM at NH
4+feeding amount of40and80mM,respectively,while the added NH4
+ Fig.3.Effects of feeding amount of NH4+on cell growth and cordycepin production by C.militaris in fed-batch culture with feeding of10,20,40 and80mM NH4+on day7,respectively.Symbols:maximal D
W( )and maximal cordycepin production( ).
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