DO I 10 .1007 /s12257 -014 -0799 -5Eva lua t ion o f Funga l Lac t ic Ac id Accumu la t ion Us ing G lycero l as the So le Carbon Source Xiaoqing Wang, Zhenhua Ruan, Webster Guan, Robert
Trang 1DO I 10 1007 /s12257 -014 -0799 -5
Eva lua t ion o f Funga l Lac t ic Ac id Accumu la t ion Us ing G lycero l as the
So le Carbon Source
Xiaoqing Wang, Zhenhua Ruan, Webster Guan, Robert Kraemer, Yuan Zhong, and Yan Liu
Received: 16 November 2014 / Revised: 28 March 2015 / Accepted: 1 Aprl 2015
© The Korean Society for Biotechnology and Bioengineering and Springer 2015
Abstract This study investigated the glycerol utization
for lactic acid accumulation by the flamentous fungus
Rhizopus oryzae 9363 and compared withthe conventional
carbon source glucose No lactic acid accumulated in the
glycerol media at 30oC, in contrast to the glucose media
Increasing the temperature from 30 to 37oC led to a 63%
decreaseinthe average growthrate of R oryzae in glycerol
media and a 61% increase in the average cell mass yield,
and the cultures on glycerol media at 37oC were able to
generate 0.6 g/L lactic acid While, raising temperature
signiicantlyinhibitedlactic acid production on glucose media
Moreover, supplementing cultures with sodium pyruvate
signiicantlyimprovedthelactic acid synthesis of R oryzae
on glycerol media, withlactic acid concentrationsreaching
1.33 g/Lat 37oC and 0.67 g/Lat 30oC, respectively.Our
results indicate that glycerol utization for lactic acid
accumulation by Rhizopus sp.isimited bythe availabity
ointracellular pyruvate, and controling pyruvatelowis a
key to enhancing the lactic acid accumulation
Keywords: Rhizopus oryzae,lactic acid, glycerol pyruvate
1 In troduc t ion
Glycerol has recently gained signiicant attention as an
aractive feedstock for value-added chemical production
due to its abundance and relatively cheap price resuling
from the rapid growth of the biodiesel industry [1,2] Comparedtothe chemical andthermal processes of glycerol
ulization, microbial fermentation provides an environ -mentallyriendly approachto conver glycerolinto value -added products Many microorganisms are ableto metabolize
glycerol as par ofine chemical production processes, such
as 1,3-propanediol productionrom Clostridium Butyricum and Klebsiella Pneumoniae, dihydroxyacetone from Glu -conobacter oxydans, and Klebsiella pneumoniae, succinic
acidrom Anaerobiospirilum succiniciproducens, hydrogen
from Enterobacter Aerogenes, and D-lactic acid from Escherichia coli [3] However, limited information is available aboutthe glycerol utization ofilamentousfungi [4-6]Thelamentousfungus Rhizopus oryzae was selected
to investigate fungal glycerol utization for lactic acid accumulation
Lactic acid has been widely usedinthefoodindustry as
an acidulantflavoring, and preservative[7] Overthe past ten years, ecological concerns have driven its use as a substrate in the production of the biodegradable plastic
poly-lactic acid, whichrequires a highly puried, preferably
L(+)lactic acid anhydrous monomer[8] Comparedtothe bacterial synthesis of lactic acid isomers, the fungus R.oryzae is able to synthesize optically pure L-(+)lactic
acidinrelatively simple media[9] Vaious carbon sources such as starch and pentose have been studied for thei
efects onlactic acid production by R oryzae [10] However
limitedinformationis availableregarding glycerol utization
by R oryzae for lactic acid production Therefore,the objectives ofthis study weretoinvestigate the glycerol conversion to lactic acid by R oryzae and to evaluate the main factors that influence the eficiency of lactic acid accumulation
X iaoq ing Wang , Zhenhua Ruan , Robe r K raeme r Yuan Zhong , Yan L iu *
B iosys tems and Ag r icu l tu ra l Eng inee r ing , M ich igan S ta Un ive rs i ty , Eas t
Lans ing , MI 48824 , USA
