molecules

Article
Medium Optimization and Fermentation Kinetics
for κ-Carrageenase Production by
Thalassospira sp. Fjfst-332
Juanjuan Guo 1,2 , Longtao Zhang 1,2 , Xu Lu 1 , Shaoxiao Zeng 1 , Yi Zhang 1 , Hui Xu 1
and Baodong Zheng 1, *
1 College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350000, Fujian, China;
gjjfst15@163.com (J.G.); zlongtao@hotmail.com (L.Z.); luxuluxu88@163.com (X.L.); zsx1111@163.com (S.Z.);
zyifst@163.com (Y.Z.); xhuifst@163.com (H.X.)
2 Fujian Provincial Technical Research Center of Marine Food and Biological Products Processing,
Fujian Agriculture and Forestry University, Fuzhou 350000, Fujian, China
* Correspondence: zbdfst@163.com; Tel.: +86-591-8370-5076

Academic Editors: Pinarosa Avato and Jianbo Xiao

Received: 11 August 2016; Accepted: 31 October 2016; Published: 5 November 2016

Abstract: Effective degradation of κ-carrageenan by isolated Thalassospira sp. fjfst-332 is reported

for the first time in this paper. It was identified by 16S rDNA sequencing and morphological
observation using Transmission Electron Microscopy (TEM). Based on a Plackett–Burman design for
significant variables, Box–Behnken experimental design and response surface methodology were
used to optimize the culture conditions. Through statistical optimization, the optimum medium
components were determined as follows: 2.0 g/L κ-carrageenan, 1.0 g/L yeast extract, 1.0 g/L
FOS, 20.0 g/L NaCl, 2.0 g/L NaNO3 , 0.5 g/L MgSO4 ·7H2 O, 0.1 g/L K2 HPO4 , and 0.1 g/L CaCl2 .
The highest activity exhibited by Thalassospira sp. fjfst-332 was 267 U/mL, which makes it the
most vigorous wild bacterium for κ-carrageenan production. In order to guide scaled-up production,
two empirical models—the logistic equation and Luedeking–Piretequation—were proposed to predict
the strain growth and enzyme production, respectively. Furthermore, we report the fermentation
kinetics and every empirical equation of the coefficients (α, β, X0 , Xm and µm ) for the two models,
which could be used to design and optimize industrial processes.

Keywords: κ-carrageenase; Thalassospira sp.; response surface methodology; kinetics model

1. Introduction
Carrageenan, as a series of hydrophilic sulfated galactan multipolymers, is extracted from the red
seaweed (Rhodophyceaea). Carrageenan consists of D-galactose residues linked by alternating linear
chains of β-1,3-D-galactose (G) and α-1,4-linked-D-galactose (DA) [1]. Based on the number and
position of sulfate ester units (S) and the presence of 3,6-anhydro galactose-bridges, carrageenan can be
classified into κ-carrageenan (DA-G4S), ι-carrageenan (DA2S-G4S) and λ-carrageenan (D2S6S-G2S) [2].
κ-Carrageenan is composed of consecutive disaccharide units of β-D-galactose-4-sulfate and
3,6-anhydro-α-D-galactose, and it has been broadly applied in food, medicines, and biomaterials.
Carrageenan oligosaccharides are typically obtained through chemical hydrolysis [3], radiation [4]
or carrageenase degradation, and they were reported to show potential bioactivities including
anticoagulant, antithrombotic, antitumor and immunoregulation [3]. Chemical hydrolysis is the
most common method to prepare carrageenan oligosaccharides. However, the method may cause
damage to sulfate ester units and produce many complicated by-products, which limits it to industrial
application. The enzymolysis approach has become a hot topic in research due to its high efficiency,
specificity, and mild reaction conditions. According to this method, the main hydrophilic products are

Molecules 2016, 21, 1479; doi:10.3390/molecules21111479 www.mdpi.com/journal/molecules

Molecules 2016, 21, 1479 2 of 17
Molecules 2016, 21, 1479 2 of 17

are disaccharide, tetrasccharide, hexose, and octasaccharide [5]. Many carrageenases have been
reported to be
disaccharide, isolated from
tetrasccharide, marine
hexose, andbacteria such as[5].
octasaccharide Alteromonas [6], Cytophaga
Many carrageenases have [7],
beenVibrio [8],
reported
Pseudomonas carrageenanovora [9], and Cellulophaga [10]. However, the low
to be isolated from marine bacteria such as Alteromonas [6], Cytophaga [7], Vibrio [8], Pseudomonas activity and yield, along
with the instability
carrageenanovora of the
[9], and carrageenases,
Cellulophaga limit the utilization
[10]. However, of this
the low activity andprocess for fermenting
yield, along with the
carrageenan oligosaccharides. Therefore, it is imperative to search for
instability of the carrageenases, limit the utilization of this process for fermenting carrageenana bacterial strain that could
produce carrageenase
oligosaccharides. steadily itand
Therefore, is efficiently.
imperative to search for a bacterial strain that could produce
carrageenase steadily and efficiently. gene modification has been taken to improve the biological
Recently, physical induction or
fermentation
Recently, of carrageenase.
physical induction Kobayashi
or gene cloned a genehas
modification from
beenoriginal
taken bacteria
to improve Pseudoalteromonas
the biological
tetraodonis JAM-K142 for an enzyme named as Cgk-K142, but the productivity
fermentation of carrageenase. Kobayashi cloned a gene from original bacteria Pseudoalteromonas of Cgk-K142 was
rather low and the enzyme was very unstable during purification
tetraodonis JAM-K142 for an enzyme named as Cgk-K142, but the productivity of Cgk-K142 was rather [11]. Another recombinant
bacterium,
low and theGgkZ,enzymeyielded
was complex products
very unstable duringwhich were difficult
purification [11].further
Another purify [1]. Another
recombinant method
bacterium,
to enhance
GgkZ, yieldedthecomplex
bacterialproducts
enzyme whichyield iswere
by adding
difficultan enzymatic
further purifyinducer, whichmethod
[1]. Another is also toa low-cost
enhance
and valid approach to improve the microbial fermentation. For
the bacterial enzyme yield is by adding an enzymatic inducer, which is also a low-cost and example, lactose was found to
valid
increase the
approach ability ofthe
to improve recombinant bacteria BL21-HTa-cgkZ
microbial fermentation. For example,tolactose
degrade wasκ-carrageenan,
found to increase with theenzyme
ability
activity increased up to 10.76 U/mL after the optimization [12]. Agarase
of recombinant bacteria BL21-HTa-cgkZ to degrade κ-carrageenan, with enzyme activity increased upcould be induced by agarose,
neoagarobiose,
to 10.76 U/mL after neoagarotetraose
the optimization and [12].
neoagarohexaose,
Agarase couldbut be was inhibited
induced by D-glucose
by agarose, and D-
neoagarobiose,
galactose [13]. Meanwhile,
neoagarotetraose maltose was but
and neoagarohexaose, the most useful inducer
was inhibited for Aspergillus
by D-glucose niger to yield
and D-galactose [13].
glucoamylase [14], so is worthwhile to screen for proper inducers
Meanwhile, maltose was the most useful inducer for Aspergillus niger to yield glucoamylase for marine bacteria fermenting
[14],
carrageenase.
so is worthwhile to screen for proper inducers for marine bacteria fermenting carrageenase.
In this
In this study,
study, aa novel
novel strain
strain that
that could
could effectively
effectively hydrolyze
hydrolyze κ-carrageenan
κ-carrageenan has has been
been isolated
isolated
from dry Chondrus crispus for the first time. We attempted to increase the
from dry Chondrus crispus for the first time. We attempted to increase the κ-carrageenase activity byκ-carrageenase activity by
statistical optimization and determining the fermentation kinetics model.
statistical optimization and determining the fermentation kinetics model. In addition, two empirical In addition, two empirical
models were
models were proposed
proposed for for designing
designing and and optimizing
optimizing the the industrial
industrial process
process in in varied
varied environments,
environments,
which were simulated under different fermentation
which were simulated under different fermentation conditions. conditions.

