Bacterial Strain Isolated from High-Salt Environments Can Produce Large Amounts of New Polyhydroxyalkanoate (PHA)

Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 4, Pages: 1268-1273

J. Environ. Treat. Tech.

ISSN: 2309-1185

Journal web link: http://www.jett.dormaj.com

https://doi.org/10.47277/JETT/8(4)1273

Bacterial Strain Isolated from High-Salt

Environments Can Produce Large Amounts of New

Polyhydroxyalkanoate (PHA)

, 2

Ahmad Gholami , Younes Ghasemi , Aboozar Kazemi , Seyyedeh Narjes Abootalebi ,

Cambyz Irajie , Aida Ireji , Navid Omidifar , Milad Mohkam

Biotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Department of Pharmaceutical Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran

Devision of Intensive Care Unit, Department of Pediatrics, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

Medicinal and natural products chemistry Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

Received: 28/05/2020

Accepted: 24/08/2020

Published: 20/12/2020

Abstract

Since the major problem connected to the industrial production of Polyhydroxyalkanoates (PHAs) is their high production price, this

study was performed to inspect the new potential bacterial species for industrial PHA production. The bacterial samples were collected

during a screening program from Pink Salt Lake as an extreme environment in the south of Iran, Fars province. The PHA-producing

bacteria were isolated. Then further studies on different morphological, cultural, and physiological characteristics of isolates were

performed. Among the isolated microorganisms in this study, 18 of 143 bacteria were selected as PHA-producer microorganisms to be

studied for analysis along with a partial sequence of the 16S rRNA gene. This study introduces two bacteria; Bacillus endophyticus

BCCS 011 and Lysobacter sp. BCCS 052 as new potential PHA producer that has not been reported previously. They could be an ideal

option for cheaper PHAs production.

Keywords: Polyhydroxyalkanoate (PHA), Bacillus, Lysobacter, 16S rRNA, Extreme environment

Introduction¹

in bacteria (18). Therefore, they have a wide range of

applications, such as in the packaging industry, pharmacy,

medicine, agriculture, and food industry (19). As a result, the

fabrication of biodegradable polymers such as PHAs from

renewable sources is the need of the today, in the face of these

environmental facts.

Polyhydroxyalkanoates (PHA), a family of biopolymers

with diverse structures, are polyoxoesters of hydroxy alkanoic

acids which are synthesized by various bacteria to overcome

environmental stress (1). Originally, they are accumulated as

carbon and energy reserves by a variety of bacterial species

under nutrient (Phosphorus, Nitrogen, or Sulfur) depleted

circumstances with excess carbon (2). Based on the monomer

structures, PHA are divided into short-chain-length (SCL) PHA

commonly consisting of 3-hydroxypropionate (3HP), 3-

Production and marketing of PHAs have been restricted in

two ways. The first cope with the ability of bacteria in

accumulation of the polymer, while more than 300 bacterial

species have been found in PHA accumulation, but

accumulation levels in many of them is very low (20). On the

other hand, species such as Alcaligenes latus, Ralstonia

eutropha (formerly known as Alcaligenes eutrophus),

Pseudomonas putida, Pseudomonas oleovorans, recombinant

Escherichia coli and Azotobacter vinelandii have been

extensively investigated (21, 22). The second aspect, which has

restricted PHA production and marketing, is associated with

the high costs of the substrate (mainly carbon source) as

compared to those of petrochemical origin (23). New strains,

hydroxybutyrate (3HB) and

medium-chain-length (MCL)

hydroxyvalerate (3HV);