Te l +517 -432 -7387 ; Fax : +517 -432 -2892
E -ma il iuyan6@msu edu
Webs te r Guan
Chem ica l Eng inee r ing , Ca l fo rn ia Ins t tu te o f Techno logy , Pasadena , CA
91125 , USA
RESEARCH PAPER
Trang 22 Ma ter ia ls and Me thods
2.1 Fungal strain and spore inoculum preparation
The Strain Rhizopus oryzae NRRL 395 (ATCC 9363)
obtainedrom American Type Culture Collection(Manassas,
VA, USA) was grown on potato dextrose agar at 30oCfor
two weeks Spores were washed as described previously
[11] andthe spore solution was stored at a concentration of
1×108 spores/mL at 4oC
2.2 Culivation
The salts used for batch culture include: KH2PO4(1 g/L)
(Malinckrodt Bakker MgCl2·6H2O(0.5 g/L)(Malinckrodt
Bakker; ZnSO4·7H2O (1.4 mg/L) (Sigma); MnSO4·H2O
(1.6 mg/L) (Sigma); CoCl2·6H2O (3.6 mg/L) (Sigma)
FeSO4·7H2O(2.7 mg/L)(Sigma) The sole nirogen source
for all experiments was 1 g/L NH4Cl (Sigma) Glucose
(Sigma) and glycerol were used as carbon sources, with
iniial concentrations of 10, 30, 50, and 70 g/L Toiniiate
the culture, 0.5 mL spore solution was inoculated into
250 mL Erlenmeyer flasks fled with 100 mL of growth
medium and grown on arotary shaker(Thermo Scientic)
wth shaking at 180 rpm at 30 or 37°C During the
culivation, CaCO3was addedtothe brothto maintainthe
pH at approximately 4 ~ 5
2.3 Analytical methods
Mycelia cel mass was collected bylration, washed with
6 mol/L HCl to neutralize the excess CaCO3in thecel
mass, and then washed with distled water The washed
cell mass was dried at 92.5 ± 0.5°C overnight untl a
constant weight was achieved Glucose, glycerol, ethanol
lactic acid and other organic acids were detected by HPLC
equipped with a Bio-rad Aminex HPX-87H organic acid
column and a refractive index detector [12] The mobile
phase was 5mmol/L sulfuric acid at alowrate of 0.6 mL/min
The column temperature was set to 65°C
2.4 Reductive lactic acid dehydrogenase activity assay
Lactic dehydrogenase(LDH) activityisradiionally assayed
at an absorbance of 340 nm to monitor the decrease in
the reduced nicotinamide adenine dinucleotide (NADH)
concentration during the reaction [13] One unit (U) of
enzymatic activity was defined as the amount of enzyme
necessary to convert 1 µmol of NADH to NAD+ per min
[13] Howeverthis methodis not suitablefor analyzingthe
activity of crude enzyme extracts, as other oxido-reductases
such as pyruvate decarboxylase and alcohol dehydrogenase
in the extracts may also affect the NADH concentration
and interfere with the spectrophotometic readings [13]
Therefore, a modiied method was developedto definethe
LDH activity based on lactic acid, the speciic product of
LDH, eliminatingthe effects of other enzymesinthe crude
extract and providing more accurate data Thereeze-dried cel mass was groundiniquid nirogen
wth a mortar and pestle The cold extraction solution containing 0.1 mol/L bis-is-propane(pH 6.8) and 1 mmol/L
dthiothreitol was addedtothe powder(1:1 w/v)to dissolve the soluble protein on ice for 1 h The suspension was centfuged at 11,400 × g a 4°Cfor 5 min, andthe supernatant was used as the crude enzyme extract [10,13] Reductive lactic acid dehydrogenase (LDH) activity (LDH is the enzymethat catalyzesthe conversion of pyruvatetolactic
acid) was determined by the total amount of lactic acid generatedrom sodium pyruvate inthe followingreaction system: a 1.0 mL reaction mixture consisted of 0.1 mol/L
bis-is-propane buffer (pH 6.8) with 20 mmol/L NADH and 40 mmol/L sodium pyruvate Reaction mixtures without
crude enzyme extracts or without substrates were used as controls Thereaction was started by adding 0.2 mL crude enzyme extracts, incubated at 30oC for 5 min, and then
terminated by 20 min incubation in a 100oC water bath One unit of reductive LDH was defined as the amount of enzymerequiredto generate 1 µmollactic acidrom pyruvate
