Journal

J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Prroductiion of Succini
S ic Acid
d: Effects of C
C:N Ratio

Tatianee Araujo Go
onzales
U
UNICAMP - University of Campiinas, DEA/F
FEA - Schoo
ol of Food E
Engineering
g,
130
083-862 Caampinas, São Paulo, Brrazil
E-mail: tatigoonzales1@y
yahoo.com.br

Lucielenn Oliveira do
os Santos
FUR
RG - Federall University
y of Rio Graande, Colleg
ge of Chemistry and Foood Engineeering,
96203-900,, Rio Grand
de, RS, Brazzil
E-mail:
E santtoslucielen@
@gmail.com
m

Beatrriz Torsani Ubeda

U
U
UNICAMP - University of Campiinas, DEA/F
FEA - Schoo
ol of Food E
Engineering
g,
130
083-862 Caampinas, São Paulo, Brrazil
E-mail: biaa_ubeda@h
hotmail.com
m

Ranuulfo Monte Alegre

A
UNICAMP - University of Campiinas, DEA/F
U FEA - Schoo
ol of Food E
Engineering
g,
130
083-862 Caampinas, São Paulo, Brrazil
E-mail: rranulfo@un
nicamp.br

Rossana Goldbeeck (Corresp

ponding autthor)
U
UNICAMP - University of Campiinas, DEA/F
FEA - Schoo
ol of Food E
Engineering
g,
130
083-862 Caampinas, São Paulo, Brrazil
Tel: 55-193
3-521-40388 E-mail: goldbeck@
g unicamp.brr
htttps://orcid.oorg/0000-00
003-2953-80
050

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 Journal
J of A
Applied Bioteechnology
ISSN 2327-0640
2
2019, Vol. 7, No. 2

Receiveed: May 30, 2019 Accepted:

A A
August 5, 2019 Pu
ublished: Auugust 8, 2019
9
Doi: 100.5296/jab.vv7i2.14867 URL: https://doi.org/10.5296
6/jab.v7i2.114867

Abstract
In this work, the effects
e of different
d carrbon:nitrogeen (C:N) ratios, carbonn source an nd initial
carbon concentration, in liquid d medium uused for sub bmerged ferrmentation, on biomasss growth,
succinicc acid prodduction and yield of prroduct in su ubstrate (YP/S) were sttudied. It was
w used
three caarbon sourcces as subsstrate such as glucose,, xylose an nd sucrose aand the C:N N ratios
studied were in thee range of 2.5-35.
2 Thee succinic acid producttion was beest at a C:N ratio of
12.5:1, as much foor the succin nic acid prooduction as for the yielld of produc
uct in relatioon to the
substratte (YP/S). For
F the biom mass growtth the C:N N ratios of 2.5:1, 5:1 aand 10:1 had h only
presenteed significaant differennce with reegards to th he too mucch assays. The substrrate that
presentss the best reesults was th
he glucose w nic acid conccentrations uup to 7.5 g..L-1.
with succin
Keywords: C:N raatio, Actinob
bacillus succcinogenes, Succinic accid
1. Introoduction
Succiniic acid, alsso known as a “amber acid”, is a C4 lineaar saturatedd dicarboxy ylic acid
produceed as an intermediat
i e of the ttricarboxyliic acid cyccle and allso as one of the
fermenttation produucts of anaeerobic metabbolism (Zeikus et al., 1999; Song & Lee, 2006).
The aciid and its derivatives
d have receiived consid derable atten ntion in thee last two decades
owing to their usse as speciialty chemiicals for ap pplications in food, ppharmaceutiical and
cosmetiic industriess (Zeikus ett al., 1999; Guettler ett al., 1996; Vemuri et aal., 2002). ItI is also
used as surfactant, detergent extender,
e anntifoam ageent, ion chellators, and iindustrially
y used as
raw maaterial for important chemicals such as: 1,4 1 butaned diol, N-metthyl pyrrollidinone,
gamma-butyrolactoone, adipic acid, tetrahhydrofuran, 2-pyrrolidiinone, succcinate salts. (Zeikus
et al., 11999; Landuucci et al., 1994; Glasssner et al., 1995). Bey yond that, suuccinic acid d can be
used inn the syntheesis of biod degradable ppolymers su uch as poly ybutyrate suuccinate (PB BS) and
polyam mides (Nylonn), and thaat increases the deman nd for this product. (SSong & Leee, 2006;
Willke & Vorlop, 2004; Hon ng et al., 22000; Sriraam & Denn nis, 1999) and variou us green
solventss (Zeikus ett al., 1999; Song
S & Leee, 2006; Milllard et al., 1996).
Succiniic acid is mainly
m prod
duced by coonversion of o maleic anhydride
a tto succinic acid by
petrochhemical-baseed processees, which liimits the usse of succin
nic acid forr the wide range
r of
applicattions. Recenntly, due to
o the enviro nmental concerns and the conceppts of sustaiinability,
researchhes are direected towarrds the pro duction of succinic accid by micrrobial fermmentation
(Lee et al., 2003a; Lee et al., 2003b; Mcckinlay et al.,
a 2005; Agarwal
A et aal., 2006). It
I is also
notable that greennhouse gas CO2 is fixeed into succcinic acid during the fermentation, thus
providinng an additiional advanntage to the pprocess (So
ong & Lee, 2006).
2
Many ddifferent microorganismms have beeen screened
d and studieed for succinnic acid pro
oduction
from vaarious carboon sources. Among theem, Anaerobiospirillumm succinicipproducens (Nghiem
(

