# Production of polyhydroxyalkanoates by Halomonas sp. HG01, a halophilic bacterium from northern Peru, using various carbon sources: metabolic and genomic analysis

César W Guzmán-Moreno, Juliana Cardinali-Rezende, Alex Di Genova, Mariana Ferrarini, Marie-France Sagot, Kelli L Rodrigues, Luiziana F Silva, Marilda K Taciro, José Gregório C Gomez

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César W Guzmán-Moreno, Juliana Cardinali-Rezende, Alex Di Genova, Mariana Ferrarini, Marie-France Sagot, et al. Production of polyhydroxyalkanoates by Halomonas sp. HG01, a halophilic bacterium from northern Peru, using various carbon sources: metabolic and genomic analysis. International Journal of Biological Macromolecules, 2026, 337, 10.1016/j.ijbiomac.2025.149399. hal-05449695

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# Production of polyhydroxyalkanoates by *Halomonas* sp. HG01, a halophilic bacterium from northern Peru, using various carbon sources: metabolic and genomic analysis

César W. Guzmán-Moreno<sup>a,b*</sup>, Juliana Cardinali-Rezende<sup>a,c*</sup>, Alex Di Genova<sup>d</sup>, Mariana Ferrarini<sup>d,e</sup>, Marie-France Sagot<sup>d</sup>, Kelli L. Rodrigues<sup>a</sup>, Luiziana F.Silva<sup>a</sup>, Marilda K. Taciro<sup>a</sup>, José Gregório C. Gomez<sup>a,**</sup>

Dr. José Gregório Cabrera Gomez

Contact: +55 11 3091-7352 / E-mail: jgcgomez@usp.br

University of São Paulo, ICB II - Av. Prof. Lineu Prestes, 1374 - Butantã, São Paulo - SP, Brazil, 05508-900

<footnote><sup>a</sup>Institute of Biomedical Sciences, University of São Paulo, São Paulo, Brazil;</footnote>

<footnote><sup>b</sup>Facultad de Ciencias Agrarias y Ambientales, Universidad Católica Sedes Sapientiae, Chulucanas, Piura, Peru</footnote>

<footnote><sup>c</sup>LGEM - Laboratory of Genetics and Metabolic Engineering, Center for Natural and Human Science, Federal University of ABC, Santo André, SP, Brazil</footnote>

<footnote><sup>d</sup>ERABLE, Inria Laboratoire de Biométrie et Biologie Évolutive, Université de Lyon, Villeurbanne, France</footnote>

<footnote><sup>e</sup>Max Planck Institute for Chemical Ecology, Jena, Germany</footnote>

<footnote>\*Shared first authorship</footnote>

<footnote>\*\*Corresponding author:</footnote>
**Abstract**

*Halomonas* sp. HG01, a moderate halophilic bacterium isolated from a northern Peru salt mine, is promising as a polyhydroxyalkanoate (PHA) producer. Experimental data and genome analysis were used to evaluate its capabilities. Shaken flask experiments revealed that HG01 can accumulate 70-86 wt.% poly(3-hydroxybutyrate) [P(3HB)] from various carbon sources, including glucose, sucrose, and fructose. In a fed-batch bioreactor, it achieved a cell dry weight (CDW) of 12.2 g/L with 63% P(3HB) content after 72 hours. The PHA synthase enzyme exhibited substrate specificity for C4 to C5 compounds. HG01 strain also produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] from propionic and valeric acids, with a maximum 3HV content of 25.53 mol% monomers into the polymer (69.40 %wt) when valeric acid was used. The complete bacterial 3.66 Mbp genome sequence revealed metabolic pathways for carbohydrate and fatty acid catabolism, PHA biosynthesis, and stress tolerance factor. This genetic information enhances our understanding of PHA synthesis and supports the development of metabolic engineering strategies, positioning *Halomonas* sp. HG01 as a promising candidate for biotechnological applications.

**Keywords:** *Halomonas* sp. HG01. Moderate halophilic. Poly(3-hydroxybutyrate). Poly(3-hydroxybutyrate-co-3-hydroxyvalerate). Fed-batch bioreactor. Genome-scale metabolic networks (GEMs)
# 1. Introduction

Polyhydroxyalkanoates (PHA) comprise a family of biodegradable polyesters synthesized by a wide variety of prokaryotic cells as energy storage compounds under conditions of excess carbon and nutrient limitation [1,2]. These biopolymers have attracted special attention due to their biodegradable nature, biocompatibility, and physicochemical properties comparable as those of petroleum-derived plastics, making them a sustainable alternative to petro-plastics [3].

Among PHA, poly(3-hydroxybutyrate) [P(3HB)] has been the most extensively studied; however, copolymers such as poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] offer better mechanical properties, namely higher flexibility and impact resistance, which broadens their potential applications [4]. Thus, P(3HB-co-3HV) can be suitable for food packaging or medical devices [5], whereas P(3HB) has been widely explored in biomedical fields including tissue engineering, bone substitutes, and drug delivery systems [6].

*Escherichia coli*, *Bacillus* spp., *Burkholderia* spp., *Cupriavidus necator* [7], and *Pseudomonas* *putida* are model bacteria used for the industrial PHA production; however, despite their great benefits, PHA production remains limited, mostly due to the high costs of the carbon source and the expensive sterilization processes of the proper bacterial culture media [8]. It is estimated that the cost of the carbon source represents 30 to 50% of production costs, while the need for sterile conditions further limits scalability [8,9].

To address these problems, halophilic bacteria emerge as potential candidates for cost-effective PHA production since it is possible to establish bioprocesses using seawater or other poor quality water sources due to high salinity (avoiding the problems of fresh water availability) which may or may not be enhanced with extra amounts of NaCl, so that the bioprocesses are less prone to contamination and medium and equipment sterilization less relevant. Species of the genus
*Halomonas* have shown great physiological versatility and ecological adaptability [10], thriving in high-salinity environments such as oceans [1,11], salterns [12], marine hydrothermal vents [13] and hypersaline lakes [14,15]. Many species of the genus *Halomonas* have shown the ability to accumulate PHA from various carbon sources such as starch hydrolysates [16] or glycerol [17], a by-product of biodiesel production. In addition, *Halomonas bluephagenesis* and *Halomonas campaniensis*, grown in high NaCl concentration, have been successfully maintained free of contamination under open, non-sterile, and continuous cultivation [18,19]. Furthermore, *Halomonas halophila* [20] and *Halomonas boliviensis* [21] have been considered very promising candidates for PHA production from sugar-based substrates.

