International Journal of Biological Macromolecules 186 (2021) 574–579

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International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Production of polyhydroxyalkanoates by a moderately halophilic bacterium

of Salinivibrio sp. TGB10
Guan-Bao Tao, Bi-Wei Tan, Zheng-Jun Li *
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China

A R T I C L E I N F O A B S T R A C T

Keywords: A moderately halophilic bacterium isolated from the water samples collected from a salt field, Salinivibrio sp.
Salinivibrio TGB10 was found capable of producing poly-3-hydroxybutytate (PHB) from various sugars. Cell dry weight
Poly-3-hydroxybutytate (CDW) of 8.82 g/L and PHB titer of 6.84 g/L were obtained using glucose as the carbon source after 24 h of
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
cultivation in shake flasks. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) was synthesized when pro­
pionate was provided as secondary carbon source. Salinivibrio sp. TGB10 exhibited favorable tolerance to pro­
pionate. The use of 8 g/L propionate and 20 g/L glucose as combinational substrates yielded 1.45 g/L PHBV with
a 3-hydroxyvalerate monomer content of 72.02 mol% in flask cultures. In bioreactor study, CDW of 33.45 g/L
and PHBV titer of 27.36 g/L were obtained after 108 h of fed-batch cultivation. The results indicated that Sal­
inivibrio sp. TGB10 is a promising halophilic bacterium for the production of PHBV with various polymer
compositions.

1. Introduction host employed [3]. For example, R. eutropha accumulates poly-3-

hydroxybutyrate (PHB) homopolymer using fructose as carbon source,
The chemical industry based on petroleum feedstock produces a while it synthesizes poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
huge amount of non-degradable plastic materials every year, which has [poly(3HB-co-3HV), PHBV] when propionate is provided as additional
caused serious white pollution problem. It is estimated that over 10% of carbon source to generate 3-hydroxyvalerate (3 HV) precursor [6].
plastic waste turned into aquatic ecosystems every year, and the waste When propionate and butyrate were simultaneously provided,
emissions would increase to 53 million tons per year by 2030 [1]. Dis­ R. eutropha harboring the synthetic PHA operon phaC2AJ produced poly
carded plastic materials cannot be decomposed even after hundreds of (3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate)
years in the environment. To solve the white pollution problem, appli­ [poly(3HB-co-3HV-co-3HHx)] terpolymer [7]. A. hydrophila 4AK4 could
cation of biodegradable plastic materials would be a feasible way. Pol­ produce poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) when culti­
yhydroxyalkanoate (PHA), polylactic acid (PLA), polybutylene vated on lauric acid [8]. Pseudomonas sp. PAMC 28620 isolated from
succinate (PBS), and starch-based plastics are the major biodegradable Arctic glacier soil was reported to synthesize medium-chain length
polymers being commercialized as alternatives to conventional (MCL) PHA polymers consisting of 8–14 carbon lengths using glycerol as
petroleum-based plastics [2]. PHA is a large group of intracellularly the sole carbon source [9]. The wild type strain of E. coli lacks PHA
accumulated polymers synthesized by microorganisms grown under synthesis or degradation pathway. Overexpressing bktB, phaB, and phaC
unbalanced nutrient condition, such as carbon excess and nitrogen/ from R. eutropha in E. coli gives recombinants capable of producing
phosphate limitation [3]. Until now, over 150 different monomers have PHBV from glucose and propionate, and further strengthening the
been polymerized and yielded a series of PHAs with various material expression of succinyl-CoA synthase increased 3 HV fraction in PHBV
properties [4,5]. copolymer [10]. Additionally, the overexpression of acetyl-CoA acetyl­
Traditional PHA producing bacteria include Ralstonia eutropha, transferase was shown to effectively enhance intracellular propionyl-
Aeromonas hydrophila, Pseudomonas putida, recombinant Escherichia coli, CoA pool size and achieved 3 HV monomer content up to 57.9 mol%
etc. The carbon sources used for PHA production could be carbohy­ [11].
drates, fatty acids, glycerol, and even carbon dioxide, depending on the Although PHA is regarded as one of the most promising biobased

* Corresponding author at: Beijing University of Chemical Technology, Mailbox 53, No. 15 Beisanhuan Donglu, Chaoyang District, Beijing 100029, China.
E-mail address: [email protected] (Z.-J. Li).

