Efficient production of sugar-derived aldonic acids by Pseudomonas fragi TCCC11892

Article abstract of DOI:10.1039/C8RA07556E

Aldonic acids are receiving increased interest due to their applications in nanotechnology, food, pharmaceutical and chemical industries. Microbes with aldose-oxidizing activity, rather than purified enzymes, are used for commercial production with limited success. Thus it is still very important to develop new processes using strains with more efficient and novel biocatalytic activities for the production of adonic acids. In the present study, Pseudomonas fragi TCCC11892 was found to be an efficient producer of aldonic acids, with the production of galactonic and l-rhamnonic acid by P. fragi reported for the first time. The semi-continuous production of maltobionic acid and lactobionic acid was developed for P. fragi TCCC11892, achieving a yield of over 90 g L^?1 for the first 7 cycles. The excellent performance of P. fragi in the production of lactobionic acid (119 g L^?1) was also observed when using waste cheese whey as an inexpensive fermentation medium. Scaling up of the above process for production of aldonic acids with P. fragi TCCC11892 cells should facilitate their commercial applications.

Full text of DOI:10.1039/C8RA07556E

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Efficient production of sugar-derived aldonic acids
by Pseudomonas fragi TCCC11892†
Cite this: RSC Adv., 2018, 8, 39897
a
b
a
c
c
b
Shuhong Mao,
Yanna Liu, Yali Hou, Xiaoyu Ma, Juanjuan Yang, Haichao Han,
b
a
ac
ab
Jianlin Wu, Longgang Jia, Huimin Qin
*
and Fuping Lu
*
Aldonic acids are receiving increased interest due to their applications in nanotechnology, food,
pharmaceutical and chemical industries. Microbes with aldose-oxidizing activity, rather than purified
enzymes, are used for commercial production with limited success. Thus it is still very important to
develop new processes using strains with more efficient and novel biocatalytic activities for the
production of adonic acids. In the present study, Pseudomonas fragi TCCC11892 was found to be an
efficient producer of aldonic acids, with the production of galactonic and L-rhamnonic acid by P. fragi
reported for the first time. The semi-continuous production of maltobionic acid and lactobionic acid was
Received 11th September 2018
Accepted 18th November 2018
ꢀ1
developed for P. fragi TCCC11892, achieving a yield of over 90 g L for the first 7 cycles. The excellent
ꢀ1
performance of P. fragi in the production of lactobionic acid (119 g L ) was also observed when using
waste cheese whey as an inexpensive fermentation medium. Scaling up of the above process for
production of aldonic acids with P. fragi TCCC11892 cells should facilitate their commercial applications.
DOI: 10.1039/c8ra07556e
rsc.li/rsc-advances
a workable approach to overcome the drawbacks associated
with chemical or enzymatic oxidation.
Aldonic acids could be produced from microorganisms with
Introduction
7–9
Many carbohydrate derivatives, such as sugar acids, are desir-
able platform chemicals and have the potential to be used as aldose-oxidizing activity found in many archaea and bacteria
1
,2
1,2,10
a source of energy. Aldonic acids belong to the class of sugar and some eukaryotes.
acids, and some aldonic acids such as lactobionic acid and much attention as an efficient microbial platform for the
Recently, P. taetrolens has gained
4,8,11
maltobionic acids have many potential applications in the food, production of lactobionic acid.
Various methodologies for
medical, pharmaceutical, cosmetics and chemical industries optimizing bio-processing conditions have been designed to
1
–6
due to their antioxidant, chelating and humectant properties.
improve lactobionic acid yields. For example, Alonso et al.
Aldonic acids can be produced via electrochemical or demonstrated the feasibility of using a novel ow cytometry
chemical oxidation, but these methods are not economically approach for optimizing lactobionic acid production based on
12
viable on an industrial scale because of the use of harmful the physiological status of P. taetrolens cells. In addition, the
7
catalysts and the generations of undesirable side-products.
pH-control mode, seed culture as well as supply of oxygen have
Production methods for aldonic acids that use puried enzymes also been shown to be important parameters for optimizing the
1
11
or microbial cells have also been reported. However, enzymatic bio-production of lactobionic acid. However, more efficient
preparation of aldonic acids is costly due to the need for enzyme processes for commercial bio-production of lactobionic acid on
purication and requirement of expensive redox mediators or industrial scale still need to be explored.