Species of the genus *Halomonas* can naturally accumulate P(3HB) or the copolymers P(3HB-*co*-3HV), upon addition of precursor substrates, however many of them have been genetically modified to produce P(3HB-*co*-3HV) and poly(3-hydroxybutyrate-*co*-4-hydroxybutyrate) [P(3HB-*co*-4HB] [10,22]. To go further to increase the *spectrum* of PHA produced and generate strains of super productive *Halomonas*, an essential requirement is to know their genome and metabolic pathways to be modified [19].

Nowadays, 53 draft genomes (https://pubmed.ncbi.nlm.nih.gov/?term=draft+genomes+of+Halomonas+species+&size=10) and nine complete genome sequences of *Halomonas* strains are available in the GenBank Database: *H. elongata* DSM 2581, *Halomonas* sp. strain KO116 [11], *Halomonas* sp. R5-57 [24], *Halomonas* sp. SF2003 [25], *Halomonas* sp. Soap Lake #7 [26], *Halomonas axialensis* strain Althf 1[1], *Halomonas sulfidaeris* ATCC BAA-803 [27], *Halomonas olivaria* strain TYRC17 [28] and *Halomonas boliviensis* strain kknpp38 [29]. Genome-scale metabolic networks (GEMs) reconstructed from genomic sequence data serve as a valuable modelling and computational tool in the expansive field of systems biology [30], since
they allow integrating high-throughput biological data - encompassing genomic, transcriptomic, metabolomic, and other datasets - facilitates the exploration of complex metabolic processes across various cell types and conditions.

Brazil, due to its enormous agricultural production, has important characteristics for the production of different bioproducts in the biorefinery scenario. In fact, the plants responsible for the production of sugar and alcohol are large biorefineries. In this environment, hexoses (sucrose, glucose and fructose) are important carbon sources for the production of PHA. Sugarcane bagasse can be an additional source of carbon for the production of ethanol. Due to the difficulty of yeasts to use xylose for the production of ethanol, a stream rich in xylose, derived from the hydrolysis of hemicellulose and estimated at 10 million tons per year, could power an important PHA production plant. Glycerol from biodiesel production in Brazil corresponds to approximately 1 million tons per year, in addition to the vinasse generated annually in the ethanol production process, which contains approximately 3.5 million tons of glycerol. These amounts of glycerol would be sufficient to promote relevant PHA production.

Lactose is an important residue in the dairy industry in Brazil.

In this context, this work evaluated the potential of *Halomonas* sp. HG01 for PHA production under high salinity conditions, using various carbon sources, especially those present in important agro-industrial residues available in relatively large amounts in Brazil. For this purpose, the PHA production by *Halomonas* sp. HG01 was evaluated using different carbon sources (glucose, sucrose, fructose, lactose, xylose, and glycerol) as well as precursor substrates for the synthesis of comonomers (propionic, butyric, valeric, hexanoic, heptanoic, octanoic, nonanoic acids, or sodium 4-hydroxybutyrate). Metabolic analysis was achieved after complete sequencing of its genome. The strain's GEM was reconstructed, and the metabolic pathways involved in the catabolism of these substrates for PHA production were also evaluated. The
results highlight the potential of *Halomonas* sp. HG01 as a candidate for sustainable and cost-effective PHA production.

## 2. Materials and methods

### 2.1. Isolation, screening, and maintenance of *Halomonas* sp. HG01

*Halomonas* sp. strain HG01 strain was isolated from a salt mine located in the San Jose District, Lambayeque, Peru (6°46'8.26" S and 79° 56'56.78" W) [31]. Briefly, salts mine samples were cultured at 30 °C in HM1 broth (in g/L: MgSO₄·7H₂O 0.25, CaCl₂·2H₂O 0.09, KCl 0.5, NaBr 0.06, yeast extract 1.0, glucose 10.0) containing 5 to 30% w/v of NaCl, followed by serial dilutions which were inoculated on HM1 agar plates. Isolates were clustered by morphology and evaluated by Sudan Black B staining for PHA detection, selecting HG01 for its intense accumulation of granules. It was observed that HG01 originated from the previous enrichment culture containing 10% NaCl. The bacterial strain was maintained as a frozen stock culture at -80°C in 20% (v/v) glycerol until the subsequent experiments.

### 2.2. Phylogenetic analysis

The phylogenetic analysis of *Halomonas* sp. HG01 was performed according to [32]. The complete sequence of the 16S rDNA *Halomonas* sp. HG01 gene was amplified by conventional PCR and sequenced using the same bacterial specific random primers 27F (5' AGAGTTTGATCCTGGCTCAG -3') and 1401R (5'CGGTGTGTACAAGAAGACCC 3') [33]. The 16S rDNA gene sequence was compared to the sequences deposited in the GenBank (https://www.ncbi.nlm.nih.gov/) and Ribosomal Database Project II (RDPII) (http://rdp.cme.msu.edu) [34]) using BLASTn to search for similar sequences. Phylogenetic relationships were inferred using MEGA 7.0 [35] and subsequently, a phylogenetic tree was
constructed based on the 16S DNA sequences available in the NCBI and RDPII Databases using
the neighbor-joining method [36] (Figure 1). The phylogenetic tree topology was evaluated with
a bootstrap analysis of 1,000 replicates.

**2.3. Complete genome sequencing and assembly**

The total genomic DNA of *Halomonas* sp. HG01 was extracted using a DNeasy blood and tissue
kit (Qiagen, Valencia, CA, USA) and quantified in a Qubit 2.0 fluorometer (Life Technologies,
USA). A paired-end sequencing library was constructed using a TruSeq DNA PCR-free LT
sample preparation kit (Illumina, San Diego, CA, USA) as previously described [37]. To obtain
the complete genome sequence, a total of 2,743,560 reads were produced after quality control
with the Galaxy software (https://usegalaxy.org/). In this program, the FastQ files were
converted to FastQ Groomer, and the reads were "trimmed" by cutting 10 bp from the 5'
extremity and 110 from the 3' extremity (Reads trimming). The poor-quality reads - all reads
where less than 90% of their bases were of qualities less than 20 - were filtered. In addition, the
forward and reverse reads were grouped and those that did not complement each other were
eliminated from the assembly (Table SM1). The reads were assembled employing the DiscoVar
de novo assembler (https://software.broadinstitute.org/software/discovar) and the contigs
obtained were subsequently assembled into 20 scaffolds using MeDuSa++ [38] with an N50
of=1,414,506 bases. Using a GC-Skew analysis, it was possible to sort the final set of scaffolds
into 1 single chromosome. After sorting the scaffolds, the remaining gaps were filled using the
original reads. The GC-Skew analysis (Figure 2) was performed to confirm if the chromosome
obtained in this work corresponded to a finished genome.