PHA containing 3-

hydroxyhexanoate (3HHx), 3 hydroxyheptanoate (3HHp) to 3-

hydroxytetradecanoate (3HTD) (3, 4). Many bacteria are

capable of producing PHAs in activated sludge, in high seas,

and extreme environments (5). PHAs are biocompatible,

biodegradable, and environmentally friendly thermoplastics as

compared to petroleum-based plastics that are harmful wastes

and take several years to degrade completely (6, 7). Because of

increasing global environmental concerns associated with

discarded petrochemical-based plastics (8-12), several studies

have been conducted on the development of an appropriate eco-

friendly material that can substitute at least some of the

commodity plastics (13-17). PHAs properties like conventional

plastics (especially polypropylene) have versatile plasticable

properties and are produced as high molecular mass polymers

using economic substrates along with having

a high

accumulation proportion, must thus be isolated to solve these

problems mentioned above. Interest has been a focus on Gram-

positive bacteria such as the genera Bacillus, where this

bacterium has chemoorganotrophic features(24-28), secretion

of a large number of amylases and proteases (29) and lack

lipopolysaccharide (LPS) (30). These properties of Bacillus

Corresponding author: Milad Mohkam Pharmaceutical Sciences Research Center, Shiraz University of Medical Sciences, Shiraz, Iran.

Tel.: +98 71 3242 6729; Fax: +98 71 3242 6729 and E-mail: miladmohkam@gmail.com

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 4, Pages: 1268-1273

spp. are of interest for exploring the possibility of utilizing

different agricultural raw materials as a carbon source for the

fabrication of various metabolites (24). Furthermore, the

genera of Bacillus are suitable model systems for the

heterologous expression of foreign genes related to PHA

manufacturing and numerous fine chemicals (31). In the

present study, PHB producing bacteria from a salty lake (Pink

Lake, Shiraz, Iran) were isolated, identified, and characterized

using morphological, biochemical, and molecular methods. To

our best knowledge, this is the first report of Bacillus

endophyticus along with Lysobacter sp., and Pantoea sp. as

PHA producers that can be industrially exploited for bioplastics

fabrication.

by using the Basic Local Alignment Search Tool (BLAST).

The nucleotide sequences of 16S rRNA genes were published

to GenBank under the accession numbers, as shown in table1.

Then the PHA extracted from all the PHA positive isolates

were analyzed by an spectroscopic methods, FT-IR.

1.4 FT-IR spectroscopy

The infrared (IR) spectrum was recorded using Bruker,

Vertex 70, FTIR spectrometer (34). The extracted PHA was

dissolved in chloroform, and the unfiltered solution was cast

onto NaCl crystal.

Results and discussion

In this study, a total of 143 bacterial isolates were examined

Materials and Methods

for isolation of PHA-producing bacteria from a salty lake (Pink

Lake). This seasonal lake also locally known as Maharloo is

located in the 27.0 km southeast of Shiraz, Iran, which is full

of potassium and other salts and looks pink (Fig. 1).

.1 Sample collection

Bacterial isolates were obtained from a salty lake "Pink

Lake" Fars province, in the south of Iran. Briefly, liquid

samples were collected and transferred in sterile tubes at the

ice. The samples were then serially diluted with sterile distilled

water, and then 200 µL of the dilution was spread on a sterile

Nutrient Agar (NA) plates. The plates were incubated at pH=7

and 30 °C for two days. Various colonies of different

morphology, including color, form, and edge appearance, were

individually picked and subcultured 3-4 times on nutrient agar

plates. Pure bacterial isolates were achieved by subculturing

individual colonies several times on a fresh NA medium to gain

single colonies. Agar slants of these colonies were kept at 4 °C

for one month.

Of these isolates, 21 of them were positive for PHA using

Sudan Black B and then confirmed by Nile Blue A. Sudan

Black B was used as the first-line screening for PHA-

accumulating bacteria when they were cultured in an

unbalanced growth medium. It was assumed that the negatively

stained of isolates with Sudan Black B did not form lipid

granules and thus also did not produce PHA due to the lipidic

feature of polyester. Sudan black B stains PHA non-

specifically as well as for other lipid bodies. In contrast, Nile

Blue A is more specific than Sudan black B for PHA detection.