per minute
2.5 Calculations of kinetic parameters The maximum speciic growthrate(µmax) was determined
fromthe slope ofthe straightine par(exponential phase)
othe growth curve by plotingthelogarthm of cel mass concentration versus fermentation time Cell mass yield (Yx /s) was calculated as grams of cell mass obtained per
gram of glucose or glycerol consumed at the end of the
batch culture [14]
3 Resu l ts and D iscuss ion
3.1 Comparison of growth kinetics and extracellular
metaboltes of R oryzae ATCC 9363 growing on glycerol and glucose at 30°C
To compare the growth and extracellular metaboltes of
R oryzae ATCC 9363 on glucose and glycerol media, spores were directly culivated in the media with various
iniial concentrations of glucose and glycerol (10, 30, 50, and 70 g/L) at aixed nirogen concentration(1 g/L NH4C The speciic growth rates of 0.6 /hremained constant as
glucose concentration varied, whereasthe highest cel mass
yield(Yx /s) of 0.3 g/g was obtained atthelowest concentration
of 10 g/L (C·N-1ratio of 17.8 mol/mol) (Fig S1A and Table 1) Lactic acid accumulation started at 44 h and peaked at 20 g/L at 89 h withthe 70 g/Lglucose concentration (C/N ratio of 124.8 mol/mol) (Fig S1E) Compared with
glucose culivation, glycerol concentration exerted diferent
Trang 3efects onthe growthrate and cel mass yield(Table 1 and
Fig S1C) The speciic growthratesremained at 0.1/hfor
the cultures with glycerol concentrations above 30 g/L,ive
times higher than the rate of 0.02/h with 10 g/Lglycerol
Howeverthe highest cel mass yield(0.4 g/g) was obtained
wth 10 g/L glycerol, which may be caused by the low
iniial C/N ratio (17.4 mol/mol) in the culture medium
The overall cell mass yields were higher on glycerol
than on glucose, althoughthe overal speciic growthrates
on glycerol were dramaticallylowerthanthose on glucose
(Table 1) Thisresul can beinterpreted by comparingthe
products obtainedrom two carbon sources.In contrastto
glucose fermentation, almost no extracellular metaboltes
such as acetate, formate, or lactic acid were detected in
glycerol cultures This meansthatthe majorty of glycerol
was utized by the strain for propagation, resuling in a
higher cell mass yield
To date, limited studies have been conducted on the
glycerol conversionforlactic acid accumulation by Rhizopus
sp, although there are reports on glycerol metabolism by
bacteria[14-16] yeast[17,18] and somefungi[4-6] These
studies concluded that there were two main metabolic
pathways of glycerol utization: 1) the glycerol is phos
-phorylated by glycerol kinasetoform glycerol-3-phosphate
(G-3-P), and the G-3-P is dehydrogenated by G-3-P
dehydrogenasetoform dihydroxyacetone phosphate; 2)the
glycerol is dehydrogenated by glycerol dehydrogenase,
followed bythe phosphorylation of dihydroxyacetone Both
pathways are connected to the formation of pyruvate and
merged into the central metabolic pathway [14,16,18,19]
Therefore,lactic acid produced by R oryzae culivated on
glycerol was expectedto be synthesizedrom pyruvate by
lactic acid dehydrogenase(LDH) similarlytothe glucose
-pyruvate-lactic acid pathway.Interestingly,the experimental
data show that no lactic acid was detected in the broth
when glycerol was used as the sole carbon source This
result might be because glycerol has a higher degree of
reduction per carbonthan glucose, withtheresulthatlactic
acid accumulation from glycerol may not be preferred
under aerobic condiions The average degrees ofreduction
per carbon in glycerol (κg lycero l) and lactic acid (lac t ic ac id)
are 4.7 and 4.0, respectively [14,20] The pathway where
lactic acid is the final product cannot achieve redox
balance like the pathway from glucose (κg lucose= 4.0) to lactic acid [20] This is also implied by Fig 1 [16] For
glucose fermentation, 1 mole of redox power synthesized