**2.4. Genome annotation and the genome-scale metabolic networks (GEMs) reconstruction**
Rapid Annotation used Subsystem Technology - RAST (http://rast.nmpdr.org/rast.cgi) and KBase predictive biology (https://narrative.kbase.us/narrative/ws.9014.obj.2) were used for automatic genome sequencing annotation and GEMs reconstruction. The refinement (or validation) of the genome sequencing annotation was manually performed with the Blastn and Blastx tools from the Database resources of the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A search for metabolic pathways, genes, and enzymes involved in carbohydrate degradation, PHA production, etc. was carried out using the GEM obtained. The analysis was based on literature data and from public databases such as Kyoto Encyclopedia of Genes and Genomes - KEGG (http://www.genome.jp/kegg/pathway.html), MetaCyc (Summary of MetaCyc, version 28.1); Biocyc (http://biocyc.org/), BRENDA (http://www.brenda-enzymes.info/) and UniProt (https://www.uniprot.org/). Annotation of the genes and pathways involved in stress regulation, resistance, carbohydrate metabolism and PHA production by *Halomonas* sp. HG01 resulted from the comparison with other *Halomonas* sp. genes available in the GenBank Database. The proteome translated from genomic data using RAST was annotated using STRING: functional protein association networks (string-db.org) under the accession on https://version-11-5 string-db.org/organism/STRG0030BYA.

**2.5. Genome Sequence Accession Number**

This Whole Genome Shotgun project has been deposited at DDBJ/ENA/GenBank under the
accession number LAEZ00000000 and the complete genome sequence described in this paper as
CP022134.

**2.6. Culture media used in this study**

Nutrient broth (NB) medium containing beef extract (3 g/L) and peptone (5 g/L) was used to
reactivate bacterial stock cultures and as inoculum. For the PHA accumulation experiments in
shaken flask, the reactivated bacterial culture was transferred from NB to mineral salts medium
(MSM) as previously described [39]. Modified mineral salts medium (MSM1) was used in the bioreactor experiments with the following composition (in g/L): KH₂PO₄, 0.65; (NH₄)₂SO₄, 3.6; MgSO₄.7H₂O, 0.31; CaCl₂.2H₂O, 0.01; ammonium ferric citrate, 0.15 and 2 mL of traces elements solution. The trace elements solution contained (g/L): H₃BO₃, 0.30; CoCl₂.6H₂O, 0.20; ZnSO₄.7H₂O, 0.10; MnCl₂.4H₂O, 0.03; NaMoO₄.2H₂O, 0.03; NiCl₂.6H₂O, 0.02; CuSO₄.5H₂O, 0.01. All culture media used in this study were adjusted to pH 7.0 using 2M NaOH and supplemented with NaCl (100 g/L).

## 2.7. PHA accumulation experiments in shaken flasks

Different carbon sources (glucose, sucrose, fructose, lactose, xylose, and glycerol) at a final concentration of 10 g/L (C/N ratio were 18.45 (glycerol), 18.88 (glucose, fructose, galactose and xylose) and 19.35 (sucrose and lactose) g/g) were used to evaluate PHA accumulation. To this purpose, 2.5 mL of a NB culture were inoculated into flasks containing 22.5 mL of MSM. To evaluate copolymer biosynthesis, the bacterial strain was cultivated in MSM with glucose (10 g/L) for 24h in different flasks for cellular growth, then 250 µL of a solution (100 g/L) of precursor substrates propionic, butyric, valeric, hexanoic, heptanoic, octanoic, nonanoic acids, or sodium 4-hydroxybutyrate were added separately to each culture to achieve a final concentration of 1 g/L, promoting copolymer production. The experiments were performed in biological triplicate and all cultures were incubated for up to 48 and 72 h at 30 °C and 150 rpm. The sampling was carried out in analytical duplicates after 72h to evaluate pH, cell dry weight (CDW), substrate consumption and PHA content.

## 2.8. P(3HB) production in fed-batch bioreactor

Production of PHA by *Halomonas* sp. HG01 in fed-batch culture was carried out in a 2 L working volume Biostat D bioreactor (BBraun). The pre-inoculum was a 25 mL culture in NB.
After 24h incubation, cells were harvested (10000 x g, 5 min, 4 °C) then re-suspended in a 0.85% NaCl solution. Twenty mL of this suspension were inoculated in a 500 mL flask containing 180 mL of MSM supplemented with glucose (10 g/L). The 200 mL resulting culture was then inoculated in a 4L bioreactor containing 1800 mL of MSM1. The pH was automatically controlled at 7.0 by adding 10M NaOH or 5M H₂SO₄. Dissolved oxygen (DO) concentration was controlled with a polarographic electrode (Ingold) and maintained at 50% of air saturation by varying the agitation speed. A constant aeration rate of 1 L/min was applied throughout the cultivation. A 50% (w/v) glucose feeding solution was supplied to maintain a glucose concentration between 5 and 10 g/L in the reactor. The following parameters were periodically determined: concentrations of CDW, carbohydrates, and ammonium, in addition to PHA content and composition.

## 2.9. Analytical methods

### 2.9.1. Cell dry weight concentration

For quantitative determination of cell biomass, cells were harvested from 10 to 20 mL culture samples by centrifugation at 10000 xg for 10 min. Cell pellets were washed once with distilled water to remove excess NaCl and then lyophilized. Cell dry weight (CDW) was determined gravimetrically.

