# Production of Poly(3-Hydroxybutyrate) byHaloarcula, Halorubrum, and Natrinema Haloarchaeal Genera Using Starch as a Carbon Source

Authors: Fatma Karray; Manel Ben Abdallah; Nidhal Baccar; Hatem Zaghden; Sami Sayadi
Journal: Archaea
 (2021)

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## Abstract

Microbial production of bioplastics, derived from poly(3-hydroxybutyrate) (PHB), have provided a promising alternative towards plastic pollution. Compared to other extremophiles, halophilic archaea are considered as cell factories for PHB production by using renewable, inexpensive carbon sources, thus decreasing the fermentation cost. This study is aimed at screening 33 halophilic archaea isolated from three enrichment cultures from Tunisian hypersaline lake, Chott El Jerid, using starch as the sole carbon source by Nile Red/Sudan Black staining and further confirmed by PCR amplification ofphaC and phaE polymerase genes. 14 isolates have been recognized as positive candidates for PHA production and detected during both seasons. The identification of these strains through 16S rRNA gene analyses showed their affiliation to Halorubrum, Natrinema, and Haloarcula genera. Among them, three PHB-producing strains, CEJ34-14, CEJ5-14, and CEJ48-10, related to Halorubrum chaoviator, Natrinema pallidum, and Haloarcula tradensis were found to be the best ones reaching values of 9.25, 7.11, and 1.42% of cell dry weight (CDW), respectively. Our findings highlighted that Halorubrum, Natrinema, and Haloarcula genera were promising candidates for PHB production using soluble starch as a carbon source under high salinity (250 g L-1 NaCl).


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## Body

## 1. Introduction

Plastic is a highly useful material, and its production is growing. Million tons of nondegradable plastics end up in our natural environment every year affecting our health, wildlife, terrestrial, and marine habitats [1]. For this reason, polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polymers which have been promoted as an alternative to conventional oil-based plastics [2, 3]. PHAs are synthesized by a wide variety of bacteria and archaea from various carbon sources and served as intracellular storage compounds to survive under unbalanced conditions [4, 5]. Extremely halophilic archaea, inhabiting hypersaline environments containing high salt concentrations, are preferred for various potential applications, including the synthesis of polyhydroxyalkanoates. Poly(3-hydroxybutyrate) (PHB) was the first bioplastic found in species of Dead Sea Halobacterium described in 1972 [6]. Since then, microorganisms including extremely halophilic archaea belonging to the genera Haloquadratum, Halorubrum, Halobacterium, Haloterrigena, Haloferax, Natronococcus, Natronobacterium, Haloarcula, Natrinema, Halogeometricum, Halopiger, Halobiforma, and Halococcus have also been found to accumulate considerable amounts of PHB [7–9] using colony/cell staining methods, molecular tools targeting PHA synthase genes, and analytical techniques such as Fourier transform infrared spectroscopy, crotonic acid assays, gas chromatography, and liquid chromatography [10].These organisms could utilize various renewable carbon sources and release the PHB easily by cell lysis in distilled water, thus lowering its high production cost [11]. Several recent investigations have shown that the productivities of PHB by archaeal strains were obtained using glucose as the sole carbon source [9, 12, 13] than with other substrates (wastes and pretreated vinasse) [14, 15]. However, few studies based on the exploration of PHB production by archaeal species using starch have been reported, despite its wide availability [16, 17]. Therefore, it was important to select strains able to use starchy substrates for PHB biosynthesis.Chott El Jerid, the largest Salt Lake, is located in the south of Tunisia. Furthermore, this lake is the biggest one in the north of Africa (5360 km2) with a salt concentration above 33% NaCl [18]. It has a thalassohaline salt composition, despite its continental origin. It may be flooded in the winter and evaporates to a desert in the dry season. These climatic conditions make the Chott an ephemeral extreme environment. Our previous studies described microbial diversity in Chott El Jerid during wet and dry seasons, using culture and molecular methods. Halophilic anaerobic fermentative bacterial strains were isolated from surface sediments [19, 20]. Culture-independent techniques, targeting 16S rRNA and functional markers encoding the dissimilatory sulfite reductase beta-subunit gene (dsrB), methyl coenzyme M reductase (mcrA), showed abundant and diverse prokaryotic communities, sulfate-reducing bacteria (SRB), and methanogens, respectively [20, 21]. Additionally, the isolation of halophilic aerobic bacterial and archaeal strains producing extracellular hydrolases have been achieved in Chott El Jerid [22]. Because Archaea outnumbered Bacteria in the studied samples, our purpose, here, is to expand the possibility of PHA synthesis by Archaea from Chott El Jerid. The objectives of this research were (1) enrichment, isolation, and screening PHA-accumulating halophilic archaea in water or water/sediment mix samples collected from Chott El Jerid in both seasons using phenotypic and molecular methods; (2) identification and characterization of PHA-producing isolates, utilizing starch as the carbon source; and (3) identification and quantification of the polymer produced.