8
cofactor regeneration. From an industrial point of view, the use
Compared with lactobionic acid, bio-production of malto-
5
of microbial cells for aldonic acid production has become bionic acid has not been studied extensively. Maltobionic acid
derived from maltose (8.9% maltose) incubated with Pseudo-
13
monas graveolens for 50 hours was documented. In addition,
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, cellobionic and L-rhamnonic acids are also considered to have
a
Tianjin University of Science & Technology, Industrial Microbiology Laboratory, great potential as replacements for certain petrochemicals in
College of Biotechnology, Tianjin Economic and Technological Development Zone,
No. 29, 13 Main Street, Tianjin 300457, China. E-mail: huiminqin@tust.edu.cn;
lfp@tust.edu.cn; Fax: +86-22-60602298; Tel: +86-22-60601958
1,14,15
the future.
In fact, the introduction of these aldonic acids
into commerce is still limited due to the low efficiency of
currently reported biocatalysts. Therefore, further screening
and exploration of strains with novel biocatalytic activities plays
16
b
State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science &
Technology, Tianjin 300457, China
c
Tianjin Key Laboratory of Industrial Microbiology, College of Biotechnology, Tianjin an important role in the development of new processes for
University of Science & Technology, Tianjin 300457, China
aldonic acid production that are both sustainable and efficient.
RSC Adv., 2018, 8, 39897–39901 | 39897
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c8ra07556e
1
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Paper
In this study, four Pseudomonas strains were investigated
regarding their ability to produce aldonic acids. A process for
semi-continuous production of lactobionic acid and malto-
bionic acid was developed. The efficient production of lacto-
bionic acid was demonstrated using the industrial waste-
product whey as the fermentation medium.
Results and discussion
P. fragi is the most versatile sugar oxidizer among the tested
Pseudomonas spp.
Some aerobic bacteria, especially members of the genus Pseu-
domonas, can oxidize a wide range of aldose sugars into their
1
corresponding aldonic acids by their dehydrogenase systems.
P. taetrolens has recently been shown to be an efficient micro-
8,9
bial platform for lactobionic acid production. In this study,
four Pseudomonas bacteria strains were found to have the ability
to produce adonic acids (ESI Fig. S1–S19†). As shown in Table 1,
Pseudomonas cuserectae TCCC11959 transformed maltose into
maltobionic acid, and Pseudomonas beteli TCCC11961 produced
maltobionic acid and lactobionic acid, respectively; and a wide
range of aldose sugars were oxidized into their corresponding
aldonic acids by cells of P. fragi TCCC11892.
Fig. 1 showed the optimal conditions for P. fragi cells cata-
lyzed aldonic acid production using various aldoses, including
ꢁ
3
g
7 C, shaking speed of 200 rpm and a cell concentration of 3
ꢀ
1
DW
L , and in which the production efficiency was: malto-
bionic > lactobionic > galactonic > cellobionic and L-rhamnonic
acid.
It is noteworthy that we found for the rst time that P. fragi
was capable of producing L-rhamnonic acid from L-rhamnose,
17
a deoxy sugar found widely in bacteria and some plants.
Although high levels of L-rhamnonic acid production were re-
ported for Candida polymorpha and Pichia robertsii using L-
rhamnose as the substrate, the other main metabolic product of
1
,2-propanediol was also observed at the same time, resulting in
18
the difficulty in L-rhamnonic acid downstream processing.
The fermentation product of cellobionic acid by P. fragi so far
15
has not been reported. Cellobionic acid is an important
intermediate during the degradation of cellulosic biomass by
19
several lignocellulolytic fungi and bacteria. Its production
Fig. 1 The effects of temperature (a), cell concentration (b) and shaker
speed (c) on the yield of aldonic acids from aldoses.
Table 1 Bioconversion of the indicated aldoses by Pseudomonas
ficuserectae TCCC11959, Pseudomonas beteli TCCC11961, Pseudo-
monas agarici TCCC11962 and Pseudomonas fragi TCCC11892
from cellobiose has been achieved using cellobiose dehydro-
TCCC11892 TCCC11959 TCCC11961 TCCC11962 genase (CDH) as well as the combined action of polysaccharide
20
monooxygenase and cellobiohydrolase. Furthermore, cello-
Glucose
Maltose
Lactose
Glucose
Galactose
Cellobiose
L-Rhamnose
Trehalose
Arabinose
Mannose
ꢀ
+
+
ꢀ
+
ꢀ
+
+
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
bionic acid was identied in the culture supernatants of F.