### 2.9.2. Carbon source determination

Carbohydrate and glycerol concentrations were determined in a Dionex Ultimate 3000 HPLC equipped with an Aminex HPX-87H Column (Bio-Rad Laboratories, Inc. Hercules, California, USA) and a Shodex RI-101 refractive index detector. Aliquots of 10 µL were injected and separated at 45 °C with a 5 mM H₂SO₄ solution at a flow rate of 0.6 mL/min. The standard curves were constructed from solutions containing up to 3 g/L of each substrate.
### 2.9.3. Organic acids concentration

Organic acids concentrations were determined in an Agilent 7890A GC System (Agilent Technologies, Santa Clara, California, USA) gas chromatograph equipped with a FFAP capillary column after sample split (1:5). Samples (800 µL) were acidified with 200 µL of a solution containing HCl (0.2 mol/L) and butyric acid (4.8 g/L). Helium (3.0 mL/min) was used as the carrier gas. The injector and flame ionization detector (FID) temperatures were 250 and 300°C, respectively. The column temperature was maintained at 130°C during the first 5 min, increasing at a rate of 10°C/min up to 185°C, which was maintained for 1 min. Organic and butyric acids were used as external and internal standards, respectively.

### 2.9.4. Ammonium determination

The ammonium concentration was determined with an ion-selective electrode (Orion 9512HPBNWP) coupled to a potentiometer (Thermo Scientific, Orion 4 Star, pH-ISE Benchtop, Singapore) after alkalinization of the sample (3 mL) with a 10 M NaOH solution (300 µL). Standard curve was obtained using $(NH_4)_2SO_4$ solutions containing nitrogen up to 500 ppm.

### 2.9.5. PHA content and composition

PHA content and composition were determined using a gas chromatographic method [40]. About 10 mg of freeze-dried cells were subjected to propanolysis [41]. The propyl esters were analyzed in a 7890A Agilent CG System Gas Chromatograph (Agilent Technologies, Santa Clara, California, USA) equipped with a HP1 capillary column (Agilent Technologies) after sample split (1:20). Helium (0.8 mL/min) was used as the carrier gas. The injector and FID temperatures were 250 and 300°C, respectively. The oven was programmed at 100°C for 1 min, increasing temperature at a rate of 8 °C/min up to 210 °C, which was maintained for 15 min. Benzoic acid
was the internal standard (Sigma-Aldrich, Saint Louis, Missouri, USA). P(3HB) (Sigma-Aldrich)
and [P(3HB-co-3HV)] (Sigma-Aldrich) were used as external standards.

**2.10. Statistical analysis**

All experiments were conducted in biological triplicate (n = 3), and the results are presented as mean ± standard deviation (SD). Statistical analyses were performed using Microsoft Excel 2021 with the Data Analysis Toolpak add-in, considering a 95% confidence level to determine significant differences among treatments. A one-way analysis of variance (ANOVA) was applied to assess the effect of different carbon sources (glucose, sucrose, and fructose) on cell dry weight (CDW) and P(3HB) content. The same analysis was used to evaluate the influence of different precursor substrates (organic acids C3-C9 and sodium 4-hydroxybutyrate) on CDW and PHA accumulation. When significant differences were detected (p < 0.05), Tukey's HSD post hoc test was applied for mean separation. In addition, a two-sample Student's t-test assuming unequal variances (Welch's test) was performed to compare the incorporation of 3-hydroxyvalerate (3HV) using valeric acid and propionic acid as precursor substrates.

**3. Results and discussion**

**3.1. Phylogenetic analysis**

Based on a 16S rRNA sequence analysis, *Halomonas* sp. HG01 belongs to the *Halomonadaceae* family, which regroups a total of 102 halophilic bacterial species, distributed into 10 genera (http://www.bacterio.net/halomonas.html) being *Halomonas*, the most important [42]. *Halomonas* sp. HG01 16S rRNA sequence presented the highest similarity to *Halomonas halophila* NR 114161 and *Halomonas salina* NR 042050 (Figure 1).
<figure><img src="image_2.png" /><figcaption>**Figure 1.** Neighbor-joining tree deduced from *Halomonas* sp. HG01 16S rRNA gene and most similar sequences obtained from the GenBank database. The numbers at the nodes indicate the percentages of occurrence in 1,000 bootstrapped trees.</figcaption></figure>

**3.2. Growth and PHA accumulation in shaken flasks**

During the bioprocess, the optimal microbial growth is provided by the culture medium which also determines the yield and bioproduct cost on an industrial scale. One of the main factors
influencing the economic feasibility of PHA production is the carbon source cost [40,43–45]. It
is estimated that about 30 to 50% of the overall cost of the PHA production process is due to
substrate value [44,46]. Therefore, the use of bacterial strains capable of using low-cost carbon
sources [47] and effective in converting substrate into product could make the process viable
[40,48]. *Halomonas boliviensis* LC1, *H. elongate* DSM 2581 and 1H9T strains, *H. salina*,
*Halomonas* sp.TD01 etc., have demonstrated the ability to produce PHA at high cell content
from diverse carbon sources [20,49–53]. In 2010, Kawata and collaborators [17] reported that
*Halomonas* sp. KM-1 produced P(3HB) from waste glycerol as the sole carbon source [17]. In
2019, Thomas and coworkers [25] sequenced the complete *Halomonas* sp. SF2003 genome and
identified genes involved in carbohydrate and fatty acids metabolism for PHA production. In the
present study, *Halomonas* sp. HG01 was tested to determine its ability to grow and produce PHA
from different carbon sources: glucose, sucrose, fructose, lactose, xylose, and glycerol. These
substrates were selected due to their low cost [46] and their role as the main component in a wide
variety of agro-industrial products and byproducts especially in Brazil [20,44,46,54,55]. In this
investigation, shaker assays demonstrated that glucose, sucrose and fructose were successfully
used by *Halomonas* sp. HG01 as the sole carbon sources during growth and P(3HB)
accumulation (Table 1). However, the strain was not able to use lactose, glycerol and xylose
efficiently (Table SM2).