https://doi.org/10.1016/j.ijbiomac.2021.07.038
Received 21 April 2021; Received in revised form 19 June 2021; Accepted 4 July 2021
Available online 8 July 2021
0141-8130/© 2021 Elsevier B.V. All rights reserved.
G.-B. Tao et al. International Journal of Biological Macromolecules 186 (2021) 574–579

polymers suitable for variety of applications in different fields, its agar plates. Finally, the purified colonies were cultivated in shake-flask
economical uncompetitiveness with conventional plastics is always a cultures using TYS medium supplemented with 20 g/L acetate to
huge barrier limiting PHA's practical applications. The feedstock espe­ determine the accumulation of PHB. Strains with PHB producing ability
cially carbon source accounts for a large portion of the total PHA pro­ were identified through 16S rRNA sequencing by PCR amplification
duction cost [12]. A series of cheap substrates including agro-industry using 27F/1492R primer pair.
waste, food processing waste, and biodiesel waste have been employed
to produce PHA [13]. In addition, metabolic engineering, synthetic 2.2. PHA production in shake-flask cultures
biology, and morphology engineering strategies were applied to explore
novel PHA producers with the aim including rapid cell growth, high The glycerol stock of Salinivibrio sp. TGB10 was inoculated into TYS
carbon yield, low energy requirements of fermentation, and ability to broth medium and cultivated aerobically for 12 h to prepare the seed
synthesize various of tailor-made biopolymers [14,15]. culture. Subsequently, the seed culture was inoculated into 250 mL
Recently, halophilic bacteria and haloarchaea have been demon­ Erlenmeyer flasks containing 50 mL TYS medium supplemented with
strated as cost-effective hosts to manufacture PHA. These halophilic different carbon sources for PHA production experiments. For the
microorganisms can grow well under high-salts culture media thus are testing of carbohydrates utilization (glucose, xylose, sucrose, maltose,
suitable for open and continuous cultivation, which would help to and soluble starch), a concentration of 20 g/L was applied. The initial
decrease the energy input of sterilization and simply the fermentation substrate concentration for volatile fatty acids (acetate, propionate, and
process [16]. For example, Halomonas bluephagenesis has been employed butyrate) was 10 g/L. To produce the PHBV copolymer, carbohydrate
for non-sterile and continuous fermentation at pilot-scale (5000 L) to and propionate were provided simultaneously, and propionate was
produce PHA [17]. Genetic manipulation tools including CRISPR/Cas9 applied at different concentrations to study its effects on PHBV
and genomic integrations were successfully developed, and accumulation.
H. bluephagenesis was proved to be a promising halophilic chassis to
synthesize PHA or commodity chemicals such as 3-hydroxypropionate 2.3. PHA production in bioreactor cultures
and ectoine [18,19]. The haloarchaea Haloferax mediterranei was re­
ported to produce PHA from low-cost raw materials including extruded The seed culture was prepared as described above and then trans­
rice bran and cornstarch, and PHA concentration of 77.8 g/L was ach­ ferred into a 5-L bioreactor (Bailun Bio, China) at 10% (v/v) inoculation
ieved in a repeated fed-batch culture [20]. Due to the presence of with a working volume of 3 L. The initial medium in bioreactor cultures
multiple propionyl-CoA-supplying pathways, H. mediterranei was able to was TYS broth supplemented with glucose or glucose/propionate. A fed
accumulate PHBV copolymer without the addition of propionate, a solution of 500 g/L glucose or 50 g/L propionate was added when
structurally-related precursor to incorporate 3 HV unit [21]. glucose concentration was lower than 10 g/L or propionate concentra­
The employment of halophiles as industrial producers also presents tion was lower than 1 g/L. Dissolved oxygen was provided by injecting
some challenges including the treatment of high salinity wastewater, the filtered air at a flow rate of 3 L/min. Stirring velocity was initially set at
corrosion of fermentation equipment, etc. [22]. Therefore, novel mild or 400 rpm and gradually increased to 800 rpm during the fermentation.
moderate halophilic PHA producers are still to be explored in nature. Fermentation temperature and pH were set at 30 ◦ C and 7.0, respec­
The changeable marine environments provide suitable resources to tively. The pH in bioreactor was maintained by automatic addition of 5
screen versatile microorganisms which can be used to synthesize unique M NaOH and 4 M HCl. Antifoam was added to the bioreactor when
enzymes, drugs, building block chemicals, and PHA. For example, a needed. The bioreactor cultures were taken every 4 h for analysis of
Vibrio proteolyticus strain isolated from the Korean marine environment carbon consumption, cell dry weight (CDW), PHA and byproduct
produced 2.7 g/L PHB from fructose in shake flasks [23]. Another ma­ accumulation.
rine bacterium Neptunomonas concharum JCM17730 effectively accu­
mulated PHB using butyrate as the carbon source, and PHB titer reached 2.4. Analytical methods
15.51 g/L in unsterile fed-batch bioreactor cultivations [24]. Moreover,
Halomonas sp. YLGW01 isolated from Korean marine culture was able to After cultivation, the cell culture was collected by centrifugation at
produce PHB up to 95 wt% of cell dry weight [25]. Whole-genome 8000 rpm and 10 min. The supernatant was filtered and then subject to
sequencing of N. concharum JCM17730 and Halomonas sp. YLGW01 high-performance liquid chromatography (HPLC) analysis to determine
revealed the carbon utilization system and would help to construct PHA the concentration of substrate consumption and byproduct accumula­
hyperproducers in further studies [26,27]. In this paper, we investigated tion. The cell pellet was washed twice with artificial seawater and
the potential of PHA production using Salinivibrio sp. TGB10, a moder­ distilled water, then followed by lyophilization to measure the cell dry
ately halophilic strain isolated from a salt field. PHB production from weight. Dried cell was applied to methanolysis in 3% (v/v) H2SO4 at
glucose, maltose, or soluble starch was observed, and the addition of 8 100 ◦ C for 4 h in a screw-capped tube, yielding methyl esters which
g/L propionate led to the accumulation of PHBV copolymer containing could be quantified by gas chromatography (GC) to determine the
72–95 mol% 3 HV monomer. Our results suggest that Salinivibrio sp. intracellular accumulated polymers. Quantification was calculated on
TGB10 was a promising moderately halophilic chassis for PHBV comparison of peak areas with a PHBV standard (Sigma-Aldrich, USA).
production. Transmission electron microscopy (TEM) was used to observe the
intracellular PHA granules. The cells collected after cultivation were
2. Materials and methods sliced and diced for microscopic examination using a JEM-2100F TEM
(JEOL, Japan) according to the methods reported previously [28]. The
2.1. Isolation of Salinivibrio sp. TGB10 proton NMR spectrum was applied to confirm the structure of polymers
produced. PHA sample purified from dried was dissolved in deuterated
Salinivibrio sp. TGB10 was isolated from water samples collected chloroform and analyzed with a JNM-ECA500 NMR spectrometer
from a salt field in Binhai District, Tianjin, China. TYS medium was (JEOL, Japan). The molecular weights of PHA polymers were deter­
employed for screening marine bacteria, and it contained (g/L): NaCl mined by gel permeation chromatography (GPC) equipped with Shodex
27.5, KCl 0.7, CaCl2⋅2H2O 1.4, MgSO4⋅7H2O 6.8, MgCl2⋅6H2O 5.4, K-804 column (Waters).
NaHCO3 0.2, yeast extract 1, and peptone 5. The pH of the medium was
adjusted to 7.0. The collected water samples were inoculated into TYS
medium containing 20 g/L acetate and cultivated aerobically for 24 h.
Then cell cultures were purified through repeatedly streaking on TYS

