Characterization of Polyhydroxybutyrate, PHB, Synthesized by Newly Isolated Haloarchaea Halolamina spp.

This work aims to characterize the haloarchaeal diversity of unexplored environmental salty samples from a hypersaline environment on the southern coast of Jeddah, Saudi Arabia, looking for new isolates able to produce polyhydroxyalkanoates (PHAs). Thus, the list of PHA producers has been extended by describing two species of Halolamina; Halolamina sediminis sp. strain NRS_35 and unclassified Halolamina sp. strain NRS_38. The growth and PHA-production were investigated in the presence of different carbon sources, (glucose, sucrose, starch, carboxymethyl cellulose (CMC), and glycerol), pH values, (5–9), temperature ranges (4–65 °C), and NaCl concentrations (100–350 g L−1). Fourier-transform infra-red analysis (FT-IR) and Liquid chromatography–mass spectrometry (LC-MS) were used for qualitative identification of the biopolymer. The highest yield of PHB was 33.4% and 27.29% by NRS_35 and NRS_38, respectively, using starch as a carbon source at 37 °C, pH 7, and 25% NaCl (w/v). The FT-IR pattern indicated sharp peaks formed around 1628.98 and 1629.28 cm−1, which confirmed the presence of the carbonyl group (C=O) on amides and related to proteins, which is typical of PHB. LC-MS/MS analysis displayed peaks at retention times of 5.2, 7.3, and 8.1. This peak range indicates the occurrence of PHB and its synthetic products: Acetoacetyl-CoA and PHB synthase (PhaC). In summary, the two newly isolated Halolamina species showed a high capacity to produce PHB using different sources of carbon. Further research using other low-cost feedstocks is needed to improve both the quality and quantity of PHB production. With these results, the use of haloarchaea as cell factories to produce PHAs is reinforced, and light is shed on the global concern about replacing plastics with biodegradable polymers.


Introduction
The concept "plastic" usually refers to numerous organic synthetic or processed materials mainly obtained from petroleum that are not biodegradable. They are mostly thermoplastic or thermosetting polymers of high molecular weight. The extensive use of plastics manufactured from petroleum (which are non-biodegradable and recalcitrant) is a major threat to the world due to their negative impact on the environment as well as on living beings. For this reason, global contamination by plastics has become the most important problem in the world, which must be radically solved [1].

Isolation, Screening, and Identification of Haloarchaeal Strains Showing PHA-Producing Capacities
In this study, low-cost carbon sources were used to screen PHA-producing haloarchaea isolated from a Solar Saltern in Jeddah, Saudi Arabia. To identify the presence of PHA granules, traditional staining methods (Sudan Black B) were used. Among twelve isolates, two potential strains, NRS 35 and NRS 38, demonstrated the presence of black granules when stained with Sudan Black B, thus corroborating their capability as PHA producers [7]. More phenotypic characteristics were explored and are shown in Table 1. 16S rRNA gene sequencing revealed that the selected strains are members of Halolamina sediminis showing 96.42% and 96.91% sequence similarity, respectively. The obtained 16S rRNA gene data have been deposited in the NCBI and GenBank nucleotide sequence databases under the accession number OL912954 for Halolamina sediminis strain NRS_35 and OL912955 for Halolamina sp. strain NRS_38 (Figure 1). Halolamina is reported to be a halophilic organism that is widely distributed in marine environments [22].

Optimization of Growth Conditions and PHA Production
The growth patterns of the two novel strains (NRS_35 and NRS_38) producing PHA granules were monitored on a PHA-production medium including glycerol as a carbon source at various temperatures, salinity, and pH values (Figures 2 and 3). The strains grew at temperatures ranging from 4 to 65 • C. The best temperature for growth was 45 • C for both strains (Figures 2A and 3A). Regarding the concentration of NaCl used during cultivation, the results are displayed in Figure 4: Both strains grew in the presence of NaCl at concentrations ranging from 10% to 35% (w/v) with optimum growth at 15% (Figures 2B and 3B). Related to pH, strains NRS_35 and NRS_38 grew at a pH range of 5-9, with an optimum of 9 ( Figures 2C and 3C).

