Enhanced a -ketoglutaric acid production in Yarrowia lipolytica WSH-Z06by an improved integrated fed-batch strategy
Zongzhong Yu a ,Guocheng Du b ,c ,Jingwen Zhou a ,b ,?,Jian Chen a ,b ,?
a
State Key Laboratory of Food Science and Technology,Jiangnan University,1800Lihu Road,Wuxi,Jiangsu 214122,China
b
The Key Laboratory of Carbohydrate Chemistry and Biotechnology,Ministry of Education,Jiangnan University,1800Lihu Road,Wuxi,Jiangsu 214122,China c
School of Biotechnology and Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,1800Lihu Road,Wuxi,Jiangsu 214122,China
a r t i c l e i n f o Article history:
Received 24December 2011
Received in revised form 5March 2012Accepted 6March 2012
Available online 14March 2012Keywords:
a -Ketoglutaric acid pH control strategy Fed-batch
Yarrowia lipolytica Overproduction
a b s t r a c t
This study aimed at enhancing a -ketoglutaric acid (a -KG)production by Yarrowia lipolytica WSH-Z06.Batch culture experiments demonstrated that CaCO 3and a relatively low pH (3.0)in the a -KG production phase contributed to a -KG https://www.doczj.com/doc/8c15039842.html,ing a two-stage pH control strategy,in which pH was buffered by CaCO 3in the growth phase and then maintained at 3.0in the a -KG production phase,the yield of a -KG reached 53.4g L à1.In the later phase of batch fermentation,the glycerol was exhausted but synthesis of a -KG did not cease.Therefore,glycerol was fed with an integrated fed-batch mode,and a -KG production increased to 66.2g L à1with a productivity of 0.35g L à1h àhttps://www.doczj.com/doc/8c15039842.html,pared to optimal batch culture,a -KG production and productivity were enhanced by 23.9%and 16.7%,respectively.The two-stage pH control strategy,constant feeding approach and lower pH in later phase would be useful for a -KG industrial production.
ó2012Elsevier Ltd.All rights reserved.
1.Introduction
a -Ketoglutaric acid (a -KG),is an important multifunctional or-ganic acid generated in the tricarboxylic acid (TCA)cycle.It has a broad range of industrial applications in the food,pharmaceutical,?ne chemistry and animal feed industries (Finogenova et al.,2005;Otto et al.,2011).As a precursor of glutamic acid,a -KG has a spar-ing effect on endogenous glutamine pools,and is closely linked with the synthesis of proline,arginine and polyamines (Matzi et al.,2007).Additionally,a -KG plays a pivotal role in the interme-diary metabolism to modulate nitrogen balance (De Bandt et al.,1998).In clinical nutrition and healthcare,a -KG can improve gut morphology and function,counteract trauma-induced dysimmuni-ty and play an important role in metabolic adaptation to injury (Cynober,1999).The L -arginine and a -KG mixture is a popular nutrition enhancer in functional beverages to improve blood ?ow to muscle,reduce catabolism and increase protein synthesis during resistance training,resulting in improved training adaptations (Campbell et al.,2006).Barrett and Yousaf (2008)proposed that a -KG could be further exploited as a poly-(triol-a -ketoglutarate)with new potential applications,e.g.tissue engineering and drug delivery.
Currently,a -KG is produced via multi-step chemical synthesis.However,it is hard to overcome shortcomings that include low yield,low purity,residual cyanides and other toxic waste (Otto et al.,2011).Biotechnological processes,as attractive alternative methods of a -KG production,have been studied for several dec-ades (Finogenova et al.,2005).Many bacteria and yeasts have been screened for a -KG overproduction.Among them,Yarrowia lipolyti-ca has been the most intensively studied non-conventional yeast due to its several advantages:e.g.wide substrate spectrum,intense secretory ability,higher product yield,waste minimization and an existing ef?cient system for genetic engineering transformation (Holz et al.,2010;Madzak et al.,2004;Mirbagheri et al.,2011).Moreover,Y.lipolytica is considered by the Food and Drug Admin-istration (USA)as nonpathogenic and many processes based on this organism are classi?ed as Generally Recognized as Safe (GRAS)(Barth and Gaillardin,1997).In recent years,there have been many signi?cant studies concerning the overproduction of a -KG (Otto et al.,2011).Chernyavskaya et al.(2000)identi?ed the principal conditions affecting a -KG oversynthesis by the mutant Y.lipolytica N1from ethanol,and a a -KG concentration of 49g L à1was achieved;however,the concentration of ethanol in the broth should be maintained at <2.5g L à1.Liu et al.(2007)found that lit-tle a -KG was detected as a byproduct in the pyruvic acid fermen-tation broth of Torulopsis glabrata .However,redistributing the metabolic ?ux to a -KG by manipulating cofactor levels resulted in pyruvic acid concentration decreasing from 69to 21.8g L à1,while a -KG concentration increasing to 43.7g L à1.Other studies
0960-8524/$-see front matter ó2012Elsevier Ltd.All rights reserved.https://www.doczj.com/doc/8c15039842.html,/10.1016/j.biortech.2012.03.021
?Corresponding authors.Address:School of Biotechnology,Jiangnan University,1800Lihu Road,Wuxi,Jiangsu 214122,China.Tel.:+8651085329031;fax:+8651085918309.