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3. Results and discussion The TEM observation results were in agreement with previous studies, in
which PHA accumulation would give visible intracellular granules [31].
3.1. PHA production by Salinivibrio sp. TGB10 on various substrates In addition, the polymers from the lyophilized cells were purified and
dissolved in deuterated chloroform for proton NMR-spectroscopic
The water samples collected from a salt field in Tianjin, China were analysis. As shown in Fig. 2A, a homopolymer of PHB was synthe­
inoculated into TYS medium containing 20 g/L acetate to screen the sized. These results further confirmed the polymer production.
potential PHA producers. A total of 34 colonies were screened and 10
colonies exhibited the ability of PHB accumulation. TGB10 achieved the 3.2. Effect of propionate addition to PHBV production
highest PHB titer of 0.38 g/L (Supplementary Table S1). The result of
16S rRNA gene sequence homology assay indicated that TGB10 was Propionate was shown to achieve PHBV accumulation, yet the
99.87% similar with Salinivibrio sp. M318 (Supplementary Fig. S1). The polymer titer was only 0.22 g/L (Table 1). Therefore, combination of
genius Salinivibrio was firstly proposed in 1996, and its representative carbohydrates and propionate was applied to cultivate Salinivibrio sp.
species Salinivibrio costicola was a moderately halophilic bacterium TGB10, and the effect of propionate addition to cell growth and PHBV
which could grow under a wide range of NaCl concentrations [29]. accumulation was investigated (Table 2). The CDW, PHBV content, and
Salinivibrio sp. M318, isolated from fermenting shrimp paste, was re­ PHBV titer were all decreased as the increase of propionate concentra­
ported to accumulate PHA from waste fish oil and glycerol [30]. tion, while 3 HV monomer content raised significantly with increased
Firstly, Salinivibrio sp. TGB10 was cultivated in TYS medium sup­ propionate addition. For example, the use of 20 g/L glucose and 2 g/L
plemented with 0–80 g/L NaCl to investigate the effect of NaCl con­ propionate obtained 8.02 g/L CDW, containing 50.88 wt% P(3HB-co-
centration on cell growth. As shown in Supplementary Fig. S2, the 27.28 mol% 3 HV), while the addition of 8 g/L propionate produced
addition of 30–50 g/L NaCl lead to the best final OD600 of 2.3. Cell 4.89 g/L CDW, containing 29.71 wt% P(3HB-co-72.02 mol% 3 HV).
growth rate of Salinivibrio sp. TGB10 was considerably reduced at NaCl Maltose was less favorable than glucose in terms of PHBV accumulation,
concentration of 80 g/L, and the maximal OD600 decreased to 1.6. both the polymer content and titer were lower than those achieved with
Lowest cell growth was observed in TYS medium without NaCl addition, glucose. However, maltose yielded higher 3 HV monomer content, and
indicating the importance of NaCl to Salinivibrio sp. TGB10. the combination 20 g/L maltose and 8 g/L propionate gave 1.16 g/L P
Subsequently, the effect of various carbon sources on the cell growth (3HB-co-95.24 mol% 3 HV) accumulation. The phenomenon that
and PHA biosynthesis by Salinivibrio sp. TGB10 was investigated maltose elevated 3 HV monomer polymerization still required to be
(Table 1). Glucose, maltose, and soluble starch were found to be suitable further deciphered.
for supporting effective cell growth and PHB accumulation, while xylose Proton NMR spectroscopic analysis was also employed to study the
and sucrose cannot be metabolized. Glucose yielded the highest cell dry structure of PHBV copolymer produced using 20 g/L glucose and 6 g/L
weight of 8.82 g/L and PHB titer of 6.84 g/L. Maltose and soluble starch propionate. As shown in Fig. 2B, the polymer sample exhibited several
produced PHB titer of 2.81 g/L and 3.28 g/L, respectively. Volatile fatty typical resonance patterns of PHBV, and the major signals were