Statistical analysis showed significant differences in both CDW (F = 1401.87, p < 0.05) and
P(3HB) content (F = 160.66, p < 0.05) among the three carbon sources evaluated. The highest
CDW (4.75 and 4.77 g/L) and P(3HB) content values (83.7 and 85.6 wt.%) were attained after
72h of cultivation of *Halomonas* sp. HG01 on sucrose or glucose, respectively. Tukey's HSD
test confirmed that glucose and sucrose resulted in statistically equivalent biomass production
(4.77 vs. 4.75 g/L, p > 0.05) and P(3HB) accumulation (85.6 vs. 83.7 wt.%, p > 0.05), forming a
homogeneous group that was statistically different from fructose. In contrast, fructose led to
significantly lower CDW (3.31 g/L) and P(3HB) content (68.6 wt.%), corresponding to
reductions of approximately 31% in biomass and 20% in polymer accumulation compared to
glucose and sucrose (p < 0.05). The higher variability observed for P(3HB) content with fructose
(SD = 1.24) relative to glucose (SD = 0.15) indicates a less stable or efficient metabolic
response, which may be related to differences in assimilation pathways or lower conversion
efficiency to metabolic intermediates.

P(3HB) content obtained by HG01 strain on sucrose was higher than those obtained by *Halomonas halophila* (75.4 wt.%) and *Halomonas smyrnensis* AAD6T (26.9 wt.%) after 144 h of cultivation [20]. When using glucose, the P(3HB) content reached by HG01 strain was higher than that of *Cupriavidus* sp. KKU38 (73.9 wt.%) and equal to that of *Halomonas* sp. SF2003 (86 wt.%) under similar conditions (Table 2). Under the conditions evaluated for HG01, about the maximum theoretical P(3HB) yield was reached when using sucrose or glucose [40,56]. An overall P(3HB) yield from glucose corresponding to around 120% of the maximum theoretical value was reached after 72 hours of culture by *Halomonas* sp. HG01. Since the calculation was based on a previously presented equation [40] where the maximum P3HB yield from glucose was metabolically estimated and not exceed 0.48 g/g, likely a residual biomass yield from glucose should exceed the 0.5 g/g used in the previously established equation, suggesting that *Halomonas* HG01 is quite efficient in converting glucose in residual biomass. Another point to be considered in relation to HG01 strain is its ability to consume sucrose observed in this work. Unlike the case of glucose, of which only 75.6% was consumed by *Halomonas* spp., *Halomonas* sp. HG01 completely consumed sucrose as substrate. We can also highlight *Halomonas cerina* YK44, which used molasses, an important byproduct of sugar production, for the production of P3HB and reached a CDW value of 7.9 g/L and 79.7 wt% of P3HB [65], although this
performance was achieved by supplying four times the amount of carbon sources supplied to *Halomonas* sp. HG01 in our experiments. Suppling 30 g/L of high fructose corn syrup (HFCS – containing fructose, glucose and sucrose), it was possible to achieve 7.95 g/L of biomass containing 67.5 wt% of P3HB with *H. cerina* YK44 [63]. These results demonstrate very good and comparable performance between *Halomonas* sp. HG01 and *H. cerina* YK44 for the production of P3HB from hexoses. *H. cerina* YK44 was also able to produce significant amounts of P3HB from galactose or a galactose-containing red algae hydrolysate (GCRAH) [64], representing an advantage over *Halomonas* sp. HG-01, which has not yet been evaluated for the production of P3HB from galactose or residues containing this hexose. Previous studies also reported P(3HB) production from sucrose by *Rhizobium elti E1* and *Pseudomonas stutzeri E114* [57,58]. However, the sucrose catabolism leads to the production of fructan, levan, or another byproduct, rather than PHA in the strains *H. smyrnensis* AAD6T, *H. salina* DSM 5928T, *H. halophila* DSM 4770T, *H. maura* DSM 13445T and *H. nitroreducens* LMG 24185T [59]. With respect to fructose, *Halomonas* sp. HG01, despite being able to use it for growth and production of P(3HB) (reaching of 68.6 wt.%), the efficiency was lower than when using sucrose or glucose (Table 1). These results highlight the statistical superiority of glucose and sucrose as carbon sources for the HG01 strain. Both substrates supported significantly higher biomass yields and P(3HB) accumulation compared to fructose, suggesting their preferential use for industrial applications in bioplastic production.
<table><caption>**Table 1.** Growth and P(3HB) accumulation by *Halomonas* sp. HG01 using different carbon sources.</caption><thead><tr><th rowspan="2">Carbon Source</th><th rowspan="2">Culture Time (h)</th><th rowspan="2">Consumed Carbon source (g/L)</th><th colspan="2">CDW (g/L)</th><th colspan="2">P(3HB)</th><th rowspan="2">Y<sup>O</sup><sub>p/s</sub> (g/g)</th><th rowspan="2">Y<sub>Max</sub> (%)</th></tr><tr><th>Total</th><th>Residual</th><th>(%CDW)</th><th>(g/L)</th></tr></thead><tbody><tr><td rowspan="4">Glucose</td><td>0</td><td>0.00 ± 0.00</td><td>0.11 ± 0.01</td><td>0.09 ± 0.01</td><td>10.01 ± 0.68</td><td>0.01 ± 0.00</td><td>0.00 ± 0.00</td><td>0.00 ± 0.00</td></tr><tr><td>24</td><td>3.34 ± 0.17</td><td>1.65 ± 0.05</td><td>0.83 ± 0.07</td><td>50.31 ± 3.93</td><td>0.84 ± 0.06</td><td>0.25 ± 0.03</td><td>102.55 ± 7.57</td></tr><tr><td>48</td><td>8.31 ± 0.09</td><td>4.18 ± 0.07</td><td>0.81 ± 0.04</td><td>80.58 ± 0.69</td><td>3.37 ± 0.03</td><td>0.41 ± 0.01</td><td>104.35 ± 2.86</td></tr><tr><td>72</td><td>8.21 ± 0.05</td><td>4.77 ± 0.02<sup>a</sup></td><td>0.69 ± 0.01</td><td>85.61 ± 0.15<sup>a</sup></td><td>4.08 ± 0.01</td><td>0.50 ± 0.00</td><td>119.75 ± 0.39</td></tr><tr><td rowspan="4">Sucrose</td><td>0</td><td>0.00 ± 0.00</td><td>0.12 ± 0.01</td><td>0.10 ± 0.00</td><td>6.29 ± 0.38</td><td>0.01 ± 0.00</td><td>0.00 ± 0.00</td><td>0.00 ± 0.00</td></tr><tr><td>24</td><td>2.55 ± 0.10</td><td>1.31 ± 0.01</td><td>0.77 ± 0.02</td><td>41.19 ± 0.92</td><td>0.54 ± 0.01</td><td>0.21 ± 0.01</td><td>100.34 ± 3.42</td></tr><tr><td>48</td><td>7.72 ± 0.13</td><td>3.93 ± 0.06</td><td>0.83 ± 0.11</td><td>79.03 ± 2.64</td><td>3.11 ± 0.09</td><td>0.40 ± 0.02</td><td>101.09 ± 0.70</td></tr><tr><td>72</td><td>9.90 ± 0.00</td><td>4.75 ± 0.06<sup>a</sup></td><td>0.78 ± 0.10</td><td>83.66 ± 1.82<sup>a</sup></td><td>3.98 ± 0.04</td><td>0.40 ± 0.00</td><td>98.35 ± 2.76</td></tr><tr><td rowspan="4">Fructose</td><td>24</td><td>1.93 ± 0.09</td><td>0.11 ± 0.00</td><td>0.10 ± 0.00</td><td>8.71 ± 0.97</td><td>0.01 ± 0.00</td><td>0.00 ± 0.00</td><td>0.00 ± 0.00</td></tr><tr><td>48</td><td>5.89 ± 0.04</td><td>0.39 ± 0.01</td><td>0.30 ± 0.01</td><td>23.17 ± 2.80</td><td>0.09 ± 0.01</td><td>0.05 ± 0.00</td><td>40.47 ± 1.42</td></tr><tr><td>72</td><td>7.94 ± 0.31</td><td>2.26 ± 0.04</td><td>0.90 ± 0.07</td><td>60.25 ± 2.54</td><td>1.36 ± 0.04</td><td>0.23 ± 0.01</td><td>79.04 ± 1.44</td></tr><tr><td></td><td></td><td>3.31 ± 0.02<sup>b</sup></td><td>1.04 ± 0.04</td><td>68.57 ± 1.24<sup>b</sup></td><td>2.27 ± 0.05</td><td>0.29 ± 0.01</td><td>86.17 ± 2.99</td></tr></tbody></table>