2. Results
2. Resultsand
andDiscussion
Discussion

2.1. Isolation and Identification of κ-Carrageenase-Producing

κ-Carrageenase-Producing Bacterium
Bacterium
κ-Carrageenan, selected as the only carbon source, was used to isolate a potential potential bacterium
bacterium
which
which could degrade κ-carrageenan effectively.
effectively. Thereinto,
Thereinto, thethe strain
strain fjfst-332 exhibited the maximum
liquefaction
liquefaction andanddepression
depressionactivity on the
activity onplate
the medium (Figure 1).
plate medium The physiological
(Figure and biochemical
1). The physiological and
characteristics (Table 1) were
biochemical characteristics investigated
(Table 1) were according
investigated to according
Bergey’s Manual and Manual
to Bergey’s were analyzed
and were by
TEM (Figure
analyzed by TEM1). The PCR1).amplification
(Figure of the 16SofrDNA
The PCR amplification the 16Sgene
rDNA yielded a 1487-bp
gene yielded sequence
a 1487-bp of the
sequence
strain fjfst-332.
of the strain The The
fjfst-332. 16S 16S
rDNArDNAsequence
sequenceof fjfst-332 showed
of fjfst-332 showed98.7%
98.7%similarity
similaritytoto Thalassospira sp.
Thalassospira sp.
(Figure 2) based
based on on the
the public
public databases
databases (GenBank
(GenBank Accession
Accession Number
Number EU440790).
EU440790). Hence, it was
concluded thatthat the
the strain
strain was Thalassospirasp.
was Thalassospira sp.and
andnumbered
numberedasasFjfst-332,
Fjfst-332,which
whichis is a novel
a novel κ-
carrageenan-degrading species.
κ-carrageenan-degrading species.

Figure 1.
Figure 1. (A)
(A) The
The appearance
appearance ofof liquefaction
liquefaction and
and depression on plate
depression on plate medium
medium and
and (B)
(B) the
the TEM
TEM image
image
of Thalassospira sp.
of sp. Fjfst-332.
Fjfst-332.
Molecules 2016, 21, 1479 3 of 17

Molecules 2016, 21, 1479 3 of 17

Table 1. Physiological and biochemical characteristics.
Table 1. Physiological and biochemical characteristics.
Feature Results Items Results
Feature Results Items Results
Morphological
Morphologicalfeature
feature Biochemical
Biochemical Characteristics
Characteristics
mycelial
mycelialmorphology
morphology rhabditiform
rhabditiform oxidase
oxidase −−
gram
gramstain
stain positive
positive hydrogen
hydrogen peroxide
peroxide −−
spore
spore −− gelatin
gelatin liquefaction
liquefaction −−
flagellum
flagellum ++ amylolysis
amylolysis ++
Cultural Characteristics carrageenan dispergation +
Cultural Characteristics carrageenan dispergation +
colonial morphology depression, adhesion
colonial morphology depression, adhesion
bacterial colony smooth
bacterial colony smooth
borderline state irregular
borderline state irregular
pigment faint yellow
pigment faint yellow
demand for oxygen facultative anaerobic
demand for oxygen facultative anaerobic
“+” represent positive; “−” represent negative.
“+” represent positive; “−” represent negative.

99 Thalassospira tepidiphila 1-1B(T)（AB265822）

97 Thalassospira profundimaris WP0211(T)（AY186195）
Thalassospira povalilytica Zumi 95(T)（AB548215）
61 99 Thalassospira sp Fjfst-332
Thalassospira xiamenensis M-5(T)（AY189753）

100 Thalassospira permensis NBRC 106175(T)（AUNC01000051）

100
61 Thalassospira xianhensis P-4(T)（EU017546）
100 Thalassospira lohafexi 139Z-12(T)（GU584152）
Thalassospira lucentensis DSM 14000(T)（AM294944）
99 Thalassospira alkalitolerans MBE61(T)（AB786710）
80 Thalassospira mesophila MBE74(T)（AB786711）
Aestuariispira insulae AH-MY2(T)（KF876014）
Marispirillum indicum B142(T)（EU642410）

100 Novispirillum itersonii subsp. itersonii LMG 4337(T)（EF612765）

100 Novispirillum itersonii subsp. nipponicum LMG 7370(T)（EF612766）

0.01

Figure Phylogenetic
2. 2.
Figure treeofofThalassospira
Phylogenetictree Thalassospira sp.
sp. Fjfst-332 basedon
Fjfst-332 based on16S
16SrDNA
rDNAsequences
sequences and
and Neighbor
Neighbor
Joining
Joining analysis.
analysis.

2.2.2.2.
TheThe Effect
Effect of ofCarbon
CarbonSources
Sourceson
onthe
theEnzyme
Enzyme Activity
Activity

TheTheresults
results(Figure
(Figure3A) 3A)showed
showed thatthat the
the concentration
concentration of of κ-carrageenan
κ-carrageenancould couldimpact
impact thethe
effectivenessofofκ-carrageenase
effectiveness κ-carrageenase production
production bybystrain fjfst-332
strain [15]. In
fjfst-332 Figure
[15]. In 3A, the maximum
Figure activity
3A, the maximum
was observed
activity at 2.0 g/L
was observed at κ-carrageenan. There was aThere
2.0 g/L κ-carrageenan. sharp was
decrease in κ-carrageenase
a sharp activity with
decrease in κ-carrageenase
further increase of κ-carrageenan concentration, probably because a high
activity with further increase of κ-carrageenan concentration, probably because a high concentration concentration of κ-
carrageenan could cause excessive viscosity (Figure 3A) and restrict normal
of κ-carrageenan could cause excessive viscosity (Figure 3A) and restrict normal bacterial growth.bacterial growth. Other
carbon sources, shown in Figure 3B, also influenced the growth status and enzyme activity of strain
Other carbon sources, shown in Figure 3B, also influenced the growth status and enzyme activity
fjfst-332. On a base of 2.0 g/L κ-carrageenan, monosaccharides, disaccharides, and soluble starch were
of strain fjfst-332. On a base of 2.0 g/L κ-carrageenan, monosaccharides, disaccharides, and soluble
able to multiply strain growth, but the enzyme activities were not affected by these carbon sources.
starch were able to multiply strain growth, but the enzyme activities were not affected by these carbon
Interestingly, FOS, saccharose, and yeast extract could enhance the biomass and enzyme activity of
sources. Interestingly, FOS, saccharose, and yeast extract could enhance the biomass and enzyme
strain fjfst-332, respectively (Figure 3B). Yeast extract has generally been used to incubate bacteria,
activity of strain fjfst-332, respectively (Figure 3B). Yeast extract has generally been used to incubate
and FOS had been found to multiply on bifidobacterium [16,17]. Micromolecule substances, such as
bacteria, and cellobiose,
sophorose, FOS had been and found
lactoseto multiply
were easily on bifidobacterium
observed during the [16,17]. Micromolecule
fermentation, especiallysubstances,
with the
such
enzyme production [18]. As shown in Figure 3C, 1 g/L FOS could induce strain fjfst-332 to especially
as sophorose, cellobiose, and lactose were easily observed during the fermentation, yield κ-
with the enzyme
carrageenase production
effectively, [18]. As almost
in amounts shownthree
in Figure 3C, 1 g/L
fold higher thanFOS couldgroup.
the blank induce strainmore,
What’s fjfst-332
we to
yield
alsoκ-carrageenase effectively,
researched the influence of in amounts
specific almost three
components of FOS fold
onhigher than theactivity
κ-carrageenase blank group.
(FigureWhat’s
3D).
more, we also researched the influence of specific components of FOS on
GF4, the typical composition of FOS, was the optimal component for the enzyme activity of the κ-carrageenase activity
(Figure 3D).
isolated GF4, the In
bacterium. typical
othercomposition
reports, Zhouofhas FOS, was thethat
published optimal
glucosecomponent for the enzyme
was the supplementary activity
carbon
of resource
the isolated
for bacterium.
strain WZUC10 In other reports,κ-carrageenan,
to degrade Zhou has published thatnot
but it did glucose
explainwas the
the supplementary
mechanism for
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Figure
Figure
Figure Figure
3.3.(A)
Figure(A)
(A)
3. (A)3. Figure
(A)
Effects
3.Effects
(A)
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Effects Effects
of
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of 3.κ-carrageenan
(A)
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○ Viscosity; (B) Effects of other carbon sources on κ-carrageenase and biomass, including α-lactose
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Figure
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biomass,
(A)
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activity,
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including
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Viscosity;
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and
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and
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and α-lactose
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Molecules 2016, 21, 1479 5 of 17