21
succinogenes S8. The reported most efficient producer of cel-
lobionic acid, Gluconobacter frateurii NBRC3285, produced cel-
lobionic acid with a yield of only about 3 mM with the addition
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
+
+
+
10
of 27.8 mM cellobiose to the culture medium. Herein,
apparent improvement of the cellobionic acid production effi-
ꢀ
ꢀ
1
ciency (50 mg L ) was achieved using P. frag with addition of
+
ꢀ
1
1
g L cellobiose in the culture. Further upgrade of the above
ꢀ
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process should have industrial applications for cellobionic acid bio-process developed for maltobionic acid will be helpful for
production which can be used as a substrate to produce other their potential applications in the marketplace.
15
valuable biochemicals.
Highly efficient production of lactobionic acid using cheese
whey
Semi-continuous production of lactobionic acid and
Industrial waste whey from cheese production was used to
produce lactobionic acid through a biotechnological process
carried out by P. fragi. The results showed that lactobionic acid
could be produced efficiently when 20% whey was used as the
fermentation medium (Fig. 3). On the basis of shake-ask
experiments, the lactobionic acid production was scaled up to
a 2 L fermentor, and the effect of the dissolved oxygen
concentration on the product yield was also investigated. The
maltobionic acid using P. fragi TCCC11892
The semi-continuous production of lactobionic acid using P.
fragi TCCC11892 was carried out over ten cycles under the same
reaction conditions. Fig. 2 indicated that the yield in the rst 7
ꢀ
1
cycles remained over 91.7 g L for lactobionic acid, while the
yield in the last 3 cycles gradually decreased. The semi-
continuous fermentation with cell recycle offers several advan-
tages over batch fermentation when applied to the later gener-
ation lactobionic acid production, and the volumetric
ꢀ
1
cell concentration reached as high as 1.6 g
lactobionic acid titer of 119 g L was obtained aer 36 h
L , and the
DW
ꢀ
1
ꢀ1
ꢀ1
productivity was 3.8 g L
h
and the process reduced to 24 h.
fermentation when the dissolved oxygen concentration was
Maltobionic acid is currently mainly obtained from the
chemical or enzymatic oxidation of maltose. Stodola and
Lockwood rst reported the aldonic acid production from
maltose and lactose by Pseudomonas graveolens. On the other
hand; the biochemical production of maltobionic acid using
whole cell biocatalysts has not been reported in the literature. In
40%. Compared with previous studies, the specic lactobionic
acid productivity was clearly improved by the strain investigated
ꢀ1
ꢀ1
in this study, with about a 1.6-fold increase (3.3 g L
h
versus
13
ꢀ
1
ꢀ1 11
2.1 g L
h ).
ꢀ1
this study, the yield of maltobionic acid is 94.7 g L , and this
Fig. 3 Effect of the dissolved oxygen concentration on the cell
Fig. 2 Semi-continuous production of lactobionic acid (a) and mal- concentration (a) and yield of lactobionic acid (b) using 20% dried
ꢁ
ꢁ
tobionic acid (b) using P. fragi in a 2 L bioreactor at 37 C, 180 rpm and powdered whey from cheese production in a 2 L bioreactor at 37 C
pH 6.5.
and pH 6.5.
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The promising commercial applications of lactobionic acid analysis and it was identied as L-rhamnonic acid by comparing
have prompted the development of a number of novel processes it with the spectral data of the commercially available L-rham-
4,11,22
for its efficient biotechnological production.
The present nonic acid reference standard (Fig. S4–S7†). This result was
study thus demonstrates the feasibility of using P. fragi to further corroborated by ESI-mass spectroscopic analysis (ESI:
+
achieve high-level bio-production of lactobionic acids from calc. [M ꢀ H] 179.13, found 179.13; Fig. S8†), which displayed
whey, providing an biocatalyst with the potential for more effi- a molecular mass larger by 16 than that of L-rhamnose, perfectly
cient industrial production of lactobionic acid.
t the calculated molecular weight of rhamnonic acid.
Experimental
Chemicals
Maltobionic acid
The ESI-MS spectrum of the presumed maltobionic acid analyte
+
Galactose ($98%), L-rhamnose monohydrate ($98%), maltose
monohydrate ($98%), lactose monohydrate ($98%), and
cellobiose ($90%) were purchased from Sangon Biotech Co.
with and the standard product of L-rhamnonic acid ($90%) and
lactobionic acid ($97%) were purchased from Sigma-Aldrich
Co.
displayed a molecular ion at m/z ¼ 357.25 (calc. [M + H]
357.25), which was larger by 16 than that of maltose (Fig. S9†).