Optimization of Growth Conditions and PHA Production
The growth patterns of the two novel strains (NRS_35 and NRS_38) producing PHA granules were monitored on a PHA-production medium including glycerol as a carbon source at various temperatures, salinity, and pH values (Figures 2 and 3). The strains grew at temperatures ranging from 4 to 65 °C. The best temperature for growth was 45•C for both strains (Figures 2A and 3A). Regarding the concentration of NaCl used during cultivation, the results are displayed in Figure 4: Both strains grew in the presence of NaCl at concentrations ranging from 10% to 35% (w/v) with optimum growth at 15% (Figures 2B  and 3B). Related to pH, strains NRS_35 and NRS_38 grew at a pH range of 5-9, with an optimum of 9 ( Figures 2C and 3C). Considering that the availability of carbon and P and the ratio C/N are crucial to optimizing the synthesis of PHA, the effect of various carbon sources on cell growth and PHA biosynthesis in both strains was studied. The results showed that both isolated strains could synthesize PHA from a series of carbon sources including starch, CMC, glucose, glycerol, and sucrose ( Figure 4). For Halolamina sediminis NRS_35, starch, CMC, sucrose, glucose, and glycerol yielded PHA contents of 33.4%, 22.5%, 14.0%, 12.5, and 11.2, respectively. Regarding the production of PHA by NRS_38, the current results revealed that PHA contents yielded for starch, CMC, glucose, glycerol, and sucrose were equal to 27.29%, 16%, 12%, 13.5%, and 10.4%, respectively. Thus, starch was the most favorable substrate for PHA accumulation by both strains NRS_35 and NRS_38, reaching a titter of 3.6457 and 2.36 g/L, respectively.

FTIR Analysis
FTIR scan analysis has been used to illustrate the chemical functional groups within biomass yield. The current FTIR pattern (at 4000-400 cm −1 ) for both Halolamina sediminis strain NRS_35 and Halolamina sp. strain NRS_38 is illustrated in Figure 5A  The yield of PHA obtained by two different species of Halolamina strains, NRS_35 and NRS_38, under similar conditions with different carbon sources is summarized in Table 2. The results revealed that the maximum production of PHA in the presence of starch by NRS_35 is equal to 41.67 ± 2.10 mg/L, which is a higher concentration than the produced by NRS_38 under the same growth conditions. Thus, starch was the most suitable carbon source for biomass production for the tested strains. Data represent mean ± SD from three replicates. In each column, a,b,c different superscript letters indicate significant differences at the p < 0.05 level (one-way ANOVA and Duncan post hoc test).

FTIR Analysis
FTIR scan analysis has been used to illustrate the chemical functional groups within biomass yield. The current FTIR pattern (at 4000-400 cm −1 ) for both Halolamina sediminis strain NRS_35 and Halolamina sp. strain NRS_38 is illustrated in Figure 5A

LC-MS/MS Analysis
As shown in Figure 6, extracted polymers produced by the NRS_35 strain displayed peaks at different retention times (5.2, 7.3, and 8.1). This peak range indicates the occurrence of PHB and biomolecules involved in its synthesis such as acetoacetyl-CoA (precursor) and the enzyme PHB synthase (PhaC). The detection of PHB in granules produced

LC-MS/MS Analysis
As shown in Figure 6, extracted polymers produced by the NRS_35 strain displayed peaks at different retention times (5.2, 7.3, and 8.1). This peak range indicates the occurrence of PHB and biomolecules involved in its synthesis such as acetoacetyl-CoA (precursor) and the enzyme PHB synthase (PhaC). The detection of PHB in granules produced by NRS_35 and NRS_38 strains using LC-MS/MS is shown in Figure 7a,b, respectively. Regarding the PHB isolated from the NRS_38 strain, peaks are detected at 8.1 min (Figure 7), and at approximately 5.2 and 7.3 min, the last two peaks corresponding to acetyl-CoA and PhaC, respectively, are also observed. Other peaks were observed, which may contain the digested cell debris and other molecules that do not bind to the Accucore C18.