2.3. The Effects of Nitrogen Sources, Metal Ions and NaCl on the Enzyme Activity
Both organic and inorganic nitrogen influenced the strain growth, and the nitrogen sources were
studied based on 2.0 g/L κ-carrageenan. As shown in Figure 3E, yeast extract was the most suitable
nitrogen source for κ-carrageenase production. The cell biomass was enhanced by increasing the
yeast extract concentration from 0 g/L to 5g/L (Figure 3F). The maximum activity was observed at
1 g/L yeast extract. However, when the concentration of yeast extract exceeded 1 g/L, the activity
of κ-carrageenase declined. Hence, the growth of the bacteria was induced by yeast extract, but the
activity of κ-carrageenase did not increase with the same trend. The results were similar to findings
on Vibrio sp. strain JT01017 [15], where polypeptone and yeast extract had the same effect on the
growth of the strain and the production of agarase. It also could be observed that when the ratio of
κ-carrageenan and yeast extract was lower than 2:1, the activity of κ-carrageenase obviously declined.
The reason may be that the κ-carrageenase was an induced enzyme for strain Thalassospira sp. Fjfst-332,
which only could be induced by the κ-carrageenan. When the concentration of yeast extract was high,
the κ-carrageenan-degrading function would be inhibited because of substrate competition.
The possibility of an artificial sea water impact on κ-carrageenase production and cell growth
was considered [19]. NaCl, NaNO3 , MgSO4 , 7H2 O, K2 HPO4 , and CaC12 were found to have
significant effects on catalyzing κ-carrageenan (Figure 3G). The results were consistent with a previous
report by Mou [20], showing the influence of these ions on κ-carrageenase production. In addition,
the concentration of NaCl also made a difference in the bacterial growth and the κ-carrageenan
hydrolysis function. The optimal concentration of NaCl was found to be 25 g/L (Figure 3H). The effect
of NaCl on strain fjfst-332 was similar to its effect on Pseudoalteromonas-like bacterium WZUC10 [2]
and Pseudoalteromonas sp. QY203 [21], where 25 g/L was the optimal concentration. Nevertheless,
there are also opposite conclusions showing that κ-carrageenase did not require NaCl for activity.
Wang reported that a new recombinant κ-carrageenase CgkS exhibited a high specific activity to
κ-carrageenan in the absence of NaCl [1].

2.4. Screening of Significant Variables by Plackett–Burman (PB) Design

A two-level PB design with 11 runs was arranged as shown in Table 2, and the results are
shown in Table 3. Figure 4 illustrates the Pareto Chart (p < 0.05) of significant effects. The t-values
of κ-carrageenan, NaCl, yeast extract, and FOS were better than the t-value limit (t > 2.36462),
which confirmed that the predicted value was significant and acceptable. According to the PB and the
Pareto Chart regression results, the lack of significant dummy factors for any of the responses indicated
that there were neither unknown variables nor systematic errors in the PB design. With respect
to enzyme activity, κ-carrageenan, NaCl, yeast extract, and FOS had significant effects (Figure 4A).
With respect to biomass, NaCl demonstrated significant negative effects (Figure 4B), which could mean
that the strain was sensitive to salt and low NaCl was ideal. In addition, κ-carrageenan, yeast extract,
and FOS were significant factors with positive effects on biomass (Figure 4B).

Table 2. Arrangement and experimental results of the PB design.

NO. A B C D E F G H D1 D2 D3 EA (U/mL) BM, (OD600 )

a
1 2.5 1.5 0.5 2 25 0.3 0.1 0.1 −1 1 −1 250.12 ± 1.27 0.428 ± 0.005 c,d
2 1 1.5 1.5 1 25 0.3 0.3 0.1 −1 −1 1 215.21 ± 1.17 f 0.402 ± 0.006 e
3 2.5 0.5 1.5 2 10 0.3 0.3 0.3 −1 −1 −1 234.12 ± 1.73 c 0.473 ± 0.011 b
4 1 1.5 0.5 2 25 0.1 0.3 0.3 1 −1 −1 181.25 ± 1.58 h 0.39 ± 0.008 e,f
5 1 0.5 1.5 1 25 0.3 0.1 0.3 1 1 −1 191.25 ± 2.93 g 0.385 ± 0.009 f
6 1 0.5 0.5 2 10 0.3 0.3 0.1 1 1 1 123.41 ± 1.38 0.418 ± 0.010 d
7 2.5 0.5 0.5 1 25 0.1 0.3 0.3 −1 1 1 245.28 ± 0.72 b 0.403 ± 0.008 e
8 2.5 1.5 0.5 1 10 0.3 0.1 0.3 1 −1 1 221.32 ± 2.01 e 0.471 ± 0.006 b
9 2.5 1.5 1.5 1 10 0.1 0.3 0.1 1 1 −1 231.55 ± 1.48 d 0.489 ± 0.012 a
10 1 1.5 1.5 2 10 0.1 0.1 0.3 −1 1 1 164.25 ± 1.53 i 0.431 ± 0.007 c
11 2.5 0.5 1.5 2 25 0.1 0.1 0.1 1 −1 1 253.32 ± 1.77 a 0.428 ± 0.010 c,d
12 1 0.5 0.5 1 10 0.1 0.1 0.1 −1 −1 −1 118.35 ± 0.71 0.419 ± 0.011 d
* Each experiment was performed in triplicate. Different small letters represent significant difference between
different groups of numbers (p < 0.05).
Molecules 2016, 21, 1479 6 of 17
Molecules 2016, 21, 1479 6 of 17

Table 3. The regression results for the PB design.