from 0.5 mole of glucose to 1 mole of pyruvate can be consumedin producing 1 mole oflactic acidrom pyruvate reduction, and the redox power can be balanced by the
glucose-pyruvate-lactic acid pathway Howeverthe pathway
from 1 mole of glycerol to 1 mole of pyruvate generates
2 moles of redox power [14,20], whereas only 1 mole of redox power is consumed in the reduction of pyruvate to lactic acid As the redox power isn’t balanced in the
glycerol-pyruvate-lactic acid pathway, this pathway is not favored.tis also diiculto balancetheredox powerrom
glycerol to the cell mass as the degree of reduction per
carbon in the fungal cell mass is 4.2 (cell mass formula,
CH1 7O0 6N0 1) [21] Therefore, to achieve the balance of redox power, under aerobic condiions glycerol utization
prefersthe pathway with CO2 astheinal product sothat the excessredox powerromthe synthesis ofthe cel mass and other housekeeping metaboltes can be completely consumed
3.2 Lactic acid dehydrogenase activity
A study on lactic acid over-production by the R oryzae
Tab le 1 G row th k ine t ics o f oryzae in g lyce ro l and g lucose med ia a t 30 ºC *
10 g /L 30 g /L 50 g /L -1 70 g /L 10 g /L 30 g /L 50 g /L 70 g /L
C /N (mo l /mo l ) 17 4 52 3 87 1 122 0 17 8 53 4 89 0 124 8
* Da po in ts rep resen t the means o f two rep l ica tes
F ig 1 Gene ra t ion and consump t ion o f reduc ing equ iva len ts du r ing the conve rs ion o f g lyce ro l and g lucose B roken l ines rep resen t
mu l ip le s teps H , NADH /FADH 2 /QH 2 ; PEP , phosphoeno lpy ruva te ; PYR , py ruva te
Trang 4mutant by Bai et al showedthatthe pyruvate node wasthe
pincipal nodeinthelactic acid pathway andindicatedthat
pyruvate-to-ethanol and pyruvate-to acetyl CoA are the
two major pathways competing withthe pyruvate-to-lactic
acid pathway for pyruvate [22] Nicotinamide adenine
dinucleotide(NAD+) dependentlactic acid dehydrogenase
(LDH, EC 1.1.127) and pyruvate dehydrogenase(PDH) are
the primary enzymes responsible for convering pyruvate
into lactic acid and acetyl CoA, respectively [23] David
e al elucidatedthatthe activity of PDH was signiicantly
up-regulated in glycerol metabolism compared to glucose
metabolism[24] Consideringthat no extracellular ethanol
was detected in glycerol culture (data not shown), it is
assumed that most pyruvate is converted into acetyl CoA
by the up-regulated PDH and that less pyruvate can be
accessed by LDH Moreover Mazumdar et al observedthat
LDH could not be activated atlow pyruvate concentrations
in vivo[3] Therefore,the LDH activity andtheintracellular
pyruvate level were predicted to be crical to the fungal
lactic acid accumulation from glycerol
As shownin Table 2,lactic acid accumulation on glucose
mediumincreased astheintracellular LDH activityincreased
The lactic acid concentration reached its maximum at
0.8 g/L, with the highest LDH activity of 104 U/g dry
biomass While R oryzae culivation on glycerol medium
did not produce any detectable lactic acid during the
fermentation, minor LDH activity(14.5 U/g) was observed
at 66.5 h of the culture (Table 2), demonstrating the
presence ofintracellular LDH enzyme atthe beginning of
the exponential growth phase(Table 2 and Fig S1c)I was
hypothesizedthat in vivo LDH may not be activated dueto
thelow concentration of accessible pyruvateresulingrom
the up-regulated pyruvate dehydrogenase complex As a
resul, no lactic acid was produced in vivo In the in vitro
assay, however, an excess of pyruvate and NADH were
supplied in the reaction system, enabling LDH to be
activated and reduce pyruvate to lactic acid with NADH
The undetectable LDH activity after 66.5 hisikely because
extremely weak activity cannot be detected due to the
limited sensiivity ofthis method Overalthe experimental
results, along with the observations from David et al and Mazumdar et al., suggest that the accessibity of LDH to