acids were also employed to cultivate Salinivibrio sp. TGB10. Acetate, consistent with the spectrum reported previously [30]. Propionate is
propionate, and butyrate were all shown to be effective for generating often used as a structurally-related precursor to generate 3 HV monomer
PHA, yet both cell dry weight and PHA titer were much lower than those for PHBV synthesis, yet it has certain toxicity to cells and the concen­
obtained using carbohydrates. As expected, PHBV could be produced tration should be maintained at low levels. Previously, the addition of 3
when propionate was employed as the carbon source. Up to now, there is g/L propionate yielded 18.36 mol% and 15.8 mol% 3 HV in Halomonas
only one report on the PHA production using Salinivibrio species [30]. TD01 and V. proteolyticus, respectively [23,28], while the addition of 2
Our results indicated that Salinivibrio sp. TGB10 is also a suitable chassis g/L propionate yielded 8.16 mol% 3 HV in Schlegelella thermodepoly­
for PHA production which could utilize versatile substrates. merans [32]. In this study, Salinivibrio sp. TGB10 was proved to be able to
Salinivibrio sp. TGB10 grown on TYS medium and TYS supplemented polymerize more 3 HV monomer under limited propionate concentra­
with glucose were subjected to TEM observation. As shown in Fig. 1, tion, and it also exhibited favorable tolerance to propionate thus ensure
there were electron-translucent inclusions observed in the cells culti­ the ability to synthesize PHBV with high 3 HV monomer content.
vated with glucose. In contrast, the control group cultivated with TYS The molecular weight of PHB and PHBV polymers produced by
medium without additional carbon source didn't contain any inclusion. Salinivibrio sp. TGB10 was determined and shown in Table 3. The Mw
varied between 300 kDa and 400 kDa, similar with those of polymers
Table 1 produced by Salinivibrio sp. M318, Halomonas, and Schlegelella species
PHA production from various carbon sources. [30,32,33]. The naturally produced PHA polymers usually possess mo­
lecular weight ranged from 200 to 30,000 kDa [34]. It is believed that
Carbon CDW (g/ PHA content PHA (g/ 3HB 3 HV
source L) (wt%) L) (mol%) (mol%) the amount and catalytic activity of PHA synthase determines the
polymerization reaction and controls the molecular weight of PHA
Glucose 8.82 ± 77.52 ± 2.21 6.84 ± 100 0
0.23 0.27 [35,36]. Establishing genetic modification methods in Salinivibrio spe­
Xylose ND ND ND ND ND cies to regulate the expression of PHA synthase is expected to synthesize
Sucrose 1.31 ± ND ND ND ND polymers with a variety of molecular weights for potential applications
0.02 in different fields.
Maltose 7.28 ± 38.79 ± 3.39 2.81 ± 100 0
0.27 0.15
Soluble 8.25 ± 39.80 ± 0.84 3.28 ± 100 0 3.3. Fed-batch cultivation for PHB production by Salinivibrio sp. TGB10
starch 0.32 0.14
Acetate 3.26 ± 6.36 ± 0.06 0.21 ± 100 0 Next, fed-batch fermentation in a 5-L bioreactor was investigated to
0.07 0.01
produce PHB using glucose as the sole carbon source (Fig. 3). Salinivibrio
Propionate 2.20 ± 9.80 ± 0.59 0.22 ± 53.38 46.62
0.06 0.01 sp. TGB10 consumed 96.03 g/L glucose and reached 30.06 g/L of CDW
Butyrate 2.49 ± 32.58 ± 0.37 0.81 ± 100 0 after 72 h cultivation, with a PHB titer of 26.34 g/L. The PHB yield YPHB/
0.16 0.06 glucose was 0.27 g PHB/ g glucose, reaching 56% of the theoretical yield.
Shake flask experiments were performed at 30 ◦ C, 200 rpm with 20 g/L of The strain secreted pyruvate as a major byproduct, and no other car­
carbohydrates or 10 g/L volatile fatty acids for 24 h. Data are expressed as av­ boxylic acids were found through HPLC analysis. Pyruvate titer reached
erages and standard deviations of three parallel experiments. CDW, cell dry 6.23 g/L at the end of fermentation. The final PHB titer (26.34 g/L) in
weight; ND, not detected. this study was lower than that achieved by Salinivibrio sp. M318 (35.6 g/