Discussion
Biodegradable polymers are an alternative to address the challenges related to plastic waste [23]. Among these biopolymers, polyhydroxyalkanoates (PHAs) are excellent candidates to be explored, mainly PHB and PHBV, due to their physicochemical characteristics [5]. Some archaea that are extremely halophilic can synthesize and accumulate PHA as granules within their cells [24]. Within this context, several previous works reported that several haloarchaeal genera can use different types of carbohydrates as sources of carbon and energy for PHB production [5]. This production can be optimized by modulating the C/N ratio and phosphate availability or/and by obtaining mutants overexpressing enzymes involved in PHA synthesis [5].
In this study, the goal was to screen haloarchaea capable of PHA-production from environmental samples taken from a Solar Saltern located in Jeddah, Saudi Arabia. To conduct this research, low-cost carbon sources have been used as nutrients in order to optimize an environmentally friendly method that could be further related to circular economy approaches. Out of ten haloarchaeal isolates, two strains, NRS_35 and NRS_38, exhibited the presence of black granules when stained with Sudan Black B, indicating their capability of PHA biosynthesis [25].
The growth profile of both strains was monitored on a PHA-production medium including glucose, among other molecules, as a carbon source and modifying several parameters such as the temperature, pH values, and salt concentration. The results obtained revealed that the optimum temperature for NRS_35 and NRS_38 growth was 45•C. Both strains grew in the presence of different salt concentrations ranging from 10% to 35%, but the optimum growth was observed at a concentration of 15% NaCl (the higher the salt concentration, the lower the growth). These results indicate that the two isolates are moderate haloarchaea compared to extreme haloarchaea such as Haloferax mediterranei or Haloferax volcanii, both requiring salt concentrations above 20% for optimum growth [5]. Apart from the C/N ratio and phosphate limitation, the appropriate concentration of NaCl has been shown to be critical to producing PHA by haloarchaea [26]. Isolated strains were also subjected to a wide pH range from acidic to alkaline conditions to examine their effect on growth and PHA production. The optimal growth of strain NRS_35 was identified at a pH value of 9, whereas strain NRS_38 grew better at a pH value of 8. Cell growth and concentrations of the total PHA produced were both reduced at the acidic pH as expected for haloarchaea (most of them thrive best at neutral or slightly alkaline pHs) [27][28][29].
Once the parameters for optimal growth were identified, the quantification and characterization of the biopolymers produced were carried out. The synthesis of PHAs by haloarchaea has been less explored than in bacterial counterparts or plants. In summary, several enzymes mediated PHA synthesis and granule formation: PhaA, PhaB, and PhaC [30]. PHA biosynthetic pathways are also linked to different pathways such as glycolysis, the Krebs Cycle, the Calvin Cycle, and β-oxidation [31]. Under nutrient-rich environments, the production of high amounts of coenzyme A from the Krebs Cycle blocks PHA synthesis by inhibiting 3-ketothiolase (PhaA) and shifts into the Krebs Cycle for energy production [31]. On the other hand, other studies analyzing the haloarchaeal genetics stated that pathways related to PHA synthesis are mainly present in those halophilic archaea with larger proteome sizes and higher GC contents [32]. More studies are still required to understand PHA biosynthesis in haloarchaea as well as the molecular mechanism involved in its regulation. During the last decade, most of the studies on PHAs in haloarchaea were focused on their production at the laboratory scale and the isolation, quantification, and characterization of biopolymers. Thus, Pramanik et al. [33] reported the production of polyhydroxybutyrate (PHB) by Haloarcula marismortui using vinasse as a carbon source, and Karray and coworkers [7] reported the production of PHB in Haloarcula tradensis using starch as a carbon source. Other Haloarcula species, such as Haloarcula japonica, Haloarcula amylolytica, and Haloarcula argentinensis, can also accumulate PHB [12,18]. With regards to previous studies of PHA production by Haloarcula sp, only two studies reported successful PHA production using starch as carbon; Haloarcula sp. IRU1 could produce 57% PHB/CDW [30] while Karray and coworkers [7] reported PHA accumulation of approximately 1.42% PHB/CDW by Haloarcula strain CEJ48-10. Other studies [13,19] described the PHA production by Haloarcula japonica, Haloarcula amylolytica, and Haloarcula argentinensis with yields obtained from glucose of 0.5, 4.4, and 6.5% (of CDW) [34]. Haloarcula sp. strain NRSA20 can accumulate PHA 23.83%, 14%, 11%, 12%, and 8% of PHB/CDW by using 10 g L −1 of starch, CMC, sucrose, glucose, and glycerol, respectively.