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et al., 1997), Acctinobacilluss succinoggenes (Glueettler et al., 1999; L Liu et al.,, 2008),
Mannheeimia succiniciproduceens (Lee et al., 2002), Enterococccus flavesceens (Agarwal et al.,
2007), Bacteroidees fragilis (Isar et all., 2006) and
a engineeered Escheerichia colii strains
(Vermuuri et al., 20002; Millard et al., 19966).
Bacteriaa A. succinoogenes and M. succinicciproducenss are the beest candidatees for succiinic acid
producttion as they produce suuccinic acid as a major fermentatio on product ((Song & Leee, 2006;
Bechthoold et al., 20008). Many ported in thee literature aabout these bacteria
y studies havve been rep
(Liu et al., 2008; Du
D et al., 200
07; Wan et al., 2008; Song
S et al., 2007).
2
Bacteriaa demand carbon, nitro ogen and othher nutrientts for syntheesis of its ceellular comp
ponents.
It is im
mportant thaat bacteria keep
k right equilibrium m between catabolism and anabolism for
their meetabolism to work effiiciently. Cattabolism prrovides enerrgy and the constituentts of the
main buuilding bloccks of the ceell, which aare in turn used for anab
bolic reactioons (Comm michau et
al., 2006).
Culturee medium iss usually immportant to the yield of
o any fermmentation prroducts, and d carbon
and nitrrogen sourcces generallly play a ssignificant role becausse these nuutrients are directly
linked wwith cell grrowth and metabolite
m bbiosynthesiss (Nghiem et al., 19977; Mao et all., 2005;
Casas LLópez et all., 2003; Leee et al., 1 999), moreeover its asssimilation is coordinaated and
controllled by the availability
y of both nnutrients (D
Doucette, et al., 2011; Commichaau et al.,
2006).
It is weell known that
t the carrbon to nitrrogen C:N ratio in thee medium iis one of the most
importaant factors that
t influen
nce the mettabolite pro oduction of many fermmentation prrocesses
(Rosfarrizan & Ariiff, 2000; Madihah
M et aal., 2001; Lin
L & Lay, 2004). How wever, the effect
e of
C:N rattio on the peerformance of succinic acid fermentation is not availablee in the literrature.
Thus beeing, the prresent work has as objeective to veerify which it is the beest C:N ratio
o on the
yield off biomass annd for the production oof succinic acid.
a
2. Mateerial and Methods
M
2.1 Miccroorganism
ms and Cultu
ure Medium
m
Actinobbacillus succcinogenes strain (CIP P 106512) was obtain ned from thhe Institute Pasteur
(Paris, FFrance). Thhe stock cullture was mmaintained in Trypticase soy agar slants at 4 °C. The
growth medium contained th he followinng (in g.L-1 of deionizzed water): yeast extraact, 5.0;
NaHCO O3, 10.0; NaaH2PO4.H2O, O 8.5; K2H HPO4, 15.5.. The pH of the mediuum was adjusted to
7.0 withh hydrochlooric acid aftfter autoclavving. Separaately autocllaved solutiion of carbo
ohydrate
(final cooncentrationn 10 g.L-1) was
w added aaseptically to t the mediu um after auutoclaving.
2.2 Cullture Condittions
Inoculuum was preppared by traansferring ccells agar sllant into 250 mL flaskk, containing
g 80 mL
of the ggrowth medium and inccubated for 16 h in an orbital shak king incubat
ator (Marconni, Mod.
MA 8300, Piracicabba, Brazil) at
a 150 rpm and 37 °C.. Batch ferm mentations wwere carrieed out in
butyl-ruubber-stopppered, 60 mL
m serum viials contain ning 30 mL medium w with CO2 as the gas