## 2. Materials and Methods

### 2.1. Sample Collection

The samples (S1-10) and (S6M-14; S6W-14) were collected in dry (October 2010) and wet seasons (January 2014) from a continental ephemeral lake, Chott El Jerid, respectively. Hypersaline water or water/sediment mix samples were collected at different locations, approximately 0-10 cm from the surface. The samples were collected in sterile bottles, brought to the laboratory within three hours, and kept aseptically at 4°C until analyses. The environmental parameters of sampling sites are listed in Table1 as previously reported [20].Table 1
Sampling locations, conditions, and distribution of halophilic isolates.
Sample typeSampleSiteGeographical locationpHSalinity (%)Temperature (°C)Total number of isolatesWater and sedimentsS1-10Site 133° 54′ 42.21″ N 8° 31′ 7.98″ E6.6134.62311Water and sedimentsS6M-14Site 233° 54′ 44.15″ N 8° 31′ 9.01″ E7.6127.61920Hypersaline waterS6W-14Site 233° 54′ 44.15″ N 8° 31′9.01″ E7.6127.6192


### 2.2. Enrichment and Isolation of Polyhydroxyalkanoic Acids (PHA) Producing Archaea

Samples were enriched by culturing in PHA-accumulating medium (per liter NaCl, 250 g; MgCl2.6 H2O, 10 g; MgSO4.7 H2O, 15 g; KCl, 4 g; CaCl2, 2 H2O, 1 g; NaHCO3, 0.5 g; yeast extract, 1 g) supplemented with 1% soluble starch at 37°C for 7 days at 180 rpm.In order to isolate PHA-producing haloarchaea, samples were serially diluted and 100μL of each dilution were plated onto agar medium (20 g L-1) as described above. Following incubation, 33 colonies from the plates were picked as a result of their pigmentation and/or morphology and were transferred onto fresh plates several times until pure culture was obtained. Colonies were differentiated by colour, shape, and edge appearance. Morphological features of cells were examined using oil immersion at 100x objective (Optika, B-500 ERGO Model, Italy).


### 2.3. Extraction of Genomic DNA

When the growth of archaeal cultures reached the exponential phase, the extraction of genomic DNA was performed using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies Sdn Bhd, Selangor DE, Malaysia) according to the manufacturer’s instructions.


### 2.4. Identification of Archaeal Strains

PCR amplification was achieved using the primer set 21F (5′-TTCCGGTTGATCCYGCCGGA-3′) [23] and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [24]. The PCR reaction was realized in a 50 μL mixture containing 1.25 U of Taq polymerase (Fermentas), 1x PCR buffer, 200 μM of dNTP, 0.2 μM of each primer, and 50 ng of genomic DNA. Thirty cycles (1 min 94°C; 1 min 55°C; 2 min 72°C) were carried out using a thermocycler (Applied Biosystems, USA). The amplified PCR products of size 1500 bp were analyzed by electrophoresis in 1% agarose gels and photographed with a Gel Doc XR Imaging system (Bio-Rad). Then, the restriction analysis was performed by digesting 10 μL of PCR products with 10 U of restriction enzymes AluI and MboI while HaeIII (8 U μL-1) and the appropriate restriction buffer in a final volume of 20 μL for 3 hours at 37°C. These enzymes (Life Technologies), frequently used in restriction analysis, gave the most significant differences between species. 16S rRNA fragments, acquired after enzymatic digestion, were separated on 3% agarose gels for 4 h at 50 V and photographed with a Gel Doc system. The original PCR products of positive isolates selected by comparing enzymatic restriction patterns and described as PHA-producing isolates in the following section were purified with PureLink® Quick Gel Extraction and PCR Purification Combo Kit (Cat. No. K220001, Invitrogen, Carlsbad, USA) following the manufacturer’s instructions prior to cloning. The purified PCR products were ligated into pGEM-T easy (Promega Corporation, Madison, WI) system as recommended by the manufacturer. The ligation mixture was transformed into DH5α competent cells. Recombinant plasmids were verified by EcoRI digestion and chosen for sequencing. Sequencing and phylogenetic analysis were performed as previously reported [25].


### 2.5. Screening of Potential Halophilic PHA Producers

All isolates were subjected to PHA screening by Sudan Black B (SBB) and confirmed by Nile Red (NR) staining (Sigma), a more specific stain. Staining of colonies was done using 0.3% alcoholic solution of Sudan Black B. PHA-producing colonies appeared black [26]. Nile Red stain (25% (w/v) stock solution in dimethylsulfoxide (DMSO)) was directly inoculated in duplicate (0.5 μg mL-1 (w/v)) in agar medium containing 1% (w/v) starch and growth cells occurred in the presence of the dye. The strain Escherichia coli was used as a negative control. Natrinema altunense strain CEJGTEA101 [KY129977] was identified as a PHA-producing isolate in our previous work [9] and was used as a positive control in this study. After 15 days of incubation at 37°C, the isolates which revealed orange fluorescence after exposure of plates to UV light were selected as PHA accumulators [27]. Staining of promising cells with Nile Red was done using a fluorescence microscope (Olympus BX51) [28].In parallel, all isolates were screened for their genetic potential for polyhydroxyalkanoate production. The genes responsible for PHA production in haloarchaea were clustered in the class III synthases which were constituted of two subunits (PhaE and PhaC). PCR technique was used for screening archaeal polyhydroxyalkanoate producers using two pairs of codehops (codehopEF/codehopER) and (codehopCF/codehopCR), according to the highly conserved regions in PhaE and PhaC, respectively [29, 30]. The primers codehopEF (5′-CGACCGAGTTCCGCGAYATHTGGYT-3′) and codehopER (5′-GCGTGCTGGCGGCKYTCNAVYTC-3′) were used to amplify the PhaE polymerase gene. The amplification of the PhaC polymerase gene was performed using the primers codehopCF (5′-ACCGACGTCGTCTACAAGGARAAYAARYT-3′) and codehopCR (5′-GGTCGCGGACGACGTCNACRCARTT-3′) [30]. The PCR condition was as follows: after initial denaturation (94°C for 5 min), 30 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s were performed, followed by a final extension (10 min, 72°C). PCR amplification was run on a thermocycler (Applied Biosystems) using 1.25 U of Taq DNA polymerase (Fermentas), 1x PCR buffer, 0.2 μM of each primer, 200 μM of DNTP, and 50 ng DNA template. The PCR products were subjected to electrophoresis using 2% agarose gels.