With this work, the list of haloarchaea able to produce PHA is extended, shedding light on this process and confirming that these two new isolates are highly versatile using different carbon sources for growth and PHA production. The yield of PHA production in this study is similar to those previously described from haloarchaea as well as from bacteria wildtype strains [35]. The main advantage of using haloarchaea instead of bacteria apart from cellular lysis with distilled water and avoiding sterilization is their genuine and versatile metabolism. This versatility could be mainly due to the ability to produce several hydrolytic enzymes such as α-amylases as it has been previously described from other haloarchaea. α-amylases catalyze the hydrolysis of starch and produce fructose and glucose [36,37]. These enzymes were found to be stable under saline concentrations ranging from 2 to 4 M NaCl, while at a concentration of 3 M NaCl, the enzyme exhibited the maximum activity at pH values ranging from 7 to 8 and temperatures between 50 and 60 • C [38]. Apart from glucose or starch, some haloarchaea can metabolize glycerol (an inexpensive carbon source) as a nutrient source for growth and produce PHAs [39]. In this regard, two pathways are used by haloarchaea to metabolize the glycerol: On the one hand, glycerol kinase phosphorylates glycerol to synthesize sn-glycerol-3-phosphate (G3P) and then oxidizes it using G3P dehydrogenase (G3PDH) to produce dihydroxyacetone phosphate (DHAP). On the other hand, glycerol can be oxidized using glycerol dehydrogenase to produce dihydroxyacetone (DHA), with the latter being phosphorylated using DHA kinase to DHAP [37].
Haloferax species are likely the better-described and most used for biotechnological purposes at the time of writing this review. The growth pattern, as well as the high productivity and quality, of PHA synthesized by the genus Haloferax was reported by several studies [40][41][42][43][44]. Thus, the production of PHBV by Haloferax mediterranei, in the presence of glucose as the only carbon source, reached up to 9%, while exceeding 20% in Halogeometricum borinquense E3 [45]. Furthermore, in the presence of direct precursors in the culture media such as volatile fatty acids, the 3HV molar fraction in Haloferax mediterranei can reach up to 99% [46]. PHBV is the most marketed biopolymer due to its physical properties, making many applications in agri-food, biomedicine, etc., possible [5]. For this reason, the characterization of more microorganisms able to produce not only PHAs, but particularly PHB or PHBV, is becoming one of the major concerns in this field of knowledge. Besides, it has also been reported that the nutritional composition of the culture media affects the composition of PHAs finally synthesized, thus making the optimization of the culture media essential to supporting a good rate of growth and significant biomass at the stationary phase of growth as well as high yields of PHV/PHBV production [5,47,48].
In order to determine the type of PHA mainly produced by these two new isolates, FTIR and LC-MS/MS analyses were carried out. The FTIR pattern (at 4000-400 cm −1 ) for both Halolamina sediminis sp. strain NRS_35 and the unclassified Halolamina sp. strain NRS_38 revealed a wide range of absorption peaks detected around 3447.71 and 3442.84 cm −1 , thus indicating the presence of -NH or -OH groups' bonds in protein [49,50]. The weak absorption at 2958.39 cm −1 in the unclassified Halolamina sp. strain NRS_38 ( Figure 4A) is related to the C-H stretching of the -CH 3 group in fatty acids, while the peaks around 2927.30 and 2925.87 cm −1 are due to symmetric stretching of the C-H bond in CH 2 groups [51,52]. The sharp peaks identified around 1628.98 and 1629.28 cm −1 could be related to the carbonyl (-C=O) group on amides and related to proteins [53] or may be associated with the -CONH-group in amino sugars and proteins [50].
Moreover, the absorption around 1214.89 and 1216.14 cm −1 represents the availability of sulfated functional groups such as C-S-O and S-O [50,54]. The weak band around 895.30 cm −1 is attributed to α-D-glucose [55]. Meanwhile, the sharp bands at 764.31 and 761.73 cm −1 are indicative of glycosidic linkage [56]. On the other hand, the peaks at 668.45 and 671.73 cm −1 are related to the C-H groups of aromatic compounds [57]. The current FTIR patterns reflect the chemical composition of S-layer glycoproteins present in haloarchaea as it is composed of polysaccharides, pseudopeptidoglycan, glycoproteins, or pure protein. On the other hand, the FTIR pattern ( Figure 5C) illustrates the recorded peaks for standard PHB.
Finally, the results obtained convincingly demonstrated, via LC-MS analysis, the presence of PHB synthase (PhaC), and PHB accumulation was significantly impacted by the presence of PhaC, which was detected at RT: 7.353 [58]. Consequently, it is possible to assume that the accumulation of PHB with high molecular weight is connected to the activity of PhaC as previously described in earlier research [34,[59][60][61][62][63].