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Fig. 1. TEM study of intracellular polymer granules by Salinivibrio sp. TGB10 cultivated in TYS/Glucose (A) and TYS (B) medium.

Fig. 2. Proton NMR spectrum of PHB (A) and PHBV (B) purified from Salinivibrio sp. TGB10.

Table 2 Table 3
Production of copolymers containing 3HB and 3 HV using propionate and Molecular weight of polymers produced by Salinivibrio sp. TGB10.
various carbohydrates.
Polymer Carbon source Mw (×103 Mn (×103 Mw/
Carbon CDW (g/ PHBV content PHBV (g/ 3HB 3 HV samples Da) Da) Mn
source L) (wt%) L) (mol%) (mol%)
PHB Glucose 344 74 4.7
G + PA(2)a 8.02 ± 50.88 ± 4.50 4.07 ± 72.72 27.28 PHBV Glucose +6 g/L 295 97 3.0
0.24 0.24 propionate
G + PA(4) 7.10 ± 45.29 ± 6.75 3.21 ± 51.65 48.35 PHBV Maltose +6 g/L 385 134 2.9
0.27 0.39 propionate
G + PA(6) 5.66 ± 40.83 ± 0.91 2.31 ± 34.87 65.13
Mw, weight-average molecular weight; Mn, number-average molecular weight.
0.47 0.16
G + PA(8) 4.89 ± 29.71 ± 4.81 1.45 ± 27.98 72.02
0.22 0.17 L) in fed-batch culture [30]. However, the final PHB content obtained by
M + PA(2) 6.38 ± 31.85 ± 1.28 2.03 ± 40.17 59.83
Salinivibrio sp. TGB10 (87.6 wt%) was much higher than that obtained
0.32 0.04
M+ PA(4) 6.44 ± 30.54 ± 1.69 1.96 ± 25.57 74.43
by Salinivibrio sp. M318 (51.5 wt%). Another salt-tolerant strain Hal­
0.37 0.04 omonas sp. YLGW01 was reported to accumulate PHB up to 94–95 wt%
M + PA(6) 6.67 ± 18.66 ± 2.52 1.24 ± 13.69 86.31 of its cell dry weight under unsterilized conditions, yielding the highest
0.63 0.21 PHB content among halotolerant bacteria [25]. The relatively high
M + PA(8) 5.24 ± 22.12 ± 3.56 1.16 ± 4.76 95.24
polymer content would significantly decrease the cost of downstream
0.02 0.19
purification process by using recovery method based on detergents
Shake flask experiments were performed at 30 ◦ C, 200 rpm with 20 g/L of instead of organic solvents [27].
carbohydrates and propionate for 24 h. Propionate was applied at different
concentrations (2 g/L, 4 g/L, 6 g/L, and 8 g/L). Data are expressed as averages
and standard deviations of three parallel experiments. G, glucose; M, maltose; 3.4. Fed-batch cultivation for PHBV production by Salinivibrio sp. TGB10
PA, propionate; CDW, cell dry weight; ND, not detected.
a
Propionate concentration, indicated as g/L. Salinivibrio sp. TGB10 was also cultivated in a 5-L bioreactor to
produce PHBV using TYS medium supplemented with glucose and
propionate as joint carbon sources (Fig. 4). The 3 HV monomer content
in PHBV copolymer decreased from 52 mol% to 31 mol% during the first

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Fig. 3. Glucose consumption, pyruvate concentration (A) and CDW, PHB production (B) profiles of Salinivibrio sp. TGB10 cultivated in TYS/glucose medium in fed-
batch fermentation.

Fig. 4. Glucose/propionate consumption, pyruvate concentration (A) and CDW, PHBV production (B) profiles of Salinivibrio sp. TGB10 cultivated in TYS/glucose/
propionate medium in fed-batch fermentation.