CDW, cell dry weight; P(3HB), poly(3-hydroxybutyrate); Y<sup>O</sup><sub>p/s</sub>, grams of P(3HB) per gram of carbon consumed; Y<sub>Max</sub>, percentage of maximum theoretical yield. Values are mean ± SD (n = 3). Different superscript letters (a, b) at 72 h indicate significant differences (p < 0.05, Tukey's HSD test).
<table><caption>**Table 2.** Comparison of PHA (homo- and copolymers) production by halophilic and non-halophilic bacteria.</caption><thead><tr><th>Microorganisms</th><th>NaCl (g/L)</th><th>Substrate</th><th>Substrate supplied (g/L)</th><th>Cultivation time (h)</th><th>CDW (g/L)</th><th>Type PHA</th><th>PHA Content (%)</th><th>Yield (g/g)</th><th>References</th></tr></thead><tbody><tr><td>Azotobacter vinelandii</td><td>-</td><td>Glucose</td><td>30</td><td>22</td><td>9.9</td><td>P(3HB)</td><td>76</td><td>NR</td><td>[60]</td></tr><tr><td>Bacillus mycoides</td><td>-</td><td>Sucrose</td><td>20</td><td>24</td><td>3.6</td><td>P(3HB)</td><td>69.4</td><td>0.21</td><td>[85]</td></tr><tr><td>B. megaterium uyuni S29</td><td>45</td><td>Glucose</td><td>15</td><td>21</td><td>5.42</td><td>P(3HB)</td><td>41</td><td>0.13</td><td>[61]</td></tr><tr><td>Burkholderia sacchari</td><td>-</td><td>Xylose</td><td>15</td><td>48</td><td>5.5</td><td>P(3HB)</td><td>58.1</td><td>0.23</td><td>[62]</td></tr><tr><td>Cupriavidus sp. KKU38</td><td>-</td><td>Glucose</td><td>20</td><td>96</td><td>4.3</td><td>P(3HB)</td><td>73.9</td><td>0.16</td><td>[46]</td></tr><tr><td>Yangia sp. ND199</td><td>45</td><td>Fructose</td><td>20</td><td>30</td><td>5.6</td><td>P(3HB)</td><td>53.8</td><td>NR</td><td>[55]</td></tr><tr><td><strong>Halomonas sp. YLGW01</strong></td><td>SW</td><td>HFCS</td><td>30</td><td>72</td><td>7.95</td><td>P(3HB)</td><td>67.4</td><td>NR</td><td>[63]</td></tr><tr><td><strong>Halomonas cerina YK44</strong></td><td>40</td><td>Galactose</td><td>20</td><td>48</td><td>11.6</td><td>P(3HB)</td><td>78.1</td><td>NR</td><td>[64]</td></tr><tr><td><strong>Halomonas cerina YK44</strong></td><td>NR</td><td>GCRAH</td><td>14.25</td><td>96</td><td>~15</td><td>P(3HB)</td><td>~30</td><td>NR</td><td>[64]</td></tr><tr><td><strong>Halomonas cerina YK44</strong></td><td>20</td><td>Molasses + SF</td><td>40 + 0.5</td><td>72</td><td>9.2</td><td>P(3HB)</td><td>79.7</td><td>NR</td><td>[65]</td></tr><tr><td><strong>Halomonas halophila</strong></td><td>66</td><td>Rhamnose</td><td>20</td><td>72</td><td>6</td><td>P(3HB)</td><td>82.8</td><td>NR</td><td>[20]</td></tr><tr><td><strong>Halomonas boliviensis</strong></td><td>45</td><td>Maltose</td><td>8</td><td>33</td><td>0.92</td><td>P(3HB)</td><td>58.8</td><td>NR</td><td>[16]</td></tr><tr><td><strong>Halomonas campisalis</strong></td><td>45</td><td>Maltose</td><td>10</td><td>24</td><td>1.4</td><td>P(3HB-co-3HV)</td><td>81</td><td>NR</td><td>[83]</td></tr><tr><td><strong>Halomonas sp. TD01</strong></td><td>60</td><td>Glucose</td><td>30</td><td>48</td><td>6</td><td>P(3HB)</td><td>69</td><td>NR</td><td>[75]</td></tr><tr><td><strong>Halomonas campaniensis</strong></td><td>40</td><td>Glucose</td><td>NR</td><td>48</td><td>8.2</td><td>P(3HB)</td><td>57</td><td>NR</td><td>[66]</td></tr><tr><td><strong>Halomonas venusta</strong></td><td>15</td><td>Glucose</td><td>20</td><td>72</td><td>3.5</td><td>P(3HB)</td><td>68.9</td><td>NR</td><td>[47]</td></tr><tr><td><strong>Halomonas elongata</strong></td><td>100</td><td>Glucose</td><td>10</td><td>48</td><td>2.5</td><td>P(3HB)</td><td>40</td><td>0.09</td><td>[84]</td></tr><tr><td><strong>Halomonas elongata 1H9T</strong></td><td>45</td><td>Glucose</td><td>50</td><td>80</td><td>-</td><td>P(3HB)</td><td>53</td><td>0.11</td><td>[53]</td></tr><tr><td><strong>Halomonas sp. SF2003</strong></td><td>11*</td><td>Glucose</td><td>20</td><td>72</td><td>2.6</td><td>P(3HB)</td><td>86</td><td>NR</td><td>[59]</td></tr><tr><td><strong>Halomonas smyrnensis AAD6T</strong></td><td>137*</td><td>Glucose</td><td>20</td><td>120</td><td>2.0</td><td>P(3HB)</td><td>45.8</td><td>NR</td><td>[57]</td></tr><tr><td><strong>Halomonas smyrnensis AAD6T</strong></td><td>137*</td><td>Sucrose</td><td>50</td><td>144</td><td>1.34</td><td>P(3HB)</td><td>26.9</td><td>NR</td><td>[57]</td></tr><tr><td><strong>Halomonas halmophila 18H</strong></td><td>96</td><td>Glucose</td><td>20</td><td>72</td><td>1.2</td><td>P(3HB)</td><td>63.72</td><td>NR</td><td>[67]</td></tr><tr><td><strong>Halomonas sp. HG01</strong></td><td>100</td><td>Glucose</td><td>10</td><td>72</td><td>4.7</td><td>P(3HB)</td><td>85.6</td><td>0.50</td><td>This study</td></tr><tr><td><strong>Halomonas sp. HG01</strong></td><td>100</td><td>Sucrose</td><td>10</td><td>72</td><td>4.7</td><td>P(3HB)</td><td>83.7</td><td>0.40</td><td>This study</td></tr><tr><td><strong>Halomonas sp. HG01</strong></td><td>100</td><td>Fructose</td><td>10</td><td>72</td><td>3.3</td><td>P(3HB)</td><td>68.6</td><td>0.29</td><td>This study</td></tr></tbody></table>