E-mail address:zhoujw1982@https://www.doczj.com/doc/8c15039842.html, (J.Zhou).
have focused on changing related enzyme activities to in?uence the amounts and ratios of the organic acids produced(Holz et al., 2010;Zhang et al.,2009).
Little literature is available on the optimization of a-KG produc-tion by batch and fed-batch fermentation in Y.lipolytica using glyc-erol as the sole carbon source.Recently,glycerol as a waste derived from hydrophobic substrates(e.g.oils,fats,fatty acids and n-alkanes)and biodiesel production has been considered as a potential substrate for a-KG production by Y.lipolytica(Hama et al.,2011; Mirbagheri et al.,2011).Glycerol was used as the sole carbon source throughout the present study to achieve the goal of a-KG industrial production.In this work,relatively low pH as well as CaCO3were key factors leading to a-KG accumulation.By a two-stage pH control strategy,a-KG production increased to53.4g Là1.Finally,a novel integrated fed-batch culture led to a signi?cant enhancement in a-KG production by a further23.9%,reaching66.2g Là1.
2.Methods
2.1.Microorganisms
The thiamine-auxotrophic Y.lipolytica WSH-Z06,screened in a previous study(Zhou et al.,2010),was used throughout.
2.2.Medium
Medium composition for slant and seed cultures was(all in g Là1):20glucose,10peptone,0.5MgSO4á7H2O and1KH2PO4 (Zhou et al.,2009).The medium initial pH was adjusted to5.5with 4mol Là1NaOH.In the slant medium,20g Là1agar was added.
The fermentation medium had the following composition(in g Là1):100glycerol,3(NH4)2SO4,3KH2PO4,1.2MgSO4á7H2O,0.5 NaCl,0.1K2HPO4,and0.4l g Là1thiamine-HCl.The medium initial pH was adjusted to4.5with4mol Là1NaOH.The thiamine-HCl was?lter-sterilized before addition to the medium.CaCO3used as pH buffer was dry-heat sterilized at160°C for30min before being added into the medium.Other components were autoclaved for15min at115°C.
2.3.Culture conditions
The seed culture was inoculated with healthy yeast,grown on an agar slant and incubated in a500-mL?ask containing50mL of seed culture medium for24h on a reciprocal shaker at200rpm.
The batch and fed-batch fermentations were performed in500-mL?asks containing50mL of fermentation medium,or in a7-L jar fermentor(BioFlo415-7L;New Brunswick Scienti?c,En?eld,CT, USA)containing4L of fermentation medium.The inoculation size was10%(v/v).The?ask cultures were grown for168h,the rotation rate controlled at200rpm,and the medium buffered by20g Là1 CaCO3.In fermentor cultures,the agitation speed and aeration rate were controlled at600rpm and2.5vvm(volume air per volume broth per minute),respectively.pH was controlled with sterile 4mol Là1NaOH solution and CaCO3according to the different de-signed strategies.All cultivations were carried out at28°C.
2.4.Analytical method
Samples were taken for off-line analysis every12h after starting the fermentations.The optical density of the culture broth was mea-sured using a spectrophotometer(Biospe-1601;Shimadzu Co, Kyoto,Japan)at570nm after an appropriate dilution,and converted to dry cell weight(DCW)according to a predetermined calibration equation,OD570:DCW(g Là1)=1:0.223.For measurement of glyc-erol,pyruvic acid and a-KG,fermentation broth was centrifuged at10,000g for10min.The supernatant was diluted50times and?l-tered through a membrane(pore size=0.22l m).a-KG,glycerol and pyruvic acid in supernatant were simultaneously determined by HPLC(Agilent1100series,Santa Clara,CA,USA)with a Aminex HPX-87H column(300mm?7.8mm;Bio-Rad Laboratories Inc., Hercules,CA,USA).The mobile phase was5mmol Là1sulfuric acid in distilled,de-ionized water?ltered to0.22l m.The mobile phase ?ow rate was0.6mL minà1.The column temperature was main-tained at35°C,and the injection volume was10l L.The a-KG and pyruvic acid were detected by UV(wavelength at210nm)detector. Glycerol was detected by a differential refraction index detector.All values are the means of three independent extraction processes. 2.5.Statistical analysis
Student’s t-test was employed to investigate statistical differ-ences,and samples with P<0.05were considered signi?cant.