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phase. M
Media weree inoculatedd with 5% (vv/v) of inocculum in the same meddium and in
ncubated
at 37 °C
C and 150 rppm for 24 h.
h
2.3 Effeect of C:N Ratio
R of Liqu
uid Medium
m on Producction of Succcinic Acid
According to the chemical
c coomposition of the med dium mentiioned abovee, ten liquid media
with different C:NN ratios, rannge 2.5-35, were prepared for eaach substratte studied (glucose,
sucrosee and xylosee). The variaation of the concentratiions of the carbon
c and nitrogen so
ources in
the com mposition of
o each med dium, in eaach run is shown
s in Table
T 1. Thee concentraations of
carbon and nitroggen of the sugars annd yeast ex xtract, respectively, wwere determmined to
calculatte the C:N ratios requ uired. The carbon con ntent of glu ucose was 40% based d on the
-1
chemicaal formula of C6H12O6, so C (g.L ) con ncentration equals thee mass of glucose
-1
(g.L )**40%. The calculations
c s for sucrosse and xylosse were don ne the samee way. The nitrogen
n
-1
content of the yeasst extract waas 12.35%. The N (g.L L ) concentrration equalls the mass of yeast
-1
extract (g.L )*12.35%. Then, the yeast extract con ncentration in the meddium was fiixed and
the conncentration of glucose, sucrose aand xylose calculated consideringg the requirred C:N
ratio.

Table 1. Chemical compositio

on of the liquuid media with
w differen
nt C:N ratioos
Glucosee and Xylosse Sucroose CCarbon Yeast Extract
E N C:N
C
(g.L-1) -1
(g.L ) ((g.L-1) -1
(g.L ) (gg.L-1) raatio
3.859 3.667 1.544 5 0.6618 2.5:1
2
7.719 7.333 33.088 5 0.6618 5:1
5
10.00 9.500 44.000 5 0.6618 6.5:1
6
15.438 14.666 66.175 5 0.6618 10:1
19.297 18.332 77.719 5 0.6618 12.5:1
23.156 21.998 99.263 5 0.6618 15:1
30.875 29.331 12.350 5 0.6618 20:1
2
38.594 36.664 15.438 5 0.6618 25:1
2
46.313 43.997 18.525 5 0.6618 30:1
54.031 51.330 221.613 5 0.6618 35:1

2.4 Anaalytical Methhods

The nittrogen conteent of the yeast
y extracct was deteerminate by
y Hach® kiit. Cell grow
wth was
measureed as opticaal density at
a 660 nm (O OD660) wiith an ultrav
violet (UV) spectropho
otometer
(HACH H, Mod. DR/4000U,
D Colorado, USA). Dry D cell weeight was determined d using
precalcuulated standdard curves by substituuting OD6600 values.
The ressidual glucoose and sucrose concenntrations were measured in the ceell-free sup
pernatant
by a gluucose oxidaase-peroxidaase methodd using an enzymatic
e kit
k (Laborcliin, Pinhais, Brazil).
The ressidual xylosse concentraation was an
analyzed by colorimetriic method oof Somogyii-Nelson
(Somoggyi, 1952; Nelson,
N 1944 4).