### 2.6. Growth Kinetics of Potential PHA Producers

The strains were cultivated in 50 mL of PHA-producing medium amended with 10 g L-1 starch. The cultures in Erlenmeyer flasks were incubated in duplicate at 37°C, 180 rpm. Absorbance at 600 nm was determined using a UV-visible spectrophotometer (Shimadzu UV-1800, Japan) each 24 h for each PHA-producing strain.


### 2.7. Determination of Cell Dry Weight (CDW)

Cultures grown to late logarithmic phase were centrifuged at 6000 rpm for 30 min; then, cells were washed twice with distilled water. The supernatant was discarded leaving the pellet, which was lyophilized and weighed.


### 2.8. Determination of PHB Content in Potential Halophilic PHA Producers by Gas Chromatography (GC)

After methanolysis, the cellular content of the polymer and its composition were assayed by gas chromatography (GC) using an Agilent Technologies 7890A chromatograph, equipped with a capillary column (30m×0.25mm×0.25μm) and a flame ionization detector (FID) as previously reported [31]. Samples were analyzed in duplicate. The standard PHB (Sigma-Aldrich, USA) was used for calibration. The PHB content in the cells was determined as massofPHB/celldrymass×100%. The peak at 4.4 min represents the 3-hydroxybutyrate methylester.


## 2.1. Sample Collection

The samples (S1-10) and (S6M-14; S6W-14) were collected in dry (October 2010) and wet seasons (January 2014) from a continental ephemeral lake, Chott El Jerid, respectively. Hypersaline water or water/sediment mix samples were collected at different locations, approximately 0-10 cm from the surface. The samples were collected in sterile bottles, brought to the laboratory within three hours, and kept aseptically at 4°C until analyses. The environmental parameters of sampling sites are listed in Table1 as previously reported [20].Table 1
Sampling locations, conditions, and distribution of halophilic isolates.
Sample typeSampleSiteGeographical locationpHSalinity (%)Temperature (°C)Total number of isolatesWater and sedimentsS1-10Site 133° 54′ 42.21″ N 8° 31′ 7.98″ E6.6134.62311Water and sedimentsS6M-14Site 233° 54′ 44.15″ N 8° 31′ 9.01″ E7.6127.61920Hypersaline waterS6W-14Site 233° 54′ 44.15″ N 8° 31′9.01″ E7.6127.6192


## 2.2. Enrichment and Isolation of Polyhydroxyalkanoic Acids (PHA) Producing Archaea

Samples were enriched by culturing in PHA-accumulating medium (per liter NaCl, 250 g; MgCl2.6 H2O, 10 g; MgSO4.7 H2O, 15 g; KCl, 4 g; CaCl2, 2 H2O, 1 g; NaHCO3, 0.5 g; yeast extract, 1 g) supplemented with 1% soluble starch at 37°C for 7 days at 180 rpm.In order to isolate PHA-producing haloarchaea, samples were serially diluted and 100μL of each dilution were plated onto agar medium (20 g L-1) as described above. Following incubation, 33 colonies from the plates were picked as a result of their pigmentation and/or morphology and were transferred onto fresh plates several times until pure culture was obtained. Colonies were differentiated by colour, shape, and edge appearance. Morphological features of cells were examined using oil immersion at 100x objective (Optika, B-500 ERGO Model, Italy).


## 2.3. Extraction of Genomic DNA

When the growth of archaeal cultures reached the exponential phase, the extraction of genomic DNA was performed using the GF-1 Nucleic Acid Extraction Kit (Vivantis Technologies Sdn Bhd, Selangor DE, Malaysia) according to the manufacturer’s instructions.


## 2.4. Identification of Archaeal Strains

PCR amplification was achieved using the primer set 21F (5′-TTCCGGTTGATCCYGCCGGA-3′) [23] and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [24]. The PCR reaction was realized in a 50 μL mixture containing 1.25 U of Taq polymerase (Fermentas), 1x PCR buffer, 200 μM of dNTP, 0.2 μM of each primer, and 50 ng of genomic DNA. Thirty cycles (1 min 94°C; 1 min 55°C; 2 min 72°C) were carried out using a thermocycler (Applied Biosystems, USA). The amplified PCR products of size 1500 bp were analyzed by electrophoresis in 1% agarose gels and photographed with a Gel Doc XR Imaging system (Bio-Rad). Then, the restriction analysis was performed by digesting 10 μL of PCR products with 10 U of restriction enzymes AluI and MboI while HaeIII (8 U μL-1) and the appropriate restriction buffer in a final volume of 20 μL for 3 hours at 37°C. These enzymes (Life Technologies), frequently used in restriction analysis, gave the most significant differences between species. 16S rRNA fragments, acquired after enzymatic digestion, were separated on 3% agarose gels for 4 h at 50 V and photographed with a Gel Doc system. The original PCR products of positive isolates selected by comparing enzymatic restriction patterns and described as PHA-producing isolates in the following section were purified with PureLink® Quick Gel Extraction and PCR Purification Combo Kit (Cat. No. K220001, Invitrogen, Carlsbad, USA) following the manufacturer’s instructions prior to cloning. The purified PCR products were ligated into pGEM-T easy (Promega Corporation, Madison, WI) system as recommended by the manufacturer. The ligation mixture was transformed into DH5α competent cells. Recombinant plasmids were verified by EcoRI digestion and chosen for sequencing. Sequencing and phylogenetic analysis were performed as previously reported [25].