3.Results and discussion
3.1.In?uence of initial glycerol concentration on a-KG production in shaker?asks
The effect of various initial glycerol concentrations of50,80, 100,150,200,250and300g Là1on cell growth and a-KG produc-tion in?asks are shown in Fig.1.When glycerol concentration was within50–200g Là1,the?nal biomass was very similar(about 11g Là1DCW)for each batch.However,cells grew a little faster in the presence of50or80g Là1glycerol,with the maximal bio-mass occurring24h earlier with these low glycerol concentrations, indicating that maintaining a low glycerol concentration was favorable for cell growth in prophase.
Using100g Là1of glycerol was found to produce the maximum a-KG concentration of36.7g Là1at168h,with the?nal biomass of 11.3g Là1DCW.With increasing initial glycerol concentration,the a-KG production declined.When initial glycerol concentration was 250g Là1,both cell growth and a-KG production were signi?cantly inhibited:?nal biomass and a-KG production were very low at8.1 and19.4g Là1,respectively.The results showed that a-KG produc-tion was more ef?cient at glycerol concentration<100g Là1.How-ever,lower initial glycerol concentrations resulted in lower total amounts of a-KG,indicating that100g Là1initial glycerol concen-tration was most suitable for a-KG production in batch
culture.
598Z.Yu et al./Bioresource Technology114(2012)597–602
3.2.Improving pH control strategy of a-KG production in batch fermentation
Three different pH control strategies were respectively applied in batch fermentations of a-KG production in a7-L fermentor (Fig.2).In strategy A,pH was maintained at4.5using4mol Là1 NaOH for the whole process(Fig.2A)(Chernyavskaya et al., 2000).The Y.lipolytica grew well and reached a?nal biomass of 9.6g Là1DCW.In the broth,there was23.8g Là1residual glycerol until168h.The maximum a-KG concentration was22.0g Là1at 156h,while pyruvic acid was up to36.9g Là1.Lower a-KG produc-tion and more pyruvic acid were obtained in7-L fermentor com-pared to500-mL?asks.In order to determine the reasons, culture conditions between?asks and fermentor were compared. The pH control strategy likely played a key role in a-KG accumula-tion,and consequently strategy B was tested.The pH was buffered with20g Là1CaCO3added into the medium just after inoculation to simulate conditions in the?asks(Fig.2B).Using strategy B,40.3 and31.8g Là1of a-KG and pyruvic acid accumulated,respectively, when glycerol was exhausted at180h.
However,the production rate of a-KG differed for the different pHs(Fig.2B).pH variation in the process of a-KG production was investigated using strategy B in an attempt to determine the opti-mal pH(Table1).During0–84h,pH declined from6.4to3.5,and biomass reached its maximum of12.8g Là1DCW,with only11.2 and16.1g Là1of a-KG and pyruvic acid produced,respectively. The accumulation rates of a-KG and pyruvic acid were0.13and 0.19g Là1hà1,respectively.Within84–120h(i.e.elapsed36h), pH continued to decline from3.5to2.7,and further16.5and 8.1g Là1of a-KG and pyruvic acid accumulated,respectively,with corresponding rates of0.46and0.23g Là1hà1.Thus accumulation rates of a-KG and pyruvic acid increased by250%and20%,respec-tively.Within120–180h,pH varied between2.7and2.4,and the accumulation rate of a-KG decreased to0.21g Là1hà1.The staged analysis of the strategy B results indicated that a relatively low pH (3.0)could signi?cantly facilitate the a-KG synthesis.
Consequently,an improved two-stage pH control strategy(strat-egy C)was proposed:pH was buffered with CaCO3in the growth phase and then maintained at3.0with4mol Là1NaOH during the a-KG production https://www.doczj.com/doc/8c15039842.html,ing strategy C,pH naturally declined to 3.0owing to the organic acids accumulation,and a-KG production phase started spontaneously.As expected,a-KG accumulated at a higher rate when maintained at pH3.0(Fig.2C).Final yield of a-KG reached to53.4g Là1,and pyruvic acid was low with 21.3g Là1.Besides,the lower pH would have many advantages in industrial production:it would reduce the need for neutralizing agents such as NaOH,thereby reduce costs in downstream process-ing and alleviate environmental pollution.Furthermore,low pH could protect Y.lipolytica from other living contaminants,which is an advantage for industrial fermentation.