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Succiniic acid concentratio

c on was qquantified by the enzymaticc kit Boehringer
Mannheeim/R-Bioppharm and high perfo formance liiquid chrom matographyy equipped with a
reverse phase column C18 (1550 × 4.6 mmm ID, 3 µm
m) with tempperature of 550 °C using
g 0.01 M
KH2PO O4 (pH 2.599, adjusted with phospphoric acid)) as mobilee phase witth flow ratee of 0.5
ml.mimm-1.
2.5 Dettermination of Kinetics Parameters
rs
The celll growth yield
y gar (YX/S), succinic accid yield on
on sug n glucose (Y YP/S), succiinic acid
yield onn biomass (Y
( P/X) and Succinic accid productiivity were calculated
c uusing the fo
ollowing
equationns:

𝑌 / (1)

𝑌 / (2)

𝑌 / (3)

𝑃 (4)

where: Xf is final biomass

b conncentration (g.L-1); Xi is initial bio
omass conccentration (gg.L-1); Sf
c on (g.L-1); S i is initial substrate
is final substrate concentratio s oncentrationn (g.L-1); SA
co Af is the
-1
final suuccinic acid concentratiion (g.L ) aand tf is the fermentatio
on time (h).
2.6 Stattistical Anallysis
Statisticca version 5.5
5 (Statsoft
ft, USA) sofftware was used to calculate the aanalysis of variance
v
(ANOV VA). Tukeyy test was used to ddeterminatee differencees betweenn samples using a
significcative level of
o 5%.
3. Resu
ults and Disscussion
According Mao ett al. (2005) and Casas López et all. (2003) th he carbohyddrates are im
mportant
carbon and energyy sources fo or the cell culture and
d their nature and quan
antity in thee culture
mediumm influence the cell gro
owth.
One of the most immportant facctors in opttimization of
o culture media
m on bioomass produ
uction is
the C:N
N ratio (Lin & Lay, 2004).
To datee the succinnic acid prod ductions wiith different C:N ratioss were not reported. Thus to
investiggate the effects of C:N ratio on succin nic acid production
p by Actino
obacillus
succinoogenes, in alla experimeents the quaantity of niitrogen in thhe medium m was held constant
and thee initial C content
c were varied uusing gluco ose, xylose or sucrose.. Nitrogen was the
limitingg nutrient inn all cases.
The Figgure 1 show
ws the maxim
mal biomasss productio
ons obtained
d in the eachh experimen
nt of the

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differennt carbon soources evalu

uated.
The maaximum bioomass produ uction from
m xylose occcurred in 48 8 h of fermeentation, su
ucrose in
36 h annd glucose inn 24 h. For all sugars, it was obseerved that th
he higher thee C: N ratio
o greater
biomasss is achieveed, indicatin
ng growth limmitation at low value of
o C:N ratioo.
The moost of the microorganis
m ms has inhiibition on cell
c grown in concentraations of thee carbon
source uup to 20 g.L-1. It veriffied, in the Table 1, thaat the carbo
on source cooncentrations used,
-1
from C:N 15:1 ratiio always are
a higher too 20 g.L , indicating
i th
hat the bactteria Actinoobacillus
-1
succinoogenes preseents no inhiibition of thhe growth in n concentraations up to 55.0 g.L , mainly
when xyylose is useed as carbonn source.
The maaximum succcinic acid production
p iin liquid medium withh different C
C:N ratios using
u the
three caarbon sourcces is shownn in the Figgure 2. The maximal succinic acidd productio ons were
-1
7.50, 4.04 and 3.993 g.L in n glucose, ssucrose and d xylose medium, resppectively and
a their
-1 -1
corresponding prodductivity weere 0.315, 00.112 and 0..08 g.L .h .