Enrichment, Isolation, and Growth Conditions
The samples were enriched via culturing in a PHA-accumulating medium described by Han et al. (2010) [19], HSM, containing, per liter, 250 g of NaCl, 20 g of MgSO 4 .7H 2 O, 20 g of KCl, 2.0 g of trisodium citrate, 3.0 g of Na 2 CO 3 , 8.0 g of KH 2 PO 4 , 37.5 mg of FeSO 4 .7H 2 O, and 50 mg of MnCl 2 .4H 2 O. The medium was supplemented with 10 g/liter of glucose and glycerol separately, as carbon sources, buffered with MOPS, and incubated at 37 • C for 7-14 days at 150 rpm. Samples were serially diluted, and 1 mL of each dilution was plated onto HSM as described above and supplemented with agar (20 g L −1 ) to isolate halophilic archaeal strains accumulating PHA. Pure isolates were obtained and maintained through repeated culture on HSM.

Screening for PHA-Producing Haloarchaeal Isolates
After two weeks of incubation at 37 • C, ten different halophilic archaeal isolates were stained with Sudan Black B (Sigma) to determine their potential as PHA producers, as described by Murray and coworkers [64]. At the early stationary phase of growth, an aliquot of culture was smeared on a clean glass slide, then heat-fixed and stained with Sudan Black B (3% w/v in 70% ethanol) for 10 min, and finally, immersed in xylene until completely decolorized. The samples were rinsed and dried after being counterstained with safranin (Sigma; 5% (w/v), aqueous solution) for 10 s. Phase contrast microscopy (Nicon Eclips E600) was used to investigate the cells. Positive strains, indicating PHA producers, were recognized as blue-black cells under the microscope, with the bacterial type of strain Escherichia coli (ATCC35218) as a negative control.

Optimization of Growth Conditions of the Potential PHA-Producing Haloarchaeal Strains
A total of 100 ul of the culture at the exponential phase of growth was used as the inoculum for 100 mL of the PHA production media: Previous media supplemented with glycerol (1% (v/v) and incubated at 37 • C with an agitation rate of 150 rpm. Cellular growth was measured every 48 h for up to two weeks using a spectrophotometer set to 600 nm. The effects of different conditions of glycerol on PHA production by the potential strains were examined. The effect of other parameters such as pH (5, 6, 7, 8, and 9), temperature (4, 20, 37, 45, 55, and 65 • C), and NaCl concentration (100, 150, 200, 250, 300, and 350 g L −1 ) were also explored. In addition, the effect of different carbon sources such as starch, carboxymethyl cellulose (CMC), sucrose, glucose, and glycerol on PHA production by the selected strains was examined under optimal growth conditions. Each carbon source was filtered separately and then added to the sterilized production medium at a final concentration of 10 g L −1 . All the conditions were tested in triplicate.