Table

Enzyme Activity Biomass

Enzyme Activity Biomass
Factor Coefficient Estimat Effects p-Value S Coefficient Estimat Effects p-Value S
Factor
A Coefficient
36.83Estimat Effects
73.66 p-Value
<0.0001 S ** Coefficient0.021
Estimat Effects
0.041 p-Value
<0.0001 S
**
BA 8.17
36.83 16.33
73.66 0.0138 ** **
<0.0001 0.0071
0.021 0.014
0.041 0.0084
<0.0001 **
**
CB 8.17
12.50 16.33
24.99 0.0138
0.0016 ** ** 0.0071
0.0066 0.014
0.013 0.0084
0.0119 **
**
DC 12.50
−1.38 24.99
−2.75 0.0016 ** 0.0066
−0.0008 0.013
−0.00017 0.0119 **
ED − 1.38
20.29 −40.57
2.75 <0.0001 ** −0.0008
−0.022 −0.00017
−0.044 <0.0001 **
FE 20.29
3.45 40.57
6.90 <0.0001 ** −0.022
0.0014 −0.044
0.0028 <0.0001 **
F 3.45 6.90 0.0014 0.0028
G 2.68 5.36 0.0011 0.0022
G 2.68 5.36 0.0011 0.0022
H
H 3.79
3.79 7.58
7.58 −0.0026
−0.0026 −0.0052
−0.0052
Dummy
Dummy −2.10
− 2.10 −−4.20
4.20 0.0021
0.0021 0.0042
0.0042
Dummy
Dummy −1.47
− 1.47 −−2.95
2.95 −0.0024
−0.0024 −0.0048
−0.0048
Dummy
Dummy 1.35 2.69
2.69 −0.0026
−0.0026 −0.0052
−0.0052
* Significant
* Significantatatpp << 0.05; ** Significant
0.05; ** Significant at at
p <p0.01.
< 0.01.

Figure 4. Pareto
Figure 4. Pareto Chart
Chart ofof Plackett-Burman
Plackett-Burman design
design for
for enzyme
enzyme activity
activity (A)
(A) and
and Biomass
Biomass (B).
(B).
A: κ-carrageenan, B: FOS, C: tryptone, E: NaCl.
A: κ-carrageenan, B: FOS, C: tryptone, E: NaCl.
In sum, κ-carrageenan, NaCl, yeast extract, and FOS were the significant variables for enzyme
In sum,
activity (EA) κ-carrageenan,
and biomass. The NaCl, yeast concentrations
optimal extract, and FOS were
were the significant
chosen for RSM variables
design as for enzyme
follows: κ-
activity (EA)(1,
carrageenan and
2, 3biomass.
g/L), NaClThe (15,optimal concentrations
20, 25 g/L), yeast extractwere(1, 2,chosen for RSM
3 g/L), and FOS (1,design as follows:
2, 3 g/L).
κ-carrageenan (1, 2, 3 g/L), NaCl (15, 20, 25 g/L), yeast extract (1, 2, 3 g/L), and FOS (1, 2, 3 g/L).
2.5. Model Statistical Analysis
2.5. Model Statistical Analysis
The RSM modeling results for EA and BM are presented in Table 4. The model’s R22 values were
The RSM modeling results for EA and BM are presented in Table 4. The model’s R values were
0.9689 (EA) and 0.9700 (BM), respectively, with a 95% confidence level. The experimental data fit the
0.9689 (EA) and 0.9700 (BM), respectively, with a 95% confidence level. The experimental data fit the
second order polynomial model (Equations (1) and (2)) well and described the relationship among κ-
second order polynomial model (Equations (1) and (2)) well and described the relationship among
carrageenan, NaCl, yeast extract, and FOS. Furthermore, the lack of fit was non-significant (EA
κ-carrageenan,
0.0888, NaCl,
BM 0.0598), yeast extract,
indicating that the and FOS.was
model Furthermore, the lack
acceptable (Table 5).of fit was non-significant (EA
0.0888, BM 0.0598), indicating that the model was acceptable (Table 5).
EA = 266.19 − 11.23X1 + 7.06X2 − 11.57X3 − 9.06X4 + 22.73X1X2 − 27.38X1X3 −
41.04X
EA = 266.19 −14.87X
1X 4 + 11.23X2X1 3++7.06X −4 11.57X
1.91X22X 3 −
− 44.73X 3X9.06X
4 − 22.03X 1 X2 −
12 − 23.43X
4 + 22.73X 22 − 41.11X
27.38X 1X323 −− (1)
41.04X1 X4 + 14.87X2 X3 + 1.91X2 X4 − 44.73X 49.78X 2 2 2
3 X4 − 22.03X1 − 23.43X2 − 41.11X3 −
4 2 (1)
49.78X4 2 3 + 0.00075X4 − 0.0009750X1X2 +
BM = 0.52 + 0.010X1 − 0.018X2 + 0.0007833X
0.00075X1X3 − 0.013X1X4 + 0.00005X2X3 + 0.012X2X4 + 0.00005X3X4 − 0.032X12 − (2)
BM = 0.52 + 0.010X1 − 0.018X 2+
0.082X 22 0.0007833X + 0.00075X
− 0.025X32 −30.013X 42 4 − 0.0009750X1 X2 +
0.00075X1 X3 − 0.013X1 X4 + 0.00005X2 X3 + 0.012X2 X4 + 0.00005X3 X4 − 0.032X1 2 − (2)
Table 4. Experiment design
2 and result2 of response 2surface analysis.
0.082X2 − 0.025X3 − 0.013X4
Run X1 X2 X3 X4 EA (U/mL) BM (OD600)
1 −1 −1 0 0 240.99 ± 1.33 0.405 ± 0.006
2 1 −1 0 0 174.26 ± 1.80 0.448 ± 0.010
3 −1 1 0 0 217.43 ± 0.86 0.386 ± 0.008
4 1 1 0 0 241.56 ± 1.71 0.39 ± 0.007
Molecules 2016, 21, 1479 7 of 17

Table 4. Experiment design and result of response surface analysis.

Run X1 X2 X3 X4 EA (U/mL) BM (OD600 )

1 −1 −1 0 0 240.99 ± 1.33 0.405 ± 0.006
2 1 −1 0 0 174.26 ± 1.80 0.448 ± 0.010
3 −1 1 0 0 217.43 ± 0.86 0.386 ± 0.008
4 1 1 0 0 241.56 ± 1.71 0.39 ± 0.007
5 0 0 −1 −1 150.97 ± 1.55 0.472 ± 0.011
6 0 0 1 −1 218.21 ± 1.42 0.481 ± 0.004
7 0 0 −1 1 217.54 ± 0.90 0.483 ± 0.023
8 0 0 1 1 105.86 ± 0.86 0.494 ± 0.006
9 −1 0 0 −1 180.70 ± 0.82 0.447 ± 0.005
10 1 0 0 −1 235.65 ± 1.38 0.488 ± 0.012
11 −1 0 0 1 250.14 ± 2.00 0.481 ± 0.008
12 1 0 0 1 140.93 ± 0.94 0.469 ± 0.008
13 0 −1 −1 0 235.93 ± 0.69 0.422 ± 0.006
14 0 1 −1 0 200.45 ± 1.07 0.387 ± 0.013
15 0 −1 1 0 188.06 ± 1.06 0.431 ± 0.020
16 0 1 1 0 212.07 ± 1.16 0.398 ± 0.009
17 −1 0 −1 0 194.38 ± 1.00 0.443 ± 0.009
18 1 0 −1 0 230.18 ± 1.21 0.451 ± 0.008
19 −1 0 1 0 220.04 ± 1.41 0.455 ± 0.015
20 1 0 1 0 146.33 ± 0.97 0.493 ± 0.021
21 0 −1 0 −1 185.89 ± 1.69 0.439 ± 0.011
22 0 1 0 −1 208.25 ± 0.49 0.380 ± 0.007
23 0 −1 0 1 163.21 ± 1.94 0.440 ± 0.008
24 0 1 0 1 193.42 ± 0.73 0.430 ± 0.005
25 0 0 0 0 270.32 ± 1.05 0.519 ± 0.018
26 0 0 0 0 271.85 ± 0.62 0.520 ± 0.020
27 0 0 0 0 269.09 ± 0.97 0.521 ± 0.015
28 0 0 0 0 260.33 ± 1.12 0.513 ± 0.011
29 0 0 0 0 259.36 ± 0.89 0.516 ± 0.017
* Each experiment was performed in duplicate.