12 h of fermentation, and kept stable at approximately 20 mol% after 36 the promising potential to produce PHBV for industrial purposes.
h. The strain exhibited slower cell growth as the toxicity of propionate.
At the end of 108 h fermentation, Salinivibrio sp. TGB10 consumed 4. Conclusions
99.09 g/L glucose and 20.17 g/L propionate, reached 33.45 g/L CDW
and 27.36 g/L poly(3HB-co-22.62 mol% 3 HV) accumulation (Fig. 4). The present study showed that a moderately halophilic strain Sali­
The conversion efficiency of propionate was 23%, indicating that a large nivibrio sp. TGB10 was capable of producing PHA from a series of car­
proportion of propionate was metabolized for cell growth. Previously, bohydrates and volatile fatty acids. In shake flask cultures, Salinivibrio
the knockout of 2-methylcitrate synthase gene prpC in Halomonas TD01 sp. TGB10 was found to effectively convert glucose to PHB, achieving
prevented the degradation of propionate and improved conversion ef­ PHB titer of 6.84 g/L. Copolymers of PHBV was synthesized when pro­
ficiency of propionate up to 96% [28]. Further engineering of the fatty pionate was added as the secondary carbon source to generate 3 HV
acid metabolic pathway in Salinivibrio sp. TGB10 would probably help to precursor. In fed-batch fermentation with glucose and glucose/propio­
increase the substrate utilization efficiency. The final pyruvate titer was nate as the substrates, the titers of PHB and PHBV reached 26.34 g/L and
5.81 g/L, similar with that in PHB fermentation. As far as we know, this 27.36 g/L, respectively. The polymer content was over 80 wt% of cell
is the highest reported PHBV production titer using Salinivibrio species as dry weight. This study highlights Salinivibrio sp. TGB10 as a promising
the host. Although the accumulated polymers titers in Salinivibrio spe­ strain for PHA production.
cies was relatively low, further optimization of fermentation process
would lead to improvements on both polymer titer and productivity. CRediT authorship contribution statement
Up to now, the halotolerant microorganisms reported for PHA pro­
duction mainly belong to several genera such as Halomonas [25,33,37], Guan-Bao Tao: Investigation, Methodology, Visualization.
Haloferax [20,21], Vibrio [23,38], and Neptunomonas [24,39]. Gener­ Bi-Wei Tan: Validation.
ally, propionate addition as assistant carbon source would lead to the Zheng-Jun Li: Supervision, Writing - Original Draft, Writing-
incorporation of 3 HV monomer and yield PHBV copolymer possessing Reviewing and Editing.
changeable material properties [12]. PHBV is more flexible and tougher
compared to PHB homopolymer, and has been commercially produced
Funding
since 1990s [40]. The halotolerant hyperproducers including Halomonas
sp. YLGW01 and N. concharum JCM17730 cannot synthesize PHBV with
This study was supported by Grants from the Ministry of Science and
the addition of propionate [24,25]. To this end, Salinivibrio sp. TGB10
Technology of China (2018YFA0900200), and the National Natural
screened in this study has favorable tolerance to propionate and shows
Science Foundation of China (31870075).