## 2.5. Screening of Potential Halophilic PHA Producers

All isolates were subjected to PHA screening by Sudan Black B (SBB) and confirmed by Nile Red (NR) staining (Sigma), a more specific stain. Staining of colonies was done using 0.3% alcoholic solution of Sudan Black B. PHA-producing colonies appeared black [26]. Nile Red stain (25% (w/v) stock solution in dimethylsulfoxide (DMSO)) was directly inoculated in duplicate (0.5 μg mL-1 (w/v)) in agar medium containing 1% (w/v) starch and growth cells occurred in the presence of the dye. The strain Escherichia coli was used as a negative control. Natrinema altunense strain CEJGTEA101 [KY129977] was identified as a PHA-producing isolate in our previous work [9] and was used as a positive control in this study. After 15 days of incubation at 37°C, the isolates which revealed orange fluorescence after exposure of plates to UV light were selected as PHA accumulators [27]. Staining of promising cells with Nile Red was done using a fluorescence microscope (Olympus BX51) [28].In parallel, all isolates were screened for their genetic potential for polyhydroxyalkanoate production. The genes responsible for PHA production in haloarchaea were clustered in the class III synthases which were constituted of two subunits (PhaE and PhaC). PCR technique was used for screening archaeal polyhydroxyalkanoate producers using two pairs of codehops (codehopEF/codehopER) and (codehopCF/codehopCR), according to the highly conserved regions in PhaE and PhaC, respectively [29, 30]. The primers codehopEF (5′-CGACCGAGTTCCGCGAYATHTGGYT-3′) and codehopER (5′-GCGTGCTGGCGGCKYTCNAVYTC-3′) were used to amplify the PhaE polymerase gene. The amplification of the PhaC polymerase gene was performed using the primers codehopCF (5′-ACCGACGTCGTCTACAAGGARAAYAARYT-3′) and codehopCR (5′-GGTCGCGGACGACGTCNACRCARTT-3′) [30]. The PCR condition was as follows: after initial denaturation (94°C for 5 min), 30 cycles of 94°C for 30 s, 55°C for 45 s, and 72°C for 45 s were performed, followed by a final extension (10 min, 72°C). PCR amplification was run on a thermocycler (Applied Biosystems) using 1.25 U of Taq DNA polymerase (Fermentas), 1x PCR buffer, 0.2 μM of each primer, 200 μM of DNTP, and 50 ng DNA template. The PCR products were subjected to electrophoresis using 2% agarose gels.


## 2.6. Growth Kinetics of Potential PHA Producers

The strains were cultivated in 50 mL of PHA-producing medium amended with 10 g L-1 starch. The cultures in Erlenmeyer flasks were incubated in duplicate at 37°C, 180 rpm. Absorbance at 600 nm was determined using a UV-visible spectrophotometer (Shimadzu UV-1800, Japan) each 24 h for each PHA-producing strain.


## 2.7. Determination of Cell Dry Weight (CDW)

Cultures grown to late logarithmic phase were centrifuged at 6000 rpm for 30 min; then, cells were washed twice with distilled water. The supernatant was discarded leaving the pellet, which was lyophilized and weighed.


## 2.8. Determination of PHB Content in Potential Halophilic PHA Producers by Gas Chromatography (GC)

After methanolysis, the cellular content of the polymer and its composition were assayed by gas chromatography (GC) using an Agilent Technologies 7890A chromatograph, equipped with a capillary column (30m×0.25mm×0.25μm) and a flame ionization detector (FID) as previously reported [31]. Samples were analyzed in duplicate. The standard PHB (Sigma-Aldrich, USA) was used for calibration. The PHB content in the cells was determined as massofPHB/celldrymass×100%. The peak at 4.4 min represents the 3-hydroxybutyrate methylester.


## 3. Results

### 3.1. Screening of PHA-Producing Isolates by Staining Procedures

A large number of orange, red, and pink colonies were picked and purified by repeating subculturing. A total of 33 extremely halophilic strains were isolated from three tested enrichment cultures and were screened for PHA accumulation (Table2). 11, 20, and 2 isolates were obtained from the water/sediment mix of the sample S1-10, water/sediment mix of the sample S6M-14, and hypersaline water of the sample S6W-14, respectively. 14 positive isolates were selected after staining by Sudan Black B (Figure S1 (a)). Additionally, they showed high fluorescence intensity with Nile Red when exposed to ultraviolet (Figure S1 (b)). The positive control strain exhibited orange fluorescence under UV light.Table 2
Screening of PHA-producing archaeal strains using phenotypic and genotypic methods.
Sample typeName of isolatesMorphologyColony staining methodCODEHOP PCRARDRASBBNRPhaEPhaCProfilesWater and sediments (2014)CEJ1-14Cocci----ICEJ2-14Cocci----IICEJ3-14Cocci++++IIICEJ4-14Cocci----ICEJ5-14Cocci++++IIICEJ6-14Cocci++++IIICEJ7-14Cocci++++IIICEJ8-14Cocci++++IIICEJ9-14Cocci++++IIICEJ10-14Cocci++++IIICEJ11-14Cocci++++IIICEJ17-14Pleomorph----ICEJ18-14Pleomorph----ICEJ21-14Cocci++++IIICEJ24-14Cocci++++IIICEJ25-14Cocci++++IIICEJ26-14Cocci----ICEJ28-14Cocci++++IIICEJ29-14Short rod----ICEJ32-14Cocci----IIWater (2014)CEJ33-14Cocci----ICEJ34-14Cocci++++IWater and sediments (2010)CEJ35-10Cocci----IVCEJ36-10Cocci----IVCEJ37-10Cocci----IVCEJ38-10Cocci----IVCEJ41-10Cocci----VICEJ42-10Cocci----VICEJ43-10Cocci----IVCEJ45-10Cocci----IVCEJ46-10Cocci----IVCEJ47-10Cocci----ICEJ48-10Cocci++++VDesignation of isolates by letters indicating first the origin of strains from Chott El Jerid (CEJ) followed by the number of the isolates, then a number indicating the year of sampling. The ARDRA pattern is indicated by Roman numeral. +, detectable; -, not detectable; SBB: Sudan Black B; NR: Nile Red.