Table 5. ANOVA for response surface polynomial model of all independent variables.

EA Biomass
Factor
SS F-Value Pr > F SS F-Value Pr > F
X1 1513.02 6.577711 0.0027 ** 0.00124 29.75 <0.0001 ***
X2 597.81 0.795231 0.0383 * 0.00382 91.54 <0.0001 ***
X3 1607.21 6.359724 0.0022 ** 0.00074 17.66 0.0009 **
X4 985.64 4.124658 0.0108 * 0.000675 16.19 0.0013 **
X1 X2 2065.79 11.36978 0.0008 ** 0.00038 9.12 0.0092 **
X1 X3 2997.89 12.10719 0.0002 ** 0.00023 5.40 0.0357 *
X1 X4 6736.72 46.20569 <0.0001 *** 0.00070 16.85 0.0011 **
X2 X3 885.00 6.160962 0.0147 * 0.000001 0.024 0.8791
X2 X4 14.64 0.560155 0.7258 0.00060 14.40 0.0020 **
X3 X4 8003.63 43.95054 <0.0001 *** 0.000001 0.024 0.6521
X1 2 3148.00 11.63744 0.0001 0.00656 157.43 <0.0001
X2 2 3562.08 23.78141 <0.0001 0.043 1041.34 <0.0001
X3 2 10,960.79 63.65951 <0.0001 0.00407 97.70 <0.0001
X4 2 16,077.00 94.12138 <0.0001 0.00115 29.75 0.0001
Model 49,077.94 19.7224 <0.0001 *** 0.054 92.78 <0.0001 ***
Residual error 1600.21 0.000583
Lack of fit 1461.78 3.162004 0.0888 0.000521 3.32 0.0598
Pure error 138.43 0.000063
Cor Total 50,678.15 0.055
* Significant at p < 0.05; ** Significant at p < 0.01; *** Significant at p < 0.001.

2.6. Effect of Independent Variables on Enzyme Activity (EA) and Biomass (BM)
The positive linear effects of X1 (κ-carrageenan) and X3 (yeast extract) on EA were found to be
highly significant (p < 0.01), and the effects of X2 (NaCl) and X4 (FOS) were significant (p < 0.05),
Molecules 2016, 21, 1479 8 of 17

which agrees with the conclusion of the PB analysis. Furthermore, all the quadratic effects of X1 2 ,
2 , X 2 , and
X2Molecules 2016, X21, 21479 8 of 17
3 4 were found to be significant (p < 0.05) and negative in value, which accounts for
the equation of the parabola function decreasing and the maximum point. The results of the analysis
of the parabola function decreasing and the maximum point. The results of the analysis of variance
of variance (ANOVA) indicated that the reciprocities of X1 X2 , X1 X3 , X1 X4 , and X3 X4 were highly
(ANOVA) indicated that the reciprocities of X1X2, X1X3, X1X4, and X3X4 were highly significant (p <
significant (p < 0.01), and the reciprocity of X2 X3 was significant (p < 0.05) for κ-carrageenase activity
0.01), and the reciprocity of X2X3 was significant (p < 0.05) for κ-carrageenase activity (Table 5). The
(Table 5). The strain biomass was most affected by κ-carrageenan and NaCl (p < 0.001), followed by
strain biomass was most affected by κ-carrageenan and NaCl (p < 0.001), followed by yeast extract
yeast
and FOS (pand
extract FOS All
< 0.05). (p <of0.05). All of the quadratics
the quadratics were significant
were significant items The
items (p < 0.01). (p < reciprocities
0.01). The reciprocities
of X1X2,
of XX11XX3,2X
, 1XX14X 3 , XX
, and 1X 4 4, were
2X
and Xhighly
2 X4 were highly(psignificant
significant < 0.01), yet(p < 0.01),
those of X2Xyet those of X2 Xnon-significant
3 and X3X4 were 3 and X3 X4 were
non-significant
(p > 0.05). (p > 0.05).
Enzyme
Enzyme activityincreased
activity increasedwith withthe
the four factors, with
four factors, withits
itspeak
peakactivity
activityapproaching
approaching thethe midpoint
midpoint
of of
thetheresponse
responseplot. plot.TheTheinteraction
interaction ofof κ-carrageenan
κ-carrageenan and andNaCl
NaClwas washighly
highlysensitive
sensitiveto to
EA, EA, indicating
indicating
that
thatEA EAincreased
increasedwhen whenthey theyincreased.
increased. However,
However, highhigh concentrations
concentrationsofofκ-carrageenan
κ-carrageenan andand NaCl
NaCl
restrained
restrainedEA EA(Figure
(Figure5A). 5A). Similarly,
Similarly, EAEA increased
increased asas yeast
yeast extract
extractand andFOSFOSincreased,
increased, butbuthighhigh
concentrationsled
concentrations ledto to low
low EAEA (Figure
(Figure5B,C,F).
5B,C,F).κ-carrageenase
κ-carrageenase activity waswas
activity negatively influenced
negatively by
influenced
byNaCl
NaCland andFOS FOS(Figure
(Figure5E). 5E).TheThebiomass
biomassfollowed
followed similar
similar trends
trendsas as
EAEA (Figure
(Figure 5G–L).
5G–L).A A highhigh
concentration of NaCl inhibited BM but made enhanced EA, so the
concentration of NaCl inhibited BM but made enhanced EA, so the optimal amount was determinedoptimal amount was determined
to to
bebe2020 g/L g/L[2].
[2].

Figure 5. (A–F)
Figure waswas
5. (A–F) the interaction effecteffect
the interaction of variables on enzyme
of variables activityactivity
on enzyme (EA); (G–L)
(EA);was the was
(G–L) interaction
the
effect of variables
interaction effecton biomass (BM).
of variables on biomass (BM).
 Molecules 2016, 21, 1479 9 of 17
Molecules 2016, 21, 1479 9 of 17