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Appendix A. Supplementary data [18] H. Ma, Y. Zhao, W. Huang, L. Zhang, F. Wu, J. Ye, G.Q. Chen, Rational flux-tuning
of halomonas bluephagenesis for co-production of bioplastic PHB and ectoine, Nat.
Commun. 11 (1) (2020) 3313.
Supplementary data to this article can be found online at https://doi. [19] X.R. Jiang, X. Yan, L.P. Yu, X.Y. Liu, G.Q. Chen, Hyperproduction of 3-hydroxy­
org/10.1016/j.ijbiomac.2021.07.038. propionate by halomonas bluephagenesis, Nat. Commun. 12 (1) (2021) 1513.
[20] T.Y. Huang, K.J. Duan, S.Y. Huang, C.W. Chen, Production of
polyhydroxyalkanoates from inexpensive extruded rice bran and starch by
References haloferax mediterranei, J. Ind. Microbiol. Biotechnol. 33 (8) (2006) 701–706.
[21] J. Han, J. Hou, F. Zhang, G. Ai, M. Li, S. Cai, H. Liu, L. Wang, Z. Wang, S. Zhang,
[1] S.B. Borrelle, J. Ringma, K.L. Law, C.C. Monnahan, L. Lebreton, A. McGivern, L. Cai, D. Zhao, J. Zhou, H. Xiang, Multiple propionyl coenzyme A-supplying
E. Murphy, J. Jambeck, G.H. Leonard, M.A. Hilleary, M. Eriksen, H.P. Possingham, pathways for production of the bioplastic poly(3-hydroxybutyrate-co-3-
H. De Frond, L.R. Gerber, B. Polidoro, A. Tahir, M. Bernard, N. Mallos, M. Barnes, hydroxyvalerate) in haloferax mediterranei, Appl. Environ. Microbiol. 79 (9)
C.M. Rochman, Predicted growth in plastic waste exceeds efforts to mitigate plastic (2013) 2922–2931.
pollution, Science 369 (2020) 1515–1518. [22] C. Liu, D.K. Baffoe, Y. Zhan, M. Zhang, Y. Li, G. Zhang, Halophile, an essential
[2] L. Zimmermann, A. Dombrowski, C. Völker, M. Wagner, Are bioplastics and plant- platform for bioproduction, J. Microbiol. Methods 166 (2019), 105704.
based materials safer than conventional plastics? In vitro toxicity and chemical [23] J.W. Hong, H.S. Song, Y.M. Moon, Y.G. Hong, S.K. Bhatia, H.R. Jung, T.R. Choi, S.
composition, Environ. Int. 145 (2020), 106066. Y. Yang, H.Y. Park, Y.K. Choi, Y.H. Yang, Polyhydroxybutyrate production in
[3] A. Anjum, M. Zuber, K.M. Zia, A. Noreen, M.N. Anjum, S. Tabasum, Microbial halophilic marine bacteria vibrio proteolyticus isolated from the korean peninsula,
production of polyhydroxyalkanoates (PHAs) and its copolymers: a review of Bioprocess Biosyst. Eng. 42 (4) (2019) 603–610.
recent advancements, Int. J. Biol. Macromol. 89 (2016) 161–174. [24] N. Pu, P. Hu, L.L. Shi, Z.J. Li, Microbial production of poly(3-hydroxybutyrate)
[4] K.W. Meereboer, M. Misra, A.K. Mohanty, Review of recent advances in the from volatile fatty acids using the marine bacterium neptunomonas concharum,
biodegradability of polyhydroxyalkanoate (PHA) bioplastics and their composites, Bioresour. Technol. Rep. 11 (2020), 100439.
Green Chem. 22 (17) (2020) 5519–5558. [25] Y.L. Park, S.K. Bhatia, R. Gurav, T.R. Choi, H.J. Kim, H.S. Song, J.Y. Park, Y.
[5] R. Tarrahi, Z. Fathi, M.Ö. Seydibeyoglu, E. Doustkhah, A. Khataee, H. Han, S.M. Lee, S.L. Park, H.S. Lee, Y.G. Kim, Y.H. Yang, Fructose based hyper
Polyhydroxyalkanoates (PHA): from production to nanoarchitecture, Int. J. Biol. production of poly-3-hydroxybutyrate from halomonas sp. YLGW01 and impact of
Macromol. 146 (2020) 596–619. carbon sources on bacteria morphologies, Int. J. Biol. Macromol. 154 (2020)
[6] S.T. Yu, C.C. Lin, J.R. Too, PHBV production by ralstonia eutropha in a continuous 929–936.
stirred tank reactor, Process Biochem. 40 (8) (2005) 2729–2734. [26] N. Pu, W. Li, Z.J. Li, Complete genome sequence of neptunomonas concharum
[7] H.R. Jung, J.M. Jeon, D.H. Yi, H.S. Song, S.Y. Yang, T.R. Choi, S.K. Bhatia, J. JCM17730T: an acetate assimilating bacterium isolated from a dead ark clam, Mar.
J. Yoon, Y.G. Kim, C.J. Brigham, Y.H. Yang, Poly(3-hydroxybutyrate-co-3- Genomics 53 (2020), 100754.
hydroxyvalerate-co-3-hydroxyhexanoate) terpolymer production from volatile [27] Y.L. Park, H.S. Song, T.R. Choi, S.M. Lee, S.L. Park, H.S. Lee, H.J. Kim, S.K. Bhatia,
fatty acids using engineered ralstonia eutropha, Int. J. Biol. Macromol. 138 (2019) R. Gurav, K. Park, Y.H. Yang, Revealing of sugar utilization systems in halomonas
370–378. sp. YLGW01 and application for poly(3-hydroxybutyrate) production with low-cost
[8] J. Jian, Z.J. Li, H.M. Ye, M.Q. Yuan, G.Q. Chen, Metabolic engineering for medium and easy recovery, Int. J. Biol. Macromol. 167 (2021) 151–159.
microbial production of polyhydroxyalkanoates consisting of high 3-hydroxyhex­ [28] X.Z. Fu, D. Tan, G. Aibaidula, Q. Wu, J.C. Chen, G.Q. Chen, Development of
anoate content by recombinant Aeromonas hydrophila, Bioresour. Technol. 101 halomonas TD01 as a host for open production of chemicals, Metab. Eng. 23 (2014)