### 3.2. Screening of PHA Synthase Genes by Degenerate Polymerase Chain Reaction

As a result, the screening of 33 strains showed the detection of 14 strains as PHA producers. The same strains, which showed positive results with phenotypic methods (Sudan Black and Nile Red), gave bands of approximately 230 bp (phaE) and 280 bp (phaC) (Table 2; Figure S2).


### 3.3. Morphological Characterization of Potential PHA Producers

The cells of all isolates were rods, cocci, and pleomorph (Table2). All selected strains as producers were round. The approximate cell dimensions were 1 to 2 μm (Figure S3).


### 3.4. Phylogenetic Analysis

33 haloarchaeal isolates were examined with ARDRA analysis. The comparison of three enzymesHaeIII, AluI, and MboI digestion patterns withisolates showed the occurrence of six different patterns for archaea (Table 2). Three profiles grouped polyhydroxyalkanoate-producing strains. The strains CEJ3-14, CEJ5-14, CEJ6-14, CEJ7-14, CEJ8-14, CEJ9-14, CEJ10-14, CEJ11-14, CEJ21-14, CEJ24-14, CEJ25-14, and CEJ28-14 were clustered into profile III. The strain CEJ34-14 was grouped into profile I. ARDRA pattern V represented the isolate CEJ48-10 (Figure S4). The phylogenetic analysis targeting the 16S rRNA genes of isolates indicated that the genera were Natrinema, Haloarcula, and Halorubrum. The isolate CEJ34-14 was related to Halorubrum chaoviator DSM 19316. The isolates CEJ3-14, CEJ5-14, CEJ6-14, CEJ7-14, CEJ8-14, CEJ9-14, CEJ10-14, CEJ11-14, CEJ21-14, CEJ24-14, CEJ25-14, and CEJ28-14 showed close relatedness to species of the most abundant genus Natrinema. Finally, the strain CEJ48-10 belonged to the genus Haloarcula (Figure 1).Figure 1
Phylogenetic tree, reconstructed by the neighbor-joining method, showing the positions of PHA-accumulating haloarchaeal isolates. Bootstrap values based on 1000 replicates are indicated. The sequence ofMethanohalobium evestigatum was used as the outgroup.


### 3.5. Quantitative Estimation of PHB Production by Positive Haloarchaeal Isolates

The data in Table3 showed that the PHB content in the cells was ranged from 0.07% up to 9.25% of CDW. Among the strains studied, the isolates CEJ34-14, CEJ5-14, and CEJ48-10 affiliated with Halorubrum chaoviator (99.7% of similarity), Natrinema pallidum (99.33% of similarity), and Haloarcula tradensis (97.72% of similarity) were found to be the best ones, respectively. Their cells were observed under a fluorescence microscope (Figure 2). The isolate CEJ34-14 should be highlighted because it exhibited a higher PHB production and a higher growth rate after 48 h of culture. Its growth increased with a logarithmic phase of 2 days and attained a long stationary phase. However, the growth of strains CEJ5-14 and CEJ48-10 requested 3 or 4 days to reach the logarithmic phase (Figure 3). Remarkably, the strains CEJ8-14, CEJ9-14, and CEJ28-14 exhibited a higher biomass production (1150–3620 mg L-1) but a lower PHB production (Table 3). Comparing to the standard PHB, the GC spectrum of PHA obtained by the isolates CEJ34-14, CEJ5-14, and CEJ48-10 showed predominant peaks at retention time at 4.17, 4.11, and 4.43 minutes corresponding to poly(3-hydroxybutyrate), respectively (Figure 4). These strains exhibited PHB content of about 9.25, 7.11, and 1.42% of the cell dry weight, respectively. The growth curves and chromatograms of the other strains were presented in supplementary materials (Figures S5 and S6).Table 3
PHB production values by isolates and its closest relatives.
StrainaCarbon sourceTimeb(h)CDW(mg L-1)PHA contentc (%)Type of PHAReferencesCEJ3-14Starch1203800.38PHBThis studyCEJ5-141685607.11CEJ6-14961100.09CEJ7-14965600.69CEJ8-149611500.07CEJ9-1414425000.1CEJ10-14721300.07CEJ11-14964800.91CEJ21-14961000.79CEJ24-14963000.89CEJ25-141206400.71CEJ28-149636200.21CEJ34-141442209.25CEJ48-101205501.42Haloarcula marismortui ATCC 43049Glucose192n.d.21PHB[12]Haloarcula amylolytica 26-39625004.4PHBV[30]Haloarcula argentinensis CGMCC 1.709433006.5PHBVHalorubrum litoreum 12-2Glucose9625002.1PHB[30]Halorubrum trapanicum CGMCC 1.2201190012.7PHBVNatrinema altunense CGMCC 1.37319658009.1PHBV[30]Natrinema pallidum JCM 8980350022.9PHBVNatrinema pellirubrum JCM 10476220011.5PHBNatrinema sp.XA3-116005.4PHBVNatrinema ajinwuensis RMG1072n.d.61PHBV[37]Natrinema pallidum 1KYS1Starchn.d.7553.14PHBV[36]Natrinema altunense CEJGTEA101Starch120802.7PHA[9]CDW: cell dry weight; PHA: polyhydroxyalkanoate; PHB: poly(3-hydroxybutyrate); PHBV: poly(3-hydroxybutyrate-co-hydroxyvalerate); n.d.: not determined.aIncubated at 37°C in PHA accumulation medium supplemented with starch or glucose. bCells were harvested at the early stationary phase for each strain. cPHA content in dried cells was determined using gas chromatography.Figure 2
Fluorescence microscopy of archaeal cells grown on PHA accumulation medium upon 144 h of cultivation for strain CEJ34-14 (a), 168 h for strain CEJ5-14 (b), and 120 h for strain CEJ48-10 (c) following staining with Nile Red.
(a)(b)(c)Figure 3
Growth curve over time of the strains CEJ34-14, CEJ5-14, and CEJ48-10. Optical density was taken every 24 h at 600 nm. Mean values from duplicate tests are shown.Figure 4
Chromatograms of PHA obtained from cultures of the isolates (a) CEJ34-14, (b) CEJ5-14, (c) CEJ48-10, and (d) PHB standard (Sigma). The peak at 4.4 min represents the 3-hydroxybutyrate methylester.
(a)(b)(c)(d)