Extraction of the Polymer
The cultures previously described were grown until the stationary phase and later centrifuged for 25 min at 5000 rpm to harvest the cells. The dry weight of the pellets was obtained, and the pellets were then treated as follows. To recover PHAs granules, the pellets were resuspended in an equal amount of 6% (w/v) sodium hypochlorite and incubated at 37 • C for 10 min. After this incubation, the mixture was centrifuged at 5000 rpm for 30 min. Before being treated with hot chloroform, the pellets containing the granules were washed in acetone and ethanol (30:70). After the pellets were dissolved in chloroform, Whatman filter paper (grade 1, Cat No 1001-110) was used to filter out the cell debris, thus making the presence of PHA in the chloroform solution possible. Finally, the filtrate was evaporated in a hot air oven at 40 • C, and the dry weight of the extracted polymer was determined [66]. The percentage of PHAs accumulation was calculated using the following formula, according to Sathiyanarayanan and Munir [67,68]: Dry weight of extracted PHB/PHA (g/mL) × 100/Cell Dry weight of biomass (CDW) Cell dry weight (CDW) = weight of falcon tube with dried pellets-the weight of empty falcon tube.
Dry weight of extracted PHA = weight of filter paper with dried filtered PHA-weight of empty filter paper.

Characterization of the Biopolymer by FTIR
The functional groups CH, CH 2 , CH 3 , C=O, C-O, and OH, which are significant determinants for the existence of PHAs in the extracted biopolymer, were subjectively identified and detected using FTIR spectroscopy (Perkin Elmer Spectrum GX Range Spectrometer, Bridgeport Avenue, Madison, USA), according to the method described by Mohapatra et al. [69].

Characterization of Biopolymer by LC-MS/MS
An extracted biopolymer from the NRS35 and NRS38 strains was identified using the LC-MS/MS systems of AB Sciex (Spectralab Scientific Inc., Markham, North America) linked to an API 5000 Triple Quadrupole mass spectrometer. The isocratic elution was carried out, with a flow rate of 0.003 mL min −1 of the mobile phase (0.001 M chloroform). A UV detector (10 AC) operating at 210 nm identified the presence of PHB. The sample was injected at a rate of 5.0 µL/s and detected over the course of 11.5 min. Using commercial PHB as the control (Sigma-Aldrich, St. Louis, MO, USA), the data were analyzed using the Mass Hunter software. The analytical column used was the Thermo Scientific™ Hypersil™ ODS (C18) Column, with a 10 µm particle size, and the temperature was maintained using a column heater [70,71].

Statistical Analysis
Three replicates were conducted for each analysis (n = 3). The measurements were displayed as the mean ± standard deviation (SD). IBM ® SPSS was used to conduct the statistical analysis utilizing an analysis of variance (ANOVA) on normally distributed datasets with a 95% confidence interval (IBM Corp. in Armonk, NY, USA. Chicago, IL, USA). p < 0.05 was considered to indicate a significant difference. The Duncan post hoc test was used to compare the significant difference between the means of the tested factor.

Conclusions
The promising haloarchaeal strains, Halolamina sediminis strain NRS_35 (OL912954) and Halolamina sp. strain NRS_38 (OL912955), were obtained in pure culture from the Solar Saltern on the southern coast of Jeddah, Saudi Arabia. Both strains were able to use starch, CMC, sucrose, glucose, and glycerol as the sole carbon sources for PHB production. The highest yield of PHB synthesis by strains NRS_35 and NRS_38 was 33.4% and 27.29%, respectively, using starch as a carbon source at 37 • C, pH 7, and 25% NaCl (w/v). This yield is similar to the average reported from other haloarchaea and bacteria, and it could be improved in the future by optimizing nutritional conditions or by designing mutants able to overproduce PHB. FTIR pattern and LC-MS analyses revealed significant groups that are typical characteristics of PHB. Future research will focus on optimization employing other low-cost feedstocks to improve both quality and PHB productivity.