Therefore, Nile Blue A was used for confirmation of PHA-

accumulating bacteria. The bacterial flora is categorized into

two groups, according to their Gram's reaction. In general, the

Gram-positive bacteria tended to dominate the salty lake,

nearly 66.67% of the total 21 PHA-producing isolates showed

Gram-positive character. The result of PCR blasted with other

sequenced bacteria in NCBI showed a similarity of more than

95% to the 16S rRNA of other bacteria. Various

microbiological and biochemical tests were carried out as a

means for the identification of native strains (Table 1). Among

21 of PHA-producing isolates, 13 isolates were belonged to

Bacillus and one isolate to Halobacterium genera along with

other Gram-negative bacteria (Table 2). These isolates

(Bacillus spp. and Halobacterium) showed strong growth in

10% (w/v) salt concentration suggesting that they could tolerate

relatively salt concentration, which is consistent with the

natural condition of this salty lake. These bacteria tend to

accumulate PHA, which is commonly consumed by the

bacterium itself when the growth circumstances are

unfavorable. The PHA-positive isolates opted after Nile blue A

staining and then grown in an E2 broth medium containing 2%

(w/v) glucose in 100-ml flasks, and were employed to extract

PHA after two days of incubation on a rotary shaker. The PHA

from the isolates was extracted by the chloroform method,

developed by Vizcaino-Caston et al. (35). The concentration

of PHA in the E2 medium and PHA% of cell dry weight along

with cell dry weight achieved for various positively stained

isolates are depicted in Table 2. These isolates produced PHA

from 0.031 to 0.34 g/l, amounting to about 2.16–23.13% PHA

of cell dry weight (Table 2).

.2 Screening the PHA-producing bacteria

All the bacterial isolates were qualitatively examined for

PHA production by Sudan Black B dye to detect the existence

of lipid granules in the bacteria (32). A positively-stained

isolate was considered a potential PHA producer and cultured

in the modified E2 medium, a nitrogen-limiting medium

containing 2% (w/v) glucose (20 g/l of Glucose), 0.9 gr/l of

2 4

Cl, 0.9 gr/l of NaCl, 5.22 gr/l of K HPO , 3.7 gr/l of

PO , 0.246 gr/l of MgSO .7H O, and 1ml of MT

microelement (1 liter of MT stock contains 2.78 gr of

FeSO .7H O, 1.98 gr of MnCl .4H O, 2.81 gr of CoCl .6H O,

.47 gr of CaCl , 0.17 gr of CuSO .5H O and 0.29 gr of

ZnSO .7H O) at 37°C for 72h. Then all the isolates were

stained by Nile blue a dye to confirm the PHA production.

.3 Identification of Bacterial isolates

The PHA-positive bacterial isolates were identified

according to conventional biochemical tests and by partially

sequencing the ribosomal 16s RNA gene. Total genomic DNA

was extracted according to Gholami et al. and was used as a

Chromosomal DNA template for the amplification of the 16s

RNA gene (33). The following oligonucleotide sequences 5'-

ACGGGCGGTGTGTAC -3' were used as forward, and 5'-

CAGCCGCGGTAATAC-3' reverse primers. The PCR

products were purified and then sequenced by CinnaGen

Company (Tehran, Iran). The resulting 16S rRNA gene

sequences were aligned and compared to the nucleotide

sequences of some known microorganisms in the GenBank

database of the National Center for Biotechnology Information

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2020, Volume 8, Issue 4, Pages: 1268-1273

Figure 1: Satellite image of salty Pink Lake

Table 1: The morphological and biochemical traits used for classification of 21 selected PHA-producer isolates