Figuure 1. Effectt of C:N rattio on biomaass production of Actinnobacillus su

succinogenees for
different caarbon sourcee (xylose), (sucrose)
( an
nd (glucose))

This lowwer acid prooduction, in

n medium w
with xylose oro sucrose, probably is due the kinnd of the
sugars uused. The sucrose is a disaccharidde that used
d to be hydrrolyzed in itts monosaccharides
before ffermented, being necesssary some metabolic effort,
e whilee the xylosee is metabo
olized by
other paathway.
Besidess, Van der Weff
W et al. (1997)
( in thheir studies showed thaat the use oof the more--reduced
sugars resulted inn the formaation of hiigher amou unts of succcinate, appproving thee results

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obtained in this woork, since itt were obtaained betterss results in the fermenntation with glucose
as substtrate than with
w xylose and sucrosee. Furtherm more, the succrose is no rreduce sugaar. Some
reports have show wed that thee initial suugar concenntration cou uld influencce cell grow
wth and
metabolites producction (Lee et
e al., 1999; Urbance et al., 2004; LiuL et al., 20008).
In the ffermentations, using glucose
g as ccarbon sourrce, the best C:N ratioos were betw ween of
12.5:1 aand 25:1 annd the higheest or lowesst C:N ratio
o resulted in
n relatively llower succiinic acid
producttion (Figuree 2). The highest
h succcinic acid production
p was obtainned at C:N ratio of
12.5:1. However, this
t was nott statisticallly different (p > 0.05) of
o the C:N ratios of 15 5:1, 20:1
and 25:1 (Figure 2).
The ressults indicatte that a hig 0-55 g.L-1) was unfavo
gh initial gluucose conceentration (40 orable to
formation of succcinic acid. Liu L et al. (2008) stud died the initial glucosse concentrration in
fermenttation by A.. succinogen nes CGMC CC1593. Thee authors ob bserved thaat concentraations up
-1
65 g.L inhibited the succinic acid prodduction and d biomass fo ormation. FFurthermoree, in this
study thhe authors used
u a differrent strain.
In the eexperiments using xylose as carbonn source, th he lower succcinic acid cconcentratio
ons were
obtained in low C: N ratio (2 2.5-5.0). Thhe others exxperiments showed
s conncentrationss around
3.8 g.L--1. Howeverr, this was not
n statisticaally differen
nt (p < 0.05)) from that oof the C:N ratios of
15:1, 200:1 and 25:11 (Figure 2)).
With reespect to thee experimen nts that the carbon sou
urce was sucrose, all exxperiments showed
little chhange in the maximum concentratiions of succcinic acid. The
T experim ments which the C:N
ratio rannged from 6.0 to 12.5 showed noo significantt difference (p < 0.05) between th hem, but
were different from m all others..
The ressults, in the Figure 2, showed thatt the tolerab ble limit of C:N
C ratio fo for the succiinic acid
producttion was noot too narrow w in view oof the best succinic
s aciid productioon was at C:N
C ratio
12.5:1, although was
w not stattistically diffferent of thet C:N ratios 15:1, 200:1, 25:1. Reduced
R
succinicc acid produuction at low
w C:N ratioo may be du ue the little available
a caarbon sourcee, and in
high C:N ratios theere is nitroggen deficienncy which liimits biomaass growth aand in consequence
the prodduction of succinic
s aciid (Bevilaqqua et al., 20015; Madih hah et al., 22001). Accoording to
Liu et al. (2008),, the nitrog gen source is responsible by thee best celluular growth h for A.
succinoogenes.
Makingg up a geneeral analysiis of all thee trials, wee can say th
hat the bestt C:N ratio
o for the
producttion of succcinic acid is in the raange of 10.0-20.0 and d the best ccarbon souurce was
glucosee.