(15) (2010) 6096–6102. 78–91.
[9] G. Sathiyanarayanan, S.K. Bhatia, H.S. Song, J.M. Jeon, J. Kim, Y.K. Lee, Y.G. Kim, [29] E. Mellado, E.R. Moore, J.J. Nieto, A. Ventosa, Analysis of 16S rRNA gene
Y.H. Yang, Production and characterization of medium-chain-length sequences of Vibrio costicola strains: description of Salinivibrio costicola gen. nov.,
polyhydroxyalkanoate copolymer from Arctic psychrotrophic bacterium comb. nov, Int. J. Syst. Bacteriol. 46 (3) (1996) 817–821.
pseudomonas sp. PAMC 28620, Int. J. Biol. Macromol. 97 (2017) 710–720. [30] D.V. Thuoc, D.N. My, T.T. Loan, K. Sudesh, Utilization of waste fish oil and glycerol
[10] S.K. Bhatia, D.H. Yi, H.J. Kim, J.M. Jeon, Y.H. Kim, G. Sathiyanarayanan, H.M. Seo, as carbon sources for polyhydroxyalkanoate production by salinivibrio sp. M318,
J.H. Lee, J.H. Kim, K. Park, C.J. Brigham, Y.H. Yang, Overexpression of succinyl- Int. J. Biol. Macromol. 141 (2019) 885–892.
CoA synthase for poly (3-hydroxybutyrate-co-3-hydroxyvalerate) production in [31] D. Tan, Q. Wu, J.C. Chen, G.Q. Chen, Engineering halomonas TD01 for the low-cost
engineered Escherichia coli BL21(DE3), J. Appl. Microbiol. 119 (3) (2015) production of polyhydroxyalkanoates, Metab. Eng. 26 (2014) 34–47.
724–735. [32] X. Kourilova, I. Pernicova, K. Sedlar, J. Musilova, P. Sedlacek, M. Kalina, M. Koller,
[11] J.M. Jeon, H.J. Kim, S.K. Bhatia, C. Sung, H.M. Seo, J.H. Kim, H.Y. Park, D. Lee, C. S. Obruca, Production of polyhydroxyalkanoates (PHA) by a thermophilic strain of
J. Brigham, Y.H. Yang, Application of acetyl-CoA acetyltransferase (AtoAD) in schlegelella thermodepolymerans from xylose rich substrates, Bioresour. Technol.
Escherichia coli to increase 3-hydroxyvalerate fraction in poly(3-hydroxybutyrate- 315 (2020), 123885.
co-3-hydroxyvalerate), Bioprocess Biosyst. Eng. 40 (5) (2017) 781–789. [33] M. Ilham, S. Nakanomori, T. Kihara, A. Hokamura, H. Matsusaki, T. Tsuge,
[12] M. Li, M.R. Wilkins, Recent advances in polyhydroxyalkanoate production: K. Mizuno, Characterization of polyhydroxyalkanoate synthases from halomonas
feedstocks, strains and process developments, Int. J. Biol. Macromol. 156 (2020) sp. O-1 and halomonas elongata DSM2581: site-directed mutagenesis and
691–703. recombinant expression, Polym. Degrad. Stabil. 109 (2014) 416–423.
[13] S.K. Bhatia, S.V. Otari, J.M. Jeon, R. Gurav, Y.K. Choi, R.K. Bhatia, A. Pugazhendhi, [34] K. Sudesh, H. Abe, Y. Doi, Synthesis, structure and properties of
V. Kumar, J.R. Banu, J.J. Yoon, K.Y. Choi, Y.H. Yang, Biowaste-to-bioplastic polyhydroxyalkanoates: biological polyesters, Prog. Polym. Sci. 25 (10) (2000)
(polyhydroxyalkanoates): conversion technologies, strategies, challenges, and 1503–1555.
perspective, Bioresour. Technol. 326 (2021). [35] T. Tsuge, Fundamental factors determining the molecular weight of
[14] Y. Zheng, J.C. Chen, Y.M. Ma, G.Q. Chen, Engineering biosynthesis of polyhydroxyalkanoate during biosynthesis, Polym. J. 48 (2016) 1051–1057.
polyhydroxyalkanoates (PHA) for diversity and cost reduction, Metab. Eng. 58 [36] S.J. Sim, K.D. Snell, S.A. Hogan, J. Stubbe, C. Rha, A.J. Sinskey, PHA synthase
(2019) 82–93. activity controls the molecular weight and polydispersity of polyhydroxybutyrate
[15] G.Q. Chen, I. Hajnal, H. Wu, L. Lv, J. Ye, Engineering biosynthesis mechanisms for in vivo, Nat. Biotechnol. 15 (1) (1997) 63–67.
diversifying polyhydroxyalkanoates, Trends Biotechnol. 33 (10) (2015) 565–574. [37] G.Q. Chen, X.R. Jiang, Next generation industrial biotechnology based on
[16] R. Mitra, T. Xu, H. Xiang, J. Han, Current developments on polyhydroxyalkanoates extremophilic bacteria, Curr. Opin. Biotech. 50 (2018) 94–100.
synthesis by using halophiles as a promising cell factory, Microb. Cell Factories 19 [38] C.C. Chien, C.C. Chen, M.H. Choi, S.S. Kung, Y.H. Wei, Production of poly-beta-
(1) (2020) 86. hydroxybutyrate (PHB) by vibrio spp. isolated from marine environment,
[17] J. Ye, W. Huang, D. Wang, F. Chen, J. Yin, T. Li, H. Zhang, G.Q. Chen, Pilot scale-up J. Biotechnol. 132 (3) (2007) 259–263.
of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production by halomonas [39] X.J. Liu, J. Zhang, P.H. Hong, Z.J. Li, Microbial production and characterization of
bluephagenesis via cell growth adapted optimization process, Biotechnol. J. 13 (5) poly-3-hydroxybutyrate by neptunomonas Antarctica, PeerJ 4 (2016), e2291.
(2018) 1800074. [40] G.Q. Chen, A microbial polyhydroxyalkanoates (PHA) based bio- and materials
industry, Chem. Soc. Rev. 38 (8) (2009) 2434–2446.

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