3.3.The effect of pH control strategy on a-KG production in batch fermentation
The results of the a-KG-producing fermentations by three dif-ferent pH control strategies are shown in Table2.Strategy A
(Fig.2A)resulted in?nal yields of pyruvic acid(36.9g Là1)being
much greater than of a-KG(22.0g Là1),suggesting that excessive pyruvic acid could be potentially redistributed into the citric acid cycle to produce a-KG.Ca2+was reported to enhance the activity of pyruvate carboxylase in mitochondria(Walajtys-Rhode et al., 1992),and CO2à
3
could be used as substrate of pyruvate carboxyl-
ase.Therefore,both Ca2+and CO2à
3
could facilitate the pyruvate car-boxylation process.So more carbon?ux could be channeled into the citric acid cycle.Liu et al.(2007)improved the rate of a-KG/ pyruvic acid using CaCO3as a pH regulating agent in T.glabrata.Strategy B(Fig.2B)resulted in more pyruvic acid being redistrib-uted into the citric acid cycle,probably due to the presence of CaCO3.As a result,a-KG production and productivity were im-proved by83.2%and84.0%,respectively.
Using strategy C(Fig.2C),the100g Là1of glycerol was ex-hausted after132h of culture.At the same time,the concentration of pyruvic acid reached its peak of41.8g Là1,and then slowly de-creased to21.3g Là1at180h.However,the Y.lipolytica still could synthesize a-KG ef?ciently for a long time.Rymowicz et al.(2010) prolongated the active citric acid biosynthesis(107g Là1)
by
Z.Yu et al./Bioresource Technology114(2012)597–602599
Y.lipolytica up to300h using cell recycle with a productivity of 1.42g Là1hà1.Afther132h,the concentration of a-KG continued to increase,and reached a maximum of54.0g Là1at168h,suggest-ing that excessive pyruvic acid accumulation may be transported into cells and used for a-KG synthesis under glycerol de?ciency (Huang et al.,2006),a likely reasonable route to reduce this byprod-uct.However,when the concentration of pyruvic acid decreased to 21.3g Là1,this was insuf?cient to meet the requirement of succes-sive a-KG accumulation,and so a-KG accumulation began to decrease.
3.4.Fed-batch fermentation by pulse feeding in?asks
In the later phase of batch fermentation(Fig.2C),the cells still had latent capacity to product a-KG,but this was limited by glyc-erol de?ciency.Glycerol was fed to investigate whether the results would change.The a-KG production by fed-batch fermentations in ?asks using20,50,80and100g Là1of initial glycerol is shown in Fig.3.There was5mL of glycerol solution(500g Là1)added to each?ask at72h.For initial glycerol of20g Là1,the biomass was only 5.8g Là1DCW at48h,and then ceased increasing (Fig.3A).After glycerol was fed at72h,cells continued to grow and reached a biomass of8.5g Là1DCW at144h.Although glyc-erol(total of70g Là1)was nearly exhausted at168h,only 23.9g Là1a-KG and13.6g Là1pyruvic acid were detected in the broth.
When the initial glycerol was50g Là1,i.e.a total of100g Là1,the biomass reached9.3g Là1DCW at72h(Fig.3B),and the?nal yields of a-KG and pyruvic acid were increased to41.9and18.8g Là1at 168h,respectively.This results illuminated that cell growth was ex-tremely important preparation for a-KG synthesis;only when high biomass was obtained in the early phase could high a-KG produc-tion be achieved.When initial glycerol was80g Là1(Fig.3C), although18.9g Là1glycerol remained in the broth,56.6and 21.2g Là1of a-KG and pyruvic acid were achieved within the same culture time,respectively.When initial glycerol was increased to 100g Là1,only3.8g Là1more a-KG was obtained,with25.3g Là1 glycerol remained in the broth.These results indicated that suf?cient glycerol,up to20g Là1,in the broth would aid a-KG production.
After glycerol was fed at72h,a-KG and pyruvic acid contents de-creased;however,they both exhibited a desirable trend in returning to a higher level after120h of culture(Fig.3).It was concluded that cells could not adapt to sudden changes in the extracellular environ-ment,and that a-KG and pyruvic acid were?rstly utilized by tem-porarily active protective mechanisms.With the expression of proteins involved in energy maintenance,transport and RNA trans-lation(van Duuren et al.,2011),a new adaptive mode may form,and thereby,a-KG production resume.In the new mode,it seemed that pyruvic acid was rapidly channeled into the TCA cycle to produce a-KG that pyruvic acid was kept at a low level.