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Figurre 2. Succinnic acid prod duction on m medium witth different C:N ratios.. * Columnss with
differeent letters reepresent sign
nificant diffference at P < 0.05 (onee-way ANO
OVA. Tukey y_HSD)

Table 2 summarizzes the yields (YP/S, Y X/S and YP/X

P ) and pro
oductivitiess obtained for
f each
carbon source evalluated.
The celll yield on sugar decreeased with an increasee of C:N raatios, probaably becausse initial
glucosee concentrattion increasee too with th
the increase of the C:N ratios. It w
was reported
d that the
osmoticc pressure caused by a high gllucose con ncentration might be detrimentall to the
metabolite biosynthhesis althou
ugh the cell growth was not inhibitted (Mao ett al., 2005).
The besst yield of product
p in reelation to thhe substratee (YP/S) had been foundd when gluccose was
used as carbon souurce with C:N ratios off 6.5:1 and 12.5:1, how wever there was not siggnificant
differennce betweenn them in the level of p < 0.05, beiing the yield d values aroound 65%.
Analyziing the Tabble 2, when n glucose w was used, high
h C:N ratios had ppresented reelatively
lower yyield than lowl C:N raatios, probaability by the
t microorrganism inhhibition. When
W the
carbon source usedd was sucro ose the yieldd YP/S decreeased with the
t increaseed of the raatio C:N,
indicatiing inhibitioon by substrate. Alreaddy in the ex
xperiments using xylosse as carbon n source
most triials in low C:N
C ratios showed
s yielld around 0.4gproduct/gsubbstrate.
It is appparent thatt the highesst succinic acid produ uction and productivity
p y were obtaained in
glucosee medium, whereas
w thee cell growtth in xylosee medium was
w preferreed. Different carbon
sourcess might have different effects of ccatabolic rep pression on the cellular
ar metabolissm (Mao
et al., 2005).
The results obtaineed from thiss study shouuld help dessign a betterr strategy foor the produ
uction of
succinicc acid by A. succinogennes.

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Table 22. Yields (Y

YP/S, YX/S and
a YP/X) aand productivities obtained for eeach carbon
n source
evaluateed

Parameeters
Succinic acid
Cell grrowth yield d Succinicc acid yield Succinic acid
yield onn biomass
on sugaar (YX/S) on glucoose (YP/S) productiviity
C:N (YP/X)
(gcell/gsuugar) (gSA/gsuggar) (gSA/gbiom
mass) (g.L-1.h-1)
Glu Xyl X Suc Glu X Xyl Suc Glu Xy yl Suc Glu Xyll Suc
2.5:1 0.11 0.05 0 0.07 0.52 00.62 1.06 4.7 12.3 14.7 0.07 0.05 0.11
5:1 0.11 0.16 0 0.07 0.54 00.43 0.55 4.9 2.8 8 7.7 0.17 0.07 0.11
6.5:1 0.10 0.12 0 0.10 0.64 00.39 0.38 6.1 3.3 3 4.0 0.26 0.08 0.10
10:1 0.08 0.14 0 0.08 0.54 00.46 0.35 6.9 3.2 2 4.2 0.25 0.08 0.10
12.5:1 0.09 0.12 0 0.08 0.64 00.39 0.28 7.2 3.3 3 3.4 0.32 0.08 0.10
15:1 0.09 0.14 0 0.07 0.59 00.43 0.26 6.6 3.0 0 3.8 0.29 0.08 0.09
20:1 0.08 0.22 0 0.07 0.47 00.58 0.21 6.3 2.5 5 3.1 0.29 0.08 0.09
25:1 0.06 0.14 0 0.05 0.40 00.37 0.15 7.1 2.7 7 3.0 0.30 0.08 0.09
30:1 0.09 0.23 0 0.06 0.52 00.60 0.21 5.1 2.5 5 3.3 0.26 0.08 0.10
35:1 0.07 0.11 0 0.06 0.37 00.29 0.16 5.2 2.5 5 2.7 0.24 0.08 0.09

4. Concclusion
The effe
fects of carbbon:nitrogenn ratios on ssuccinic aciid productio
on by submeerged cultiv
vation of
Actinobbacillus succcinogenes were invesstigated. Acccording to the results, the variatioon in the
C:N rattio on the biiosynthesis of succinicc acid showeed that a C:N ratio 12.55:1 is most suitable
for maxximum prodduction of su uccinic acidd in the three carbon soource studiedd.
Acknow
wledgmentts
The autthors acknoowledge to the
t CNPq (C Conselho Nacional
N de Desenvolviimento Cienntífico e
Tecnolóógico)/Brazil for the financial
f suupport. Thiss study wass financed in part by CAPES
finance code 001.
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nces
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