## 3.1. Screening of PHA-Producing Isolates by Staining Procedures

A large number of orange, red, and pink colonies were picked and purified by repeating subculturing. A total of 33 extremely halophilic strains were isolated from three tested enrichment cultures and were screened for PHA accumulation (Table2). 11, 20, and 2 isolates were obtained from the water/sediment mix of the sample S1-10, water/sediment mix of the sample S6M-14, and hypersaline water of the sample S6W-14, respectively. 14 positive isolates were selected after staining by Sudan Black B (Figure S1 (a)). Additionally, they showed high fluorescence intensity with Nile Red when exposed to ultraviolet (Figure S1 (b)). The positive control strain exhibited orange fluorescence under UV light.Table 2
Screening of PHA-producing archaeal strains using phenotypic and genotypic methods.
Sample typeName of isolatesMorphologyColony staining methodCODEHOP PCRARDRASBBNRPhaEPhaCProfilesWater and sediments (2014)CEJ1-14Cocci----ICEJ2-14Cocci----IICEJ3-14Cocci++++IIICEJ4-14Cocci----ICEJ5-14Cocci++++IIICEJ6-14Cocci++++IIICEJ7-14Cocci++++IIICEJ8-14Cocci++++IIICEJ9-14Cocci++++IIICEJ10-14Cocci++++IIICEJ11-14Cocci++++IIICEJ17-14Pleomorph----ICEJ18-14Pleomorph----ICEJ21-14Cocci++++IIICEJ24-14Cocci++++IIICEJ25-14Cocci++++IIICEJ26-14Cocci----ICEJ28-14Cocci++++IIICEJ29-14Short rod----ICEJ32-14Cocci----IIWater (2014)CEJ33-14Cocci----ICEJ34-14Cocci++++IWater and sediments (2010)CEJ35-10Cocci----IVCEJ36-10Cocci----IVCEJ37-10Cocci----IVCEJ38-10Cocci----IVCEJ41-10Cocci----VICEJ42-10Cocci----VICEJ43-10Cocci----IVCEJ45-10Cocci----IVCEJ46-10Cocci----IVCEJ47-10Cocci----ICEJ48-10Cocci++++VDesignation of isolates by letters indicating first the origin of strains from Chott El Jerid (CEJ) followed by the number of the isolates, then a number indicating the year of sampling. The ARDRA pattern is indicated by Roman numeral. +, detectable; -, not detectable; SBB: Sudan Black B; NR: Nile Red.


## 3.2. Screening of PHA Synthase Genes by Degenerate Polymerase Chain Reaction

As a result, the screening of 33 strains showed the detection of 14 strains as PHA producers. The same strains, which showed positive results with phenotypic methods (Sudan Black and Nile Red), gave bands of approximately 230 bp (phaE) and 280 bp (phaC) (Table 2; Figure S2).


## 3.3. Morphological Characterization of Potential PHA Producers

The cells of all isolates were rods, cocci, and pleomorph (Table2). All selected strains as producers were round. The approximate cell dimensions were 1 to 2 μm (Figure S3).