pyruvate is the key to achieving lactic acid accumulation
from glycerol Thus,the effects of culturetemperature and
pyruvate concentration onfungal glycerol metabolism were
studiedtofurther verfytherole of pyruvate and LDH during lactic acid accumulation
3.3 Effects of temperature on glycerol utization and lactic acid synthesis under aerobic condiions
The optimal culturetemperature of R oryzae ATCC 9363
for lactic acid accumulation on glucose medium has been reported to be approximately 27 ~ 30ºC [7] As most enzymes in vivo are sensiive to temperature changes, the expression and activity of the intracellular enzymes LDH and PDH were expectedtorespond diferentlyto changes
in temperature Our hypotheses were that under elevated temperature, LDH and PDH might beregulated at diferent levels and directthe glycerol pathwaystowardslactic acid accumulation Totestthis hypothesis, R oryzae was cultured
on both glycerol and glucose media at an elevatedtemperature
of 37oC
Inthe R oryzae culture on glucose medium,the highest speciic growthrate µmax was 0.2/h, andthe maximum cel mass yield Yx /so 0.3 g/gwas obtained attheiniial glucose concentration of 10 g/L(iniial C/Nratio of 17.8 mol/mol (Fig 2A and Table 3) The cell mass yield at 37oC on
glycerolremained similar comparedto glycerol cultures at
30oC whereasthe average growthratein glucose medium
signiicantly decreasedto only 21% ofthat at 30oC(Tables 1 and 3) A signiicant diference was also observedinlactic
acid accumulation between the two temperatures The maximum lactic acid concentration of 11 g/L observed at
37oC was only 55% of the highest concentration (20 g/L)
at 30oC (Figs S1E and 2C)
Similarly to glucose fermentation, higher temperature considerablyreducedthe average growthrate of R oryzae
in glycerol medium by nearly 63%(Tables 1 and 3, Figs 2D and S1C) Interestingly, at 37°C, the cell mass yield was improved by 33, 50, and 100% and maximum lactic acid
Tab le 2 LDH reduc t ive ac t iv i ty o f R oryzae in g lyce ro l cu l tu re a t 30 ºC *
In t race l lu la r LDH ac t iv i ty in 10 g g lyce ro l /L med ium
-In t race l lu la r LDH ac t iv i ty in 10 g g lucose /L med ium
LDH reduc t ive ac t iv i ty (U⋅ g -1 ) - - 17 2 51 9 104 0
* Da po in ts rep resen t the means o f two rep l ica tes
Trang 5concentrations were 0.36, 0.6and 0.26 g/Lin 30, 50and
70 g/Lglycerol media,respectively(Fig 2F) Afterreaching
the maximum, lactic acid concentrations leveled of
(Fig 2F) with continued glycerol consumption (Fig 2E)
suggesting that lactic acid can act as a carbon source for
R oryzae [13]and can be used simultaneously with glycerol
Signiicantly increased cell mass yields and decreased
growth rates in glycerol media at 37oC indicate that less
F ig 2 Inves t iga t ion o f the g row th k ine t ics , subs t ra te u t iza t ion , and lac t ic ac id p roduc t ion o f R oryzae in g lucose and g lyce ro l med ia a t
37 ºC (A ) G row th cu rve in g lucose med ium , (B ) g lucose u t iza t ion, (C ) lac t ic ac id accumu la t ion in lucose med ium , (D ) g row th c u rve in
g lyce ro l med ium , (E ) g lyce ro l u t iza t ion , (F ) lac t ic ac id accumu la t ion in g lyce ro l med ium Da ta po in ts rep resen t the means o f two rep l ica tes
Tab le 3 G row th k ine t ics o f oryzae in g lyce ro l and g lucose med ia a t 37 ºC *
10 g /L 30 g /L 50 g /L 70 g /L 10 g /L 30 g /L 50 g /L 70 g /L
In i ia l C /N (mo l /mo l ) 17 4 52 3 87 1 122 0 17 8 53 4 89 124 8
µ max /h ) 0 03 0 03 0 03 0 03 0 2 0 1 0 1 0 1
Y x /s (g /g ) 0 4 0 4 0 36 0 6 0 3 0 1 0 1 0 1
* Da po in ts rep resen t the means o f two rep l ica tes
Trang 6carbon from glycerol was oxidized to carbon dioxide at
highertemperature andthatthe TCA cycle andrespiratory
chain were parially inhibited at higher temperature [25]