The RSM results showed that the ideal EA was 267.851 U/mL with the medium composition:
The RSM results showed that the ideal EA was 267.851 U/mL with the medium composition:
1.76 g/L κ-carrageenan, 20.04 g/L NaCl, 0.96 g/L yeast extract, and 1.02 g/L FOS. The maximum
1.76 g/L κ-carrageenan, 20.04 g/L NaCl, 0.96 g/L yeast extract, and 1.02 g/L FOS. The maximum
predicted value of BM was 0.521281 (OD600) at 2.17 g/L κ-carrageenan, 19.47 g/L NaCl, 1.09 g/L yeast
predicted value of BM was 0.521281 (OD600 ) at 2.17 g/L κ-carrageenan, 19.47 g/L NaCl, 1.09 g/L yeast
extract, and 1.08 g/L FOS. Combined with the actual operation and Plackett–Burman design, the test
extract, and 1.08 g/L FOS. Combined with the actual operation and Plackett–Burman design, the test
was executed five times using the simplified medium: 2 g/L κ-carrageenan, 20 g/L NaCl, 1 g/L yeast
was executed five times using the simplified medium: 2 g/L κ-carrageenan, 20 g/L NaCl, 1 g/L yeast
extract and 1 g/L FOS, and the average EA was 265 ± 1.56 U/mL. Compared with bacteria investigated
extract and 1 g/L FOS, and the average EA was 265 ± 1.56 U/mL. Compared with bacteria investigated
in previous reports—Pseudoalteromonas sp. WZUC10, 50 U/mL[2] and ALAB-001, 170 U/mL [8], strain
in previous reports—Pseudoalteromonas sp. WZUC10, 50 U/mL [2] and ALAB-001, 170 U/mL [8],
fjfst-332 showed the highest crude enzyme activity (EA).
strain fjfst-332 showed the highest crude enzyme activity (EA).
2.7. Verification
2.7. Tests
Verification Tests
ToToverify
verifythe thereliability
reliabilityofofthetheRSM
RSMmodels,
models,the thestrain
strainfjfst-332
fjfst-332enzyme
enzymeproduction
production andandcell
cell
growth curve were investigated under the optimized medium (Figure 6).
growth curve were investigated under the optimized medium (Figure 6). Strain fjfst-332 reached Strain fjfst-332 reached the
logarithmic phase at
the logarithmic 20 h and
phase increased
at 20 to the stationary
h and increased to the phase at 24 h.
stationary Consideration
phase the fermentationthe
at 24 h. Consideration
forms, the fermentation
fermentation forms, themodel of strainmodel
fermentation fjfst-332 was part
of strain of growth-related
fjfst-332 type II (α ≠ 0,
was part of growth-related β ≠II0).
type (α In
6= 0,
detail, the fastigia of bacterial growth (20 h) and metabolite accumulation
β 6= 0). In detail, the fastigia of bacterial growth (20 h) and metabolite accumulation (28 h) were not (28 h) were not
synchronous,
synchronous, andandthethe
fastigium
fastigium of of
biomass
biomass growth
growth waswasreached
reached faster than
faster thanthat
thatof of
κ-carrageenase.
κ-carrageenase.
After the stationary phase (32 h), the growth of metabolite (oligosaccharide)
After the stationary phase (32 h), the growth of metabolite (oligosaccharide) was smooth was smooth and steady,
and steady,
eventually accompanied by the catabolite stacking and κ-carrageenan
eventually accompanied by the catabolite stacking and κ-carrageenan consumption. The growth consumption. The growth ofof
κ-carrageenan oligosaccharide increased in stability in 58 h. After 62 h both
κ-carrageenan oligosaccharide increased in stability in 58 h. After 62 h both the enzyme production the enzyme production
and
andthethe
cellcell
biomass
biomass went
wentintointo
thethe
decline phase.
decline It might
phase. be the
It might beeffect of substrate
the effect consumption.
of substrate consumption.At
the fastigium, the maximum EA and BM yields were 266.84 U/mL and
At the fastigium, the maximum EA and BM yields were 266.84 U/mL and 0.519 (OD600 ), respectively,0.519 (OD 600), respectively,

which
whichmatched
matchedthe the predicted values with
predicted values withrelative
relativeerror
errorlessless
thanthan
2%.2%.Thus,Thus, the RSM
the RSM regression
regression models
models could predict the EA and BM yields for the medium composition
could predict the EA and BM yields for the medium composition of κ-carrageenan, NaCl, yeast extract,of κ-carrageenan, NaCl,
yeast
andextract,
FOS. and FOS.

300 0.5
280
260
240 0.4
220
Enzyme activity (U/mL)

200
180 0.3
Biomass (OD600)

160
140
120 0.2
100
80
60 0.1
40
20
0 0.0
0 10 20 30 40 50 60 70
Time (/h)

Figure
Figure 6. The
6. The cellcell
growth (▼)(Hand
growth ) and enzyme
enzyme activity
activity (curve
(■) ) curve
ofof strain
strain Thalassospira
Thalassospira sp.
sp. fjfst-332.
fjfst-332.

2.8.
2.8. Fermentation
Fermentation Kinetics
Kinetics
InIn order
order to to investigate
investigate thethe
cellcell growth
growth curve
curve and and product
product formation
formation curvecurve of strain
of strain fjfst-332,
fjfst-332, the
the logistic
logistic equation
equation and Luedeking–Piret
and Luedeking–Piret equation
equation were utilized
were utilized to the
to predict predict the experimental
experimental data, whichdata,
which are shown in Figure 7. The traditional fitting method appeared to be deficient because
are shown in Figure 7. The traditional fitting method appeared to be deficient because of only for of only
for each individual case, while the empirical model could be applied in various
each individual case, while the empirical model could be applied in various environments. environments.
Molecules 2016, 21, 1479 10 of 17

Figure 7. (A) is the model fitting of revolving speed relative to biomass, altering variables 80r/min,
Figure 7. (A) is the model fitting of revolving speed relative to biomass, altering variables 80r/min,
130 r/min and 180 r/min, quantifying temperature 30 ◦ C, and inoculum size 3 mL; (B) was the model
130 r/min and 180 r/min, quantifying temperature 30 °C, and inoculum size 3 mL; (B) was the model
fitting of temperature relative to biomass, altering variables 10 ◦ C, 20 ◦ C and 30 ◦ C, quantifying
fitting of temperature relative to biomass, altering variables 10 °C, 20 °C and 30 °C, quantifying
revolvingrevolving
speed 130 r/min,
speed and and
130 r/min, inoculum
inoculumsize 3 3mL;
size mL;(C) wasthe
(C) was themodel
model fitting
fitting of inoculum
of inoculum size relative
size relative
to biomass, altering
to biomass, variables
altering 2 mL,
variables 3 mL
2 mL, 3 mLandand4 4mL,
mL,quantifying revolving
quantifying revolving speed
speed 130 r/min
130 r/min and and
temperature 30 ◦ C. 30 °C.
temperature

2.9. Logistic Cell Growth Model

Molecules 2016, 21, x; doi: www.mdpi.com/journal/molecules
The non-linear regression analysis was used to estimate the logistic model, and the data are
shown in Table 6. Under the different conditions (stirring speed, temperature, and bacterial load),
the parameters (X0 , Xm , µm ) varied. The results showed that all of the R2 > 0.96, indicating that
the experimental data had a high degree of fit with the logistic equation. Furthermore, different
conditions (RS, T, and IS) led to different parameters (X0 , Xm , µm ) in accordance with the logistic
equation. SPSS software was used to fit the nonlinear regression connection of parameters with RS,
T, and IS, which conformed to the polynomial equation. The empirical models are demonstrated in
Molecules 2016, 21, 1479 11 of 17

Equations (4)–(6). Establishing a system model to investigate cell growth in all kinds of fermentation
environments would be well utilized in directing scale production (Figure 7). Goudar created a
mathematical model according to logistic equations, which were helpful in the development of the
early- and late-stage fed-batch process [22]:

X0 X m e µ m t
X= (3)
X m − X0 + X0 e µ m t

X0 = 0.00004 − 0.000036I − 0.00000628T + 0.000053T2 − 0.00000018R2 (4)

Xm = −0001 − 0.002I + 0.002T + 0.006R − 0.002I × T + 0.005I2 − 0.001T2 − 0.000052R2 (5)