## 3.4. Phylogenetic Analysis

33 haloarchaeal isolates were examined with ARDRA analysis. The comparison of three enzymesHaeIII, AluI, and MboI digestion patterns withisolates showed the occurrence of six different patterns for archaea (Table 2). Three profiles grouped polyhydroxyalkanoate-producing strains. The strains CEJ3-14, CEJ5-14, CEJ6-14, CEJ7-14, CEJ8-14, CEJ9-14, CEJ10-14, CEJ11-14, CEJ21-14, CEJ24-14, CEJ25-14, and CEJ28-14 were clustered into profile III. The strain CEJ34-14 was grouped into profile I. ARDRA pattern V represented the isolate CEJ48-10 (Figure S4). The phylogenetic analysis targeting the 16S rRNA genes of isolates indicated that the genera were Natrinema, Haloarcula, and Halorubrum. The isolate CEJ34-14 was related to Halorubrum chaoviator DSM 19316. The isolates CEJ3-14, CEJ5-14, CEJ6-14, CEJ7-14, CEJ8-14, CEJ9-14, CEJ10-14, CEJ11-14, CEJ21-14, CEJ24-14, CEJ25-14, and CEJ28-14 showed close relatedness to species of the most abundant genus Natrinema. Finally, the strain CEJ48-10 belonged to the genus Haloarcula (Figure 1).Figure 1
Phylogenetic tree, reconstructed by the neighbor-joining method, showing the positions of PHA-accumulating haloarchaeal isolates. Bootstrap values based on 1000 replicates are indicated. The sequence ofMethanohalobium evestigatum was used as the outgroup.


## 3.5. Quantitative Estimation of PHB Production by Positive Haloarchaeal Isolates

The data in Table3 showed that the PHB content in the cells was ranged from 0.07% up to 9.25% of CDW. Among the strains studied, the isolates CEJ34-14, CEJ5-14, and CEJ48-10 affiliated with Halorubrum chaoviator (99.7% of similarity), Natrinema pallidum (99.33% of similarity), and Haloarcula tradensis (97.72% of similarity) were found to be the best ones, respectively. Their cells were observed under a fluorescence microscope (Figure 2). The isolate CEJ34-14 should be highlighted because it exhibited a higher PHB production and a higher growth rate after 48 h of culture. Its growth increased with a logarithmic phase of 2 days and attained a long stationary phase. However, the growth of strains CEJ5-14 and CEJ48-10 requested 3 or 4 days to reach the logarithmic phase (Figure 3). Remarkably, the strains CEJ8-14, CEJ9-14, and CEJ28-14 exhibited a higher biomass production (1150–3620 mg L-1) but a lower PHB production (Table 3). Comparing to the standard PHB, the GC spectrum of PHA obtained by the isolates CEJ34-14, CEJ5-14, and CEJ48-10 showed predominant peaks at retention time at 4.17, 4.11, and 4.43 minutes corresponding to poly(3-hydroxybutyrate), respectively (Figure 4). These strains exhibited PHB content of about 9.25, 7.11, and 1.42% of the cell dry weight, respectively. The growth curves and chromatograms of the other strains were presented in supplementary materials (Figures S5 and S6).Table 3
PHB production values by isolates and its closest relatives.
StrainaCarbon sourceTimeb(h)CDW(mg L-1)PHA contentc (%)Type of PHAReferencesCEJ3-14Starch1203800.38PHBThis studyCEJ5-141685607.11CEJ6-14961100.09CEJ7-14965600.69CEJ8-149611500.07CEJ9-1414425000.1CEJ10-14721300.07CEJ11-14964800.91CEJ21-14961000.79CEJ24-14963000.89CEJ25-141206400.71CEJ28-149636200.21CEJ34-141442209.25CEJ48-101205501.42Haloarcula marismortui ATCC 43049Glucose192n.d.21PHB[12]Haloarcula amylolytica 26-39625004.4PHBV[30]Haloarcula argentinensis CGMCC 1.709433006.5PHBVHalorubrum litoreum 12-2Glucose9625002.1PHB[30]Halorubrum trapanicum CGMCC 1.2201190012.7PHBVNatrinema altunense CGMCC 1.37319658009.1PHBV[30]Natrinema pallidum JCM 8980350022.9PHBVNatrinema pellirubrum JCM 10476220011.5PHBNatrinema sp.XA3-116005.4PHBVNatrinema ajinwuensis RMG1072n.d.61PHBV[37]Natrinema pallidum 1KYS1Starchn.d.7553.14PHBV[36]Natrinema altunense CEJGTEA101Starch120802.7PHA[9]CDW: cell dry weight; PHA: polyhydroxyalkanoate; PHB: poly(3-hydroxybutyrate); PHBV: poly(3-hydroxybutyrate-co-hydroxyvalerate); n.d.: not determined.aIncubated at 37°C in PHA accumulation medium supplemented with starch or glucose. bCells were harvested at the early stationary phase for each strain. cPHA content in dried cells was determined using gas chromatography.Figure 2
Fluorescence microscopy of archaeal cells grown on PHA accumulation medium upon 144 h of cultivation for strain CEJ34-14 (a), 168 h for strain CEJ5-14 (b), and 120 h for strain CEJ48-10 (c) following staining with Nile Red.
(a)(b)(c)Figure 3
Growth curve over time of the strains CEJ34-14, CEJ5-14, and CEJ48-10. Optical density was taken every 24 h at 600 nm. Mean values from duplicate tests are shown.Figure 4
Chromatograms of PHA obtained from cultures of the isolates (a) CEJ34-14, (b) CEJ5-14, (c) CEJ48-10, and (d) PHB standard (Sigma). The peak at 4.4 min represents the 3-hydroxybutyrate methylester.
(a)(b)(c)(d)