3.5.Fed-batch fermentation by constant feeding in a7-L fermentor
To avoid potential delays or inhibition resulting from the sud-den increases of glycerol,a constant feeding approach was applied in fed-batch culture(Kim et al.,2009;Zhu et al.,2010).Of150g Là1 total glycerol,124.3g Là1was used(Fig.3D).Thus,80g Là1initial glycerol was chosen,and50g Là1was https://www.doczj.com/doc/8c15039842.html,bining the pH con-trol of strategy C resulted in an improved fed-batch culture mode (Fig.4)(Tang et al.,2009).The delay was then negligible and both a-KG and pyruvic acid accumulated during the feeding time (Fig.4).Consequently,66.2and26.1g Là1of a-KG and pyruvic acid were obtained at192h,respectively.The total130g Là1of glycerol was exhausted and the maximal biomass was11.4g Là1DCW.The biomass reached9.0g Là1DCW12h earlier(at60h)in comparison with the batch mode,i.e.a shorter growth phase,with the a-KG production phase beginning12h earlier.This indicated that the higher nutrient concentrations in the initial medium increased the speci?c growth rate.In the fed-batch culture process with con-stant feeding,glycerol was generally maintained at30–50g Là1 during the feeding course.a-KG production was signi?cantly en-hanced,and a-KG productivity attained0.35g Là1hà1–a16.7%in-crease compared with the optimal batch culture(Fig.2C).The?nal yield of pyruvic acid was very similar to that of the batch mode.For the purpose of lower pyruvic acid and higher a-KG production,ge-netic modi?cation could redistribute the carbon?ux from the pyruvic acid node to a-KG and also block further metabolism of a-KG(Zhang et al.,2009).
Fed-batch culture is a preferred operational mode,using feeding substrate into a batch culture in an appropriate way,and has advantages of achieving a higher cell density by overcoming sub-strate inhibition and increasing production of the desired product (Li et al.,2010).From an economic point of view,the integrated fed-batch mode has huge advantages.In fed-batch mode(Fig.4), 12.8g Là1more a-KG was obtained compared to batch culture (Fig.2C)by feeding30g Là1glycerol–a cheap raw material.Addi-tionally,much energy consumption and labor would be saved when an equal amount of a-KG is produced by fed-batch mode compared to batch culture(Akerberg and Zacchi,2000).These advantages could signi?cantly facilitate low-cost industrial a-KG production.
Table1
pH variation in the process of a-KG production by strategy B.
pH Culture time(h)a-KG(g Là1)PA a(g Là1)Accumulation rate of a-KG(g Là1hà1)Accumulation rate of PA(g Là1hà1)
6.4–3.50–8411.216.10.130.19
3.5–2.784–12016.58.10.460.23
2.7–2.4120–18012.67.60.210.13
a PA:pyruvic acid.
Table2
Comparison of three pH control strategies.
pH control strategy DCW(g Là1)a-KG(g Là1)PA a(g Là1)r a-KG/PA a(g gà1)r a-KG/glycerol a(g gà1)a-KG productivity(g Là1hà1)
A9.622.036.90.590.290.13
B12.840.331.8 1.270.420.24
C11.553.421.3 2.510.530.30
a PA:pyruvic acid;r a
-KG/PA :the ratio of?nal a-KG production to?nal PA production;r a-KG/glycerol:ratio of?nal a-KG production to exhausted glycerol.
600Z.Yu et al./Bioresource Technology114(2012)597–602
4.Conclusions
In the present study,pH was identi?ed as a key factor in a -KG production by Y.lipolytica WSH-Z06.The production of a -KG was enhanced by applying a two-stage pH control strategy,with ?nal production of 53.4g L à1.In addition,a novel integrated fed-batch
mode of combining pH-shift and substrate-feeding strategies was used,and the maximum a -KG production was improved to 66.2g L à1,with a yield of glycerol to a -KG reaching 0.51g g à1.The lower pH of the fermentation process could signi?cantly de-crease the risk of living contaminants and the usage of neutralizing agents.
Acknowledgements
This work was supported by grants from the Major State Basic Research Development Program of China (973Program,No.2012CB720806),the Key Program of National Natural Science Foundation of China (No.31130043),the National Natural Science Foundation of China (No.31171638),the Priority Academic Pro-gram Development of Jiangsu Higher Education Institutions and the 111Project (No.111-2-06).
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