NR, Not reported; CDW, Cell dry weight; SW, Sea water, HFCS– high fructose corn syrup (containing fructose, glucose and sucrose); GCRAH, galactose-containing red algae hydrolysate, SF – soybean flour. * Sea salt
### 3.3. Organic acids for copolymer biosynthesis by *Halomonas* sp. HG01

P(3HB) is the most common type of PHA synthesized by a wide variety of bacteria [68,69]. The controlled synthesis of PHA containing 3HB as the main constituent and other monomers has been extensively used as the approach to obtain polymers with variable thermomechanical properties and attending the performance for different applications [70,71]. Monomeric composition of PHA can be controlled according to the carbon sources supplied, the metabolic pathway involved [55,69,72], the microbial strain [49,55,69,73], and the monomer specificity of the PHA synthase (PhaC) [68,69,72]. *Halomonas* sp. HG01 was capable of synthesizing PHA with 3HB monomers using different carbon sources in this study, a feature commonly observed in *Halomonas* spp. [16,17,74,75]. Most microorganisms require a 3HV precursor for P(3HB-co-3HV) synthesis as for example *Halomonas* sp. SF2003, which produced P(3HB) and P(3HB-co-3HV) copolymers using fructose, galactose, and glucose mixed with one fatty or organic acid. *H. bluephagenesis* TD01 and *H. profundus* produced P(3HB-co-3HV) when glucose was supplemented with propionic acid or valeric acid [18,76]. Similarly, *H. marina* HMA103 accumulated P(3HB-co-3HV) with 12.8 mol% 3HV in glucose medium provided additionally with valeric acid [77].