Um = −0.0000705 − 0.0000803I + 0.003T + 0.005R + 0.004I × T − 0.002I × R + 0.000044T
(6)
× R + 0.012I2 − 0.001T2 − 0.0000036R2

2.10. The Luedeking–Piret Model

The Luedeking–Piret equation is a nonlinear regression equation used to simulate the relationship
between bacterial growth concentration and product formation [23]. The verification test was
carried out at optimal medium (fermentation conditions 125 r/min, 25 ◦ C, and 3 mL inoculum
size), and preliminary analysis was performed on the batch pattern owing to the growth type (II).
The results were shown in Table 6. Particularly, the coefficients of α 6= 0, β = 0 were fitted under the
condition of 10 ◦ C, which showed that the cell growth concentration and product formation pattern
fell under growth type I (α 6= 0, β = 0) and R2 > 0.990. In other conditions, the fermentation patterns
were all type II, and R2 > 0.960 (Figure 8). This pattern has been seen infrequently in other reports.
The explanation for the results may be that κ-carrageenan was easily coagulated at low temperature,
so that it influenced the cell growth normally. Finally, the empirical models regarding α, β with X0 ,
Xm , and µm were simulated using a polynomial equation in the same way (Equations (9) and (10)):

dP dX
=α + βX (7)
dt dt

X0 X m e µ m t X m − X0 + X0 e µ m t
 
Xm
P=α +β ln (8)
X m − X0 + X0 e µ m t µm Xm
α = −4.15 − 0.91I − 0.18T + 7.21R − 4.72I × T − 2.29I ×R − 0.12T × R + 66.84I2 + 0.47T2 +
(9)
0.004R2

β = 0.14 − 0.09I + 0.42T + 0.03R + 1.30I × T + 0.23I × R − 0.001T × R − 11.78I2 − 0.07T2 −

(10)
0.001R2

Table 6. Logistic’ model parameters Um (OD600 /h), X0 (/OD600 ), Xm (/OD600 ) and Luedeking-Piret’
model parameters α and β coefficients of determination (R2 ) of fitted models for all the treatments.

Biomass Enzyme Activity

Value Um X0 Xm R2 α β R2
80 0.186 0.017 0.396 0.976 352.453 6.770 0.979
RS (r/min) 130 0.201 0.021 0.567 0.978 95.433 42.558 0.969
200 0.214 0.017 0.451 0.988 106.824 46.014 0.971
10 0.198 0.009 0.329 0.96 458.271 0.000 0.993
T (/◦ C) 20 0.257 0.008 0.553 0.972 300.319 20.764 0.961
30 0.217 0.014 0.577 0.974 280.334 23.532 0.97
2 0.255 0.007 0.504 0.978 341.826 17.908 0.96
IS (/mL) 3 0.214 0.016 0.655 0.976 330.606 16.432 0.981
4 0.190 0.026 0.726 0.968 262.767 13.541 0.979
Molecules 2016, 21, 1479 12 of 17
Molecules 2016, 21, x 2 of 17

Figure 8. (A) was

Figurethe model
8. (A) was thefitting of revolving
model fitting speed
of revolving speed relative to enzyme
relative to enzyme activity,
activity, altering variables
altering variables
80 r/min, 130 80r/min
r/min, and 180 and
130 r/min r/min, quantifying
180 r/min, quantifyingtemperature 30 ◦and
temperature 30 °C, C, inoculum
and inoculum
size 3 mL;size 3 mL; (B) was
(B) was
the model fitting of temperature relative to enzyme activity, altering variables 10 °C, 20 °C◦and 30 °C
the model fitting of temperature relative to enzyme activity, altering variables 10 C, 20 ◦ C, and 30 ◦ C,
quantifying revolving speed 130 r/min, and inoculum size 3 mL; (C) was the model fitting of inoculum
quantifying revolving speed
size relative 130activity,
to enzyme r/min,altering
and inoculum size
variables 2 mL, 3 mL;
3 mL and 4(C)
mL,was the model
quantifying fitting
revolving speedof inoculum
size relative to130
enzyme
r/min andactivity, altering
temperature 30 °C. variables 2 mL, 3 mL and 4 mL, quantifying revolving speed
130 r/min and temperature 30 ◦ C.

3. Materials and Methods

3.1. Chemicals and Materials

κ-Carrageenan (BR) and fructo-oligosaccharide (BR) were purchased from Solarbio Company
(Beijing, China); Kestose (GF2), nystose (GF3) and GF4 were purchased from Sigma-Aldrich
(St. Louis, MO, USA); other chemical reagents were all BR grade.

3.2. Bacterial Isolation

The dried fragments of red seaweed Chondrus ocellatus (Zhangzhou Lvqi Colloid Food Company
of Fujian Province, Zhangzhou, China) that is the raw material for extracting carrageenan was chosen
to isolate κ-carrageenan-hydrolyzing bacterial strains. The plate medium (15 g/L NaCl, 15 g/L
Molecules 2016, 21, 1479 13 of 17

κ-carrageenan, 2 g/L NaNO3 , 0.5 g/L MgSO4 ·7H2 O, 1 g/L K2 HPO4 , 0.l g/L CaC12 , pH = 7.5, 28 ◦ C)
was screened for bacterial colonies that manifested plate depression or liquefaction-forming activity.
Pure cultures of the depression-forming bacterial isolates were obtained through repeated subculture
at 28 ◦ C for 24 h (the medium was supplemented by a nutritional ingredient with 5 g/L peptone).

3.3. Identification of Strain Fjfst-332

The cellular morphology and physiology of the strain were determined using a transmission
electron microscope (TEM, JEM-1400 120kV, JEOL, Beijing, China) and Bergey’s Manual of
Determinative Bacteriolog [24]. Genomic DNA was extracted using an Ezup Column Bacteria
Genomic DNA Purification Kit (cat. NO. SK8255, Sangon Biotech, Shanghai, China). The 16S
rDNA gene was amplified using the primer pair 7F (50 -CAGAGTTTGATCCTGGCT-30 ) and 1540R
(50 -AGGAGGTGATCCAGCCGCA-30 ). The PCR products were sequenced and analyzed using a
sequencing system (ABI 337 DNA, Model: 3730XL, Applied Biosystems, Foster City, CA, USA).
Using the purified sequence as the query sequence, similar 16S rDNA sequence data were looked up
in GenBank and downloaded from the NCBI online tool (http://rdp.cme.msu.edu/index.jsp; BLAST;
Accession Number EU440790).

3.4. Selection of Significant Variables via Plackett–Burman (PB) Design

Based on the single-factor experiment results, the Plackett–Burman design [25] was used to
investigate the influence of κ-carrageenan (A), FOS (B), tryptone (C), NaCl (E), K2 HPO4 (F), MgSO4
(G), and CaCl2 (H) on the strain enzyme activity and biomass. Three dummy factors (D1, D2, D3) were
introduced to investigate the systematic error.