1
This suggested the oxidation of maltose (342.3). Both H and
1
3
C data of the product of maltobionic acid (Fig. S10 and S11†)
7,23
were in agreement with those published in the literature.
Screening of aldonic acid-producing strains
Lactobionic acid
Pseudomonas cuserectae TCCC11959, Pseudomonas beteli ESI-MS analysis indicated that the product obtained from the
TCCC11961, Pseudomonas agarici TCCC11692 and Pseudomonas oxidation of lactose was larger by 16 than lactose (Fig. S12;†
fragi TCCC11892 were cultivated in YPD medium (without agar) 357.22 vs. 342.3) suggesting the insertion of one atom of oxygen
ꢁ
for 10 h at 28 C and 220 rpm. The cells were harvested and re- into lactose. This product was further identied as lactobionic
1
13
suspended in physiological saline with the nal cell concen- acid based on the H NMR and C NMR analysis, which showed
tration of 3 gDW
ꢀ
1
L
. Then the biotransformation of solutions the same spectra as the commercially available lactobionic acid
comprising 10% (w/w) galactose, L-rhamnose, maltose, lactose, reference standard (Fig. S13–S16†).
and cellobiose, separately, was carried out using resting cells
individually. Ca CO (1 g/30 mL) was used to adjust the acidity
of the medium. The concentrations of the substrate and
product were determined by HPLC.
2
3
Cellobionic acid
In the case of the presumed cellobionic acid product, the
molecular ion peak at 357.16 ([M ]) observed in the MS spectra
+
Separation and identication of aldonic acids
was larger by 16 than that of the substrate, and this again
suggested the oxidation of cellobiose (342.3; Fig. S17†). The H
1
Aer biotransformation, the fermentation medium was cen-
NMR spectrum of cellobionic acid is shown in Fig. S18.† The H
signal from the terminal carbon present in the substrate dis-
appeared in the product, conrming the oxidation of the
trifugated at 4500 g for about 10 min, and the supernatant was
ꢁ
evaporated at 80 C. The resulting concentrated solution was
precipitated with an equal volume of 95% ethanol, and the
1
3
ꢁ
substrate. Additionally, as can be seen from the C NMR of the
cellobionic acid product (Fig. S19†), an obvious downeld shi
of the terminal carbon signal at 178 ppm was observed, while
the signal of the corresponding carbon disappeared at the same
time, which suggests the introduction of a carboxylic acid group
at the terminal carbon.
precipitate was ltered and dried at 60 C for about 24 h, aer
which the crude product was washed twice with ethanol and
2
then ltered and dried. The nal product was dissolved in D O
1
13
(
99.9%; Sigma, USA) and analyzed by H NMR and C NMR on
an AV III 400 MHz instrument (Bruker). Each product obtained
from the biotransformation was also determined by means of
ESI-mass spectroscopic analysis.
Semi-continuous production of lactobionic and maltobionic
acid using P. fragi
Galactonic acid
+
A molecular ion at m/z ¼ 195.08 (calc. [M ꢀ H] 195.08) was
Semi-continuous production of lactobionic acid and malto-
bionic acid using P. fragi was carried out with 10% lactose (or
maltose) in the 2 L bioreactor equipped with a microltration
membrane for cell recycling. In the rst cycle, the cells were
recycled back into the fermentation by microltration
membrane unit aer 24 h bioconversion, and the product
stream was obtained at the same time. Then 10% lactose or
maltose solutions was pumped to the bioreactor for the next
found in the ESI-MS spectrum of the presumed galactonic acid
sample, which was larger by 16 than that of galactose (180.16)
(Fig. S1†), indicate oxidation. The structure of galactonic acid
1
13
was conrmed by comparing its H and C NMR spectral data
with reported values (Fig. S2 and S3†).
7
L-Rhamnonic acid
The product obtained from the biotransformation of L-rham- batch of bioconversion, and the product obtained from each
1
13
nose was determined by means of H NMR and C NMR batch was analyzed by HPLC.
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Bio-production of lactobionic acid by P. fragi using cheese
whey in a 2 L bioreactor
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Acknowledgements
This work was nancially supported by grants from the Natural
Science Foundation of China (No. 21878233), the Tianjin
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