## 4. Discussion

The development of biodegradable plastics represents an alternative way to respond to problems associated with plastic waste. Polyhydroxyalkanoates are considered to be excellent candidates for biodegradable plastics. Extremely halophilic archaea have the ability to synthesize and accumulate PHA as inclusions in their cells [11, 32]. In this study, an effort has been taken to search the PHB-producing archaea isolated from the hypersaline lake, Chott El Jerid, using a starchy substrate. A total of 33 isolates were obtained from three enrichment cultures. PHB was detected in 14 strains grouped in three haloarchaeal genera Haloarcula, Halorubrum, and Natrinema and clustered within the phylum Euryarchaeota including Haloarculaceae, Halorubraceae, and Natrialbaceae families, respectively. They were screened via staining means (Sudan Black B and Nile Red) which were widely used for halophilic bacteria but also successfully used for Halococci, Haloarcula, Haloferax, Halorubrum, Natronococcus, Halogeometricum, Halobacterium genera, and other haloarchaeal strains [8, 13]. In parallel, it was evident that the detection of phaC and phaE genes were shown in the 14 archaeal cells as described above with staining methods, confirming the PHB biosynthesis. To our knowledge, few studies based on molecular characterization of the genes involved in PHB synthesis in the domain of Archaea have been investigated [33]. Importantly, one group of extremely halophilic archaea with great biotechnological importance was Haloarcula which was genetically well understood. Han et al. [12] identified two adjacent genes phaEHm and phaCHm encoding two subunits of PHA synthase (class III) and showed that these genes are required for PHB synthesis in Haloarcula marismortui (cultivated on 2% glucose, production of 21% PHB of CDW) [12]. Later, Han et al. [30] confirmed the detection of phaEC genes in 18 PHB or poly(3-hydroxybutyrate-co-hydroxyvalerate) PHBV producers, including Haloarcula, Halorubrum, Natrinema, and other genera by utilizing carbohydrates either glucose or fructose [30]. More recently, it was reported that these two genes were detected in the genome of Natrinema altunense CEJGTEA101, isolated from Chott El Jerid [9].The present study is a continuity of our previous work [9], proposing the possibility to enlarge our knowledge about PHB secretion by a large number of haloarchaeal strains from Chott El Jerid due to their several advantages: firstly, their growth at high salinity minimized microbial contamination. Secondly, the high osmotic pressure in their cells facilitated the PHB recovery. Finally, their ability to consume a wide range of low-cost carbon sources reduced the PHB production cost. Among the tested isolates, three strains CEJ34-14, CEJ5-14, and CEJ48-10 affiliated with Halorubrum chaoviator (99.7% of similarity), Natrinema pallidum (99.33% of similarity), and Haloarcula tradensis (97.72% of similarity) have been considered as the best PHB producers 9.25%, 7.11%, and 1.42% of its CDW, respectively. It was important to note that these three genera were found to be PHA accumulators in other hypersaline environments [7], but only a few species were able to utilize starch to secrete large amounts of PHB. With regard to Haloarcula investigations, no PHB was accumulated by Haloarcula species using starch except Haloarcula. sp. IRU1 which could produce 57% PHB/CDW [17]. This species isolated from Urmia lake has been shown to produce important quantities of PHB (63% of CDW) from petrochemical wastewater as a carbon source containing multiple hydrocarbons such as linear alkylbenzenes [34]. Other Haloarcula species such as Haloarcula japonica, Haloarcula amylolytica, and Haloarcula argentinensis can accumulate PHB, and their yields obtained from glucose were 0.5, 4.4, and 6.5% (of CDW), respectively [30, 35]. Although production of PHB has been reported from starch, glucose, and waste materials, members of Haloarcula were observed for the first time in southern Tunisian salt lakes as PHB producers [15]. Currently, there is no evidence of PHB accumulation by the Halorubrum species when starch was used as a carbon source [11]. As previously stated, two species affiliated with Halorubrum which produced PHB or PHBV (2.1-12.7% of CDW) were able to use glucose as the sole carbon source [30]. On the other hand, the majority of members of Natrinema were found to be PHBV producers when cultivated on a medium with glucose [30] or starch [36] as the carbon sources.In our previous work [9], 20 extremely halophilic archaea belonging to the genera Halorubrum (17 strains), Natrinema (2 strains), and Haloterrigena (1 strain) and isolated from the sample S1-10 collected from Chott El Jerid in the dry season were screened for PHA production in the same PHA-accumulating medium used in this study. Among them, only two strains belonging to Natrinema and Haloterrigena genera have been shown to accumulate 7% of PHB and 3.6% of poly(3-hydroxyvalerate) (PHV), respectively, in a medium supplemented with 2% glucose. In this current study, enrichment cultures with the same sample (S1-10) and supplemented with starch showed the selection of only one PHB producer related to the genus Haloarcula. However, enrichment cultures with samples collected in the wet season and supplemented with the same carbon source revealed the presence of one species Halorubrum and a high number of Natrinema species (12 strains) with PHB-producing ability from the samples S6W-14 and S6M-14, respectively. These findings displayed that isolation method, carbon source, season, and sampling location could influence the selection of PHB-producing archaeal species.


## 5. Conclusions

On the basis of data obtained, different archaeal isolates were obtained in pure cultures from the Tunisian desert and were able to consume starch as the sole carbon source for the PHB biosynthesis. However, the yields of PHB production in flasks batch cultures within the cells were low in comparison with other closest relatives. Therefore, future studies on fermentations optimization in batch, fed-batch, and continuous cultures using starchy and other low-cost feedstock’s as well as the metabolic engineering strategies to improve both the quality and PHB productivity will be investigated.



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*Source: 8888712-2021-01-27.xml*