3.5. Statistical Optimization

Many variables, such as the carbon source, nitrogen source, metal ions, etc., are known to
affect enzyme activity and strain biomass. In the present study, the influence of κ-carrageenan and
other auxiliary carbon sources was investigated. Organic and inorganic nitrogen sources were also
investigated, and various valence metal ions were considered. Medium components with significant
effects were screened based on PB experiments. All of the experiments for media components were
carried out in triplicates.
A Box-Behnken experimental design and response surface methodology (RSM) were used to
evaluate the relationship between the medium composition (κ-carrageenan, NaCl, yeast extract,
FOS) and κ-carrageenase activity (EA). A three-level, four-factor Central Composite Design (CCD),
consisting of 29 runs including 24 analytical points, was used. The four independent variables were
arranged at three levels (−1, 0, +1) (Table 7). The test data were fitted to the generalized second order
polynomial model equation (Equation (11)), which was used in the RSM analysis:
k k k −1 k
Y = β0 + ∑ βi Xi + ∑ βii Xii 2 + ∑ ∑ βij Xi Xj (11)
i =1 i =1 i j

where, Y was the response variable (EA); Xi and Xj were the independent variables. β0 was the
constant coefficient, βi was the linear coefficient, βii was the quadratic coefficient, and βij was the cross
product coefficient.

Table 7. Factors and levels of response surface analysis.

Factor
Level
X1 : κ-Carrageenan g/L X2 : NaCl g/L X3 : Yeast Extract g/L X4 : FOS g/L
−1 1 15 0.5 0.5
0 2 20 1 1
1 3 25 1.5 1.5
Molecules 2016, 21, 1479 14 of 17

3.6. Evaluation of κ-Carrageenase Activity (EA)

κ-Carrageenase activity (EA) was determined by using the 3,5-dinitrosalicylic acid (DNS)
method [26]. The incubation conditions were as follows: 5 mL κ-carrageenan solution
(0.2% κ-carrageenan in deionized water) was degraded using 1 mL crude κ-carrageenase at 45 ◦ C
for 60 min, then mixed with 1 mL DNS; 1 mL resulting mixture was heated at 100 ◦ C for 5 min.
The absorbance of the mixture (10 times dilution) was read at 540 nm. One unit of κ-carrageenase
activity (EA) is equivalent to an enzyme that produces 1 µg of D-galactose per minute under the given
conditions. κ-Carrageenase activity was calculated using the formula:

( A × 640.5 + 13.99) × V × n
EA(U/mL) = (12)
v×T

where A is the absorbance under OD540 , 640.5 and 13.99 are the constants for calculating liberated
galactose, V is the total reaction volume (mL), n is the dilution factor, v is the volume of enzyme (mL),
and T is the length of digestion time (min).

3.7. Determination of Cell Biomass (BM)

Turbidimetry was used to determine the fjfst-332 strain biomass (BM). A 250 mL Erlenmeyer flask
containing 30 mL optimized fermentation medium with 3 mL seed culture was incubated at 125 r/min
and 30 ◦ C for 72 h. Samples were taken every 4 h and the turbidity at OD600 was measured.

3.8. Detemination of Viscosity

A NDJ-7 Rotary Viscosimeter (Beijing ZKDH, Beijing, China) was used to determine the viscosity.
The initial medium with 1 g/L~5 g/L κ-carrageenan were determined under the room temperature.
The viscosity was calculated using the formula: η = kα, where η is the viscosity; k is the coefficient of
the rotor; α is the reading number.

3.9. Mathematical Model of Fermentation Process

3.9.1. Determination of Cell Growth Dynamics

In order to assess the growth pattern of strain fjfst-332, a logistic equation (Equation (13)) was
used to predict the cell growth model at log phase and stable phase. The logistic equation describes the
growth of a simple population in a confined space, where resources are limited [22]. Due to the bacteria
having different growth curves in different fermentation environments [27], the dynamics equation
was fitted at the conditions of revolving speed (RS 80 r/min, 130 r/min, 200 r/min), temperature
(T 10 ◦ C, 20 ◦ C, 30 ◦ C), and bacterial load (IS 2 mL, 3 mL, 4mL), based on the optimal medium:
 
dX X
= µm X 1 − (13)
dt Xm

Equation (13) integrated into the following algebraic equation:

X0 X m e µ m t
X= (3)
X m − X0 + X0 e µ m t

where X0 is the initial cell biomass, Xm is the maximum cell biomass, µm is the maximum specific
growth rate, and t is the fermentation time.

3.9.2. Analysis of Product Formation Kinetic

Gaden [28] generalized three fermentation forms: I. growth-related type; II. growth part-related
type; III. non-growth-related type. Luedeking and Piret [29] proposed the Luedeking–Piret equation
(Equation (7)) according Gaden’s conclusion, which considered the relationship of cell growth to
Molecules 2016, 21, 1479 15 of 17

product formation. When the cells, or some constituent of cells that is proportional to cell mass, are
the product, the rate of product formation directly relates to the rate of growth. The relationship of
product concentration with cell concentration can be rewritten into Equation (8):

dP dX
=α + βX (7)
dt dt
where P is the product concentration, X is the concentration of cells, t is the fermentation time, and α
and β are the coefficients. I: α 6= 0, β = 0; II: α 6= 0, β 6= 0; III: α = 0, β 6= 0.

X0 X m e µ m t X m − X0 + X0 e µ m t
 
Xm
P=α +β ln (8)
X m − X0 + X0 e µ m t µm Xm

where X0 is the initial cell biomass, Xm is the maximum cell biomass, µm is the maximum specific
growth rate, t is the fermentation time, and α and β are the coefficients.

3.10. Statistical Analysis

The statistical analysis systems DPS (V9.50, Ruifeng Information Company, Hangzhou, China)
and Origin Pro (V8.5, OriginLab, Wellesley Hills, WA, US) were used to analyze the data. A one-factor
analysis of variance (ANOVA) was performed for each parameter. Values given below are the means of
repeatedly measured values. The PB and RSM analysis were performed using Design-Expert (V8.0.6,
State-East Company, Minneapolis, MN, USA). The fitting of two kinetic models (cell growth model,
logistic equation, and product formation model, Luedeking–Piret equation) to the experimental data
of each sample was carried out using IBM SPSS Statistics (V21.0, IBM, Armonk, NY, USA). The R2
coefficient was used to evaluate the accuracy of the experimental data’s fit to the models.

4. Conclusions
The strain Thalassospira sp. fjfst-332 isolated from dry Chondrus crispus was shown for the first
time to degrade κ-carrageenan. According to statistical analysis, the optimal fermentation components
for strain fjfst-332 were determined to be 2 g/L κ-carrageenan, 20 g/L NaCl, 1 g/L yeast extract,
1 g/L FOS, 2 g/L NaNO3 , 0.5 g/L MgSO4 ·7H2 O, 1 g/L K2 HPO4 , and 0.l g/L CaC12 , resulting in
the strain’s highest enzyme activity of 267 U/mL. Compared with previous reports, fjfst-332 was the
most effective wild bacterium for obtaining κ-carrageenan oligosaccharides. We also identified an
inducer (FOS) to make strain fjfst-332 more prolic and enable it to hydrolyze κ-carrageenan more
effectively. What’s more, our research revealed three much more adaptive empirical models to describe
the microorganism growth and product formation. All of this work will be useful as a foundation for
the industrial production of κ-carrageenan oligosaccharide.

Acknowledgments: We gratefully acknowledge the financial support from the Science and Technology Major
Project of Fujian Province (2015NZ0001-1); Science and Technology Plan Major Project of Fujian Province
(2014I0008); National Marine Public Welfare Industry Special Scientific Research Projects (201505033).
Author Contributions: All the authors have mad substantive intellectual contributions to the study and given
approval to the final version of the manuscript. Baodong Zheng, Shaoxiao Zeng and Yi Zhang mainly contributed
to the study design and manuscript revision; Juanjuan Guo and Xu Lu contributed to the experimental studies
and data analysis; Hui Xu, Longtao Zhang contributed to the manuscript editing.
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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