Therefore, the abity of LDH to compete with other
pyruvate branch point enzymes such as PDHfor pyruvate
may beindirectlyimproved Correspondingly,thelactic acid
accumulation was enhanced
3.4 Enhancement oflactic acid accumulation on glycerol
medium by increasing the intracellular pyruvate level
The results of the previous section also suggest that the
intracellular pyruvate concentration would be crical for
fungal lactic acid synthesis by metabolizing glycerol To
confrm, sodium pyruvate was dosedintothe culture media
every 24 h atthreelevels of 0, 40, and 80 mmol/Lfor both
30 and 37°C cultures (Fig 3)
When sodium pyruvate was added, lactic acid was
detectedinthe 30oC culture beginning at 70 h Atthe end
of the fermentation (146 h), the lactic acid concentrations
in the cultures supplied with 40 and 80 mmol/L sodium
pyruvatereached 0.67 and 0.44 g/L(Fig 3A)respectively
Forthe 37oC culture,thelactic acid accumulation occurred
a 50 hforthe culture containing sodium pyruvate, around one day earier than the control culture without sodium
pyruvate The maximumlactic acid concentrationsreached
1.33 and 1.0 g/Lfor 40 and 80 mmol/L sodium pyruvate
treatments, which were enhanced by 2 and 1.4imesrom the control, respectively (Fig 3C) At both temperatures, the culture with 40 mmol/L sodium pyruvate exhibited better lactic acid synthesis than 80 mmol/L sodium pyruvate,
which might be due to the negative impact of the high sodiumion concentration onfungal cells Moreover adding sodium pyruvate improved glycerol utization at both temperatures(Figs 3B and 3D) paricularly at 37oC where
glycerol consumption wasincreased by 17.5 and 12.3%in the presence of 40 and 80 mmol/L sodium pyruvate compared
wththe controls,respectively.In addiion, R oryzae culture
on pyruvate as the sole carbon source yielded almost no
growth at both temperatures (data not shown) Therefore, the intracellular level of pyruvate is crical for lactic acid synthesis from glycerol by R oryzae These results also indicate that lactic acid production could be improved by
ether increasingthe LDH expression level to divert more
pyruvatetolactic acidrom other branch points andimprove
F ig 3 Inves t iga t ion o f lac t ic ac id accumu la t ion when sod ium py ruva te is added to the cu l tu re T rea tmen t A : 40 mM sod ium py ruva te was added to the b ro th eve ry 24 h T rea tmen t B : 80 mM sod ium py ruva te was added to the b ro th eve ry 24 h T rea tmen t C : con t ro l
t rea tmen t w i thou t the add i ion o f sod ium py ruva te The same med ium was used fo r a l t rea tmen ts (A ) Lac t ic ac id accumu la t ion a t 30 ºC , (B ) g lyce ro l u t iza t ion a t 30 ºC , (C ) lac t ic ac id accumu la t ion a t 37 ºC , (D ) g lyce ro l u t iza t ion a t 37 ºC The da ta po in ts rep resen t the means
o two rep l ica tes
Trang 7the conversion eficiency oflactic acid accumulation[23]
or by parially inhibiing PDH by knocking down certain
genes so that more pyruvate is available for LDH to
produce lactic acid
Thistudy evaluatedthe possibity oflactic acid production
from glycerol bythelamentousfungus oryzae Dueto
the higher degree of reduction per carbon of glycerol, the
TCA cycle and the respiratory are preferred in glycerol
metabolism over the pathway of lactic acid accumulation,
andthe LDH accessibity of pyruvate might beimited By
increasingthe culturetemperaturerom 30to 37°C,lactic
acid accumulation was achieved on glycerol medium,
probably duetothe parialinhibiion ofthe TCA cycle and
therespiratory chain at an elevatedtemperature The addiion
of sodium pyruvate enhanced the lactic acid production
by the culture on glycerol medium, demonstrating the
importance of an accessibleintracellular pyruvate poolfor
LDH in lactic acid synthesis
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