Morphology

Gelatinase production

Catalase production

Oxidase production

Lipase production

Hippurate hydrolysis

Esculin hydrolysis

Cell shape

Cell size

Motility

Gram staining

Endospore

Spore shape

Acid production from

Spore position

Cultural characteristics

Colony shape

Optimum pH

Glucose

Galactose

Fructose

Mannitol

Maltose

Sucrose

Optimum temperature

Growth on nutrient agar

Growth on McConkey agar

Growth on Eosin methylene blue agar

Growth at 5, 20 and 50 °C

Growth in NaCl 2.5–7.0%

Utilization of

Succinate

Citrate

Resistance to antibiotics

Erythromycin

Neomycin

Biochemical properties

Urease production

Nitrate reduction

Voges-Proskauer

Arginine hydrolysis

Casein hydrolysis

Lecithinase production

HCN production

Novabiocin

Tetracycline

Kanamycin

Chloramphenicol

Ampicillin

Tetracycline

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2020, Volume 8, Issue 4, Pages: 1268-1273

Table 2: The amount (mg/ml) and the weight yield (w %) of PHA of production, the accession numbers and the concentration of

obtained from the screened bacteria which were isolated from Maharlu Lake.

Bacteria

Dry cell weight (gr/l)

1.36

PHA.con (gr/l)

0.24

% PHA (CDW)

Bacillus endophyticus BCCS 011

Bacillus sp. BCCS 060

17.64

8.39

8.63

3.9

0.786

0.926

3.24

0.066

0.08

Bacillus subtilis BCCS 028

Bacillus subtilis BCCS 033

Bacillus sp. BCCS 036

0.126

0.128

0.09

0.926

0.973

1.4

13.82

9.4

Halobacterium sp. BCCS 030

Bacillus subtilis BCCS 031

Bacillus endophyticus BCCS 024

Bacillus pumilus BCCS 002

Bacillus subtilis BCCS 005

Bacillus subtilis BCCS 012

Pantoea sp. BCCS 053

0.033

0.034

0.10

2.3

1.36

2.5

0.686

1.03

15.4

0.04

0.66

0.085

0.05

0.613

1.14

8.4

Escherichia coli BCCS 054

Escherichia coli BCCS 055

Aeromonas sp. BCCS 056

Bacillus sp. BCCS 057

0.045

0.106

0.038

0.05

3.93

11.4

1.8

0.933

2.166

0.793

1.866

1.286

1.46

6.4

Aeromonas sp. BCCS 058

Bacillus sp. BCCS 059

0.07

0.113

0.026

0.046

8.8

Bacillus sp. BCCS 061

1.8

Lysobacter sp. BCCS 052

1.466

3.2

FTIR spectroscopy of the polymer producing bacteria was

investigated along with Poly(R)-3-hydroxybutyric acid (PHB)

prepared from Sigma-Aldrich (cat no: 363502). The polymer

extracted illustrated the intense absorption characteristic for

ester carbonyl (C=O) stretching groups at 1720, 2325 and 2985

cm corresponding to the –CH group in comparison with the

PHB (Fig. 2).

An important finding was that all Bacillus spp could able

to produce a high amount of PHA in comparison to other

bacterial species. To our knowledge, this study introduces a

new species of Bacillus named Bacillus endophyticus that has

a high ability to produce PHA (23.13% of CDW) as compared

to other isolates. The Bacillus spp. are critical industrial

bacteria for the production of such enzymes and also for PHAs

production.

The

properties,

including

lack

lipopolysaccharides (LPS) and the ability to consume a wide

range of cheap carbon sources, made this bacterium a suitable

candidate for the production of PHAs, especially for medical

implant purposes (33). Moreover, recovery of approximately

Figure 2: FT-IR spectra of standard PHB and PHA produced by

isolated bacterial strain

3 Conclusion

0 to 90% of bacterial dry biomass as PHA production is

In conclusion, different bacterial strains were isolated from

an extremely salty lake and screened for polyhydroxyalkanoate

production, and bacterial isolates were identified and

characterized using morphological, biochemical, and

molecular methods in this study. Within this extremely

halophilic environment, most PHA-producing strains were

from genus bacillus. Besides, other strains such as Pantoea sp.,

Halobacterium sp., and Lysobacter sp. have a high potential for

the conversion of carbohydrates into PHB. Among the isolated

strains, Bacillus endophyticus was able to produce the highest

amount of polyhydroxy butyrate (23.13% of the cell dry mass,

which is very important from an industrial point of view.

Presently, the selected microorganisms are further considered

to enhance the production of polyhydroxyalkanoates by the

optimization of the process factors. In order for the industrial

potentially enough for determining an economically feasible

process (2).Most interestingly, this research also identifies a

novel bacterium Lysobacter, which has not been reported as a

PHA producer so far. This bacterium is considered a rich source

for the fabrication of novel antibiotics, such as macrocyclic

lactams, β-lactams containing substituted side chains and

macrocyclic peptides (36). The ease of genetic manipulation

and able to occupy a wide range of ecological niches, including

a broad range of extreme environments, provides this

bacterium an ideal candidate for PHA production (37).

Therefore, such studies are needed for the development of these

new potential strains for commercial PHA production.

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Journal of Environmental Treatment Techniques

2020, Volume 8, Issue 4, Pages: 1268-1273

production of this environmentally valuable product, it is

critical to produce more cost-effectively and to achieve greater

competitiveness with similar petroleum products that severely

pollute the environment.

bacteria growth, physical and mechanical properties. Polymers

from Renewable Resources. 2017;8(4):177-96.

1. Mousavi SM, Hashemi SA, Jahandideh S, Baseri S, Zarei M,

Azadi S. Modification of phenol novolac epoxy resin and

unsaturated polyester using sasobit and silica nanoparticles.

Polymers from Renewable Resources. 2017;8(3):117-32.

Acknowledgment

12. Mousavi SM, Zarei M, Hashemi SA, Lai CW, Bahrani S. K-Ion

Battery Practical Application Toward Grid-Energy Storage.

Potassium-ion Batteries: Materials and Applications. 2020:43.

This work was supported by the Research Council of Shiraz

University of Medical Sciences, Shiraz, Iran.

3. Bahrani S, Hashemi SA, Mousavi SM, Azhdari R. Zinc-based

metal–organic frameworks as nontoxic and biodegradable

platforms for biomedical applications: review study. Drug

metabolism reviews. 2019;51(3):356-77.

4. Goudarzian N, Hashemi S, Mirjalili M. Unsaturated polyester

resins modified with cresol novolac epoxy and silica nanoparticles:

processing and mechanical properties. Int J Chem Pet Sci.

Ethical issue

Authors are aware of, and comply with, best practice in

publication ethics specifically with regard to authorship

(avoidance of guest authorship), dual submission, manipulation

of figures, competing interests and compliance with policies on

research ethics. Authors adhere to publication requirements

that submitted work is original and has not been published

elsewhere in any language.

016;5(1):13-26.

5. Hashemi SA, Mousavi SM, Ramakrishna S. Effective removal of

mercury, arsenic and lead from aqueous media using Polyaniline-

Fe3O4-silver diethyldithiocarbamate nanostructures. Journal of

Cleaner Production. 2019;239:118023.

6. Mousavi S, Zarei M, Hashemi S. Polydopamine for biomedical

application and drug delivery system. Med Chem (Los Angeles).

Competing interests

The authors declare that there is no conflict of interest that

would prejudice the impartiality of this scientific work.

018;8:218-29.

7. Mousavi SM, Hashemi SA, Salahi S, Hosseini M, Amani AM,

Babapoor A. Development of clay nanoparticles toward bio and

medical applications. Current Topics in the Utilization of Clay in

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polyhydroxyalkanoate thermoplastics substituting xenobiotic

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Authors’ contribution

All authors of this study have a complete contribution for

data collection, data analyses and manuscript writing.

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