To evaluate the ability of *Halomonas* sp. HG01 to synthesize PHA copolymers, different organic acids (C3 to C9), and sodium 4-hydroxybutyrate were added as co-substrates after 24 hours of the culture using glucose as the main carbon source (Table 3). ANOVA revealed highly significant differences in both CDW (F = 1115.17, p < 0.05) and PHA content (F = 750.29, p < 0.05) among the different precursors tested. The higher the carbon backbone extent of co-substrate, the higher is the inhibitory effect on growth and PHA production. Tukey's HSD post hoc test was used to group the precursors according to their effect on biomass production. Three distinct groups were identified. The first group included precursors that maintained high biomass levels, similar to the control (>4.0 g/L; valeric acid, 4-
hydroxybutyric, and propionic acid; p > 0.05). The second group consisted of precursors that moderately inhibited growth, with biomass concentrations between 2.0 and 3.5 g/L (hexanoic and butyric acids). The third group included precursors that strongly inhibited growth, with values below 1.5 g/L (heptanoic, octanoic, and nonanoic acids), corresponding to reductions greater than 70% compared to the control (p < 0.05) (Table 3). Clear inhibitory effects on PHA accumulation occurred with the addition of all organic acids except 4-hydroxybutyric acid (Table 3). A negative correlation was observed between fatty acid chain length and both cell growth and PHA accumulation, with acids ≥C7 causing severe inhibition likely associated with cellular toxicity or membrane disruption. In the future, additional investigations should be carried out to identify the phenomena the resulted in the inhibitory effects caused by the longer fatty acids, however in bioreactor cultures it is possible to control the concentration of this organic acids by a feeding approach making this inhibitory effects less relevant.
<table><caption>**Table 3.** Growth and PHA accumulation by *Halomonas* sp. HG01 using different organic acids and sodium 4-hydroxybutyrate as precursors.</caption><thead><tr><th rowspan="2">Precursor Substrates</th><th rowspan="2">Culture time (h)</th><th colspan="2">CDW (g/L)</th><th colspan="3">PHA</th><th rowspan="2">Y<sub>p/s</sub><sup>a,b</sup><br>(g/g)</th><th rowspan="2">Y<sub>MAX</sub><br>(%)</th></tr><tr><th>Total</th><th>Residual</th><th>(g/L)</th><th>(%CDW)</th><th>3HV (mol%)</th><th>4HB (mol%)</th></tr></thead><tbody><tr><td rowspan="2">None</td><td>48</td><td>3.98 ± 0.00</td><td>1.24 ± 0.00</td><td>2.74 ± 0.00</td><td>68.76 ± 0.00</td><td>ND</td><td>ND</td><td>0.35 ±</td><td>103.73 ± 0.00</td></tr><tr><td>72</td><td>4.40 ± 0.06<sup>a</sup></td><td>1.27 ± 0.05</td><td>3.13 ± 0.05</td><td>71.23 ± 0.51<sup>a</sup></td><td>ND</td><td>ND</td><td>0.40 ± 0.00<sup>*</sup></td><td>115.05 ± 1.45</td></tr><tr><td rowspan="2">Propionic acid</td><td>48</td><td>3.00 ± 0.00</td><td>1.26 ± 0.00</td><td>1.74 ± 0.00</td><td>58.06 ± 0.00</td><td>1.33 ± 0.00</td><td>ND</td><td>0.04 ± 0.00<sup>**</sup></td><td>3.04 ± 0.00</td></tr><tr><td>72</td><td>4.15 ± 0.01<sup>a,b</sup></td><td>1.42 ± 0.02</td><td>2.73 ± 0.02</td><td>65.84 ± 0.43<sup>b</sup></td><td>1.21 ± 0.03<sup>b</sup></td><td>ND</td><td>0.04 ± 0.00<sup>**</sup></td><td>2.81 ± 0.16</td></tr><tr><td rowspan="2">Butyric acid</td><td>48</td><td>2.17 ± 0.00</td><td>1.34 ± 0.00</td><td>0.83 ± 0.00</td><td>38.14 ± 0.00</td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>72</td><td>2.31 ± 0.12<sup>c</sup></td><td>1.26 ± 0.06</td><td>1.04 ± 0.06</td><td>45.27 ± 0.40<sup>c</sup></td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td rowspan="2">Valeric acid</td><td>48</td><td>2.76 ± 0.00</td><td>1.16 ± 0.00</td><td>1.60 ± 0.00</td><td>57.83 ± 0.00</td><td>22.03 ± 0.00</td><td>ND</td><td>0.89 ± 0.00<sup>**</sup></td><td>90.82 ± 0.00</td></tr><tr><td>72</td><td>4.45 ± 0.05<sup>a</sup></td><td>1.36 ± 0.06</td><td>3.09 ± 0.05</td><td>69.40 ± 1.25<sup>a</sup></td><td>25.53 ± 0.87<sup>a</sup></td><td>ND</td><td>0.81 ± 0.06<sup>**</sup></td><td>82.65 ± 5.62</td></tr><tr><td rowspan="2">Hexanoic acid</td><td>48</td><td>2.73 ± 0.00</td><td>1.19 ± 0.00</td><td>1.54 ± 0.00</td><td>56.57 ± 0.00</td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>72</td><td>3.14 ± 0.02<sup>b</sup></td><td>1.21 ± 0.04</td><td>1.93 ± 0.02</td><td>61.35 ± 1.03<sup>b</sup></td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td rowspan="2">Heptanoic acid</td><td>48</td><td>1.46 ± 0.00</td><td>0.92 ± 0.00</td><td>0.54 ± 0.00</td><td>36.84 ± 0.00</td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>72</td><td>1.45 ± 0.08<sup>d</sup></td><td>0.89 ± 0.05</td><td>0.56 ± 0.05</td><td>38.58 ± 1.85<sup>c</sup></td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td rowspan="2">Octanoic acid</td><td>48</td><td>1.29 ± 0.00</td><td>0.84 ± 0.00</td><td>0.45 ± 0.00</td><td>34.56 ± 0.00</td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>72</td><td>1.20 ± 0.09<sup>d</sup></td><td>0.75 ± 0.05</td><td>0.45 ± 0.04</td><td>37.74 ± 1.17<sup>c</sup></td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td rowspan="2">Nonanoic acid</td><td>48</td><td>1.43 ± 0.00</td><td>0.96 ± 0.00</td><td>0.50 ± 0.00</td><td>33.05 ± 0.00</td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td>72</td><td>1.43 ± 0.11<sup>d</sup></td><td>0.94 ± 0.07</td><td>0.50 ± 0.04</td><td>34.59 ± 0.42<sup>c</sup></td><td>ND</td><td>ND</td><td>-</td><td>-</td></tr><tr><td rowspan="2">4-hydroxybutyric</td><td>48</td><td>4.41 ± 0.00</td><td>1.44 ± 0.00</td><td>2.97 ± 0.00</td><td>67.41 ± 0.00</td><td>ND</td><td>1.19 ± 0.00</td><td>0.04 ± 0.00</td><td>3.54 ± 0.00</td></tr><tr><td>72</td><td>4.32 ± 0.00<sup>a</sup></td><td>1.60 ± 0.00</td><td>2.72 ± 0.00</td><td>63.00 ± 0.00<sup>a,b</sup></td><td>ND</td><td>1.36 ± 0.00</td><td>0.04 ± 0.00</td><td>3.70 ± 0.00</td></tr></tbody></table>

CDW, Cell dry weight; Residual cell dry weight (Cell biomass without polymer); 3HV, 3-hydroxyvalerate; ND, Not detected. Values are mean ± SD (n = 3). Different superscript letters (a-d) at 72 h indicate significant differences (p < 0.05, Tukey's HSD test). 3HV incorporation from valeric vs. propionic acid was compared by Student's t-test (p < 0.05).*; Y<sub>3HB/Gluc</sub>, Yield coefficient of 3HB produced per gram of glucose consumed.

**; Y<sub>3HV/Prop</sub> or Y<sub>3HV/Val</sub>, respectively, 3HV yields from propionic or valeric acids.
Odd-chain organic acids were added to the cultures to evaluate if *Halomonas* sp. HG01 was able to produce PHA containing odd-chain monomers (3HV, 3HHp, etc). In this investigation, *Halomonas* sp. HG01 demonstrated the ability to biosynthesize the 3HV monomer from propionic and valeric acids (Table 3) which was consistent with studies evaluating other *Halomonas* strains [18,43,75-77]. When the strain HG01 was cultured on glucose with propionic acid, the P(3HB-co-3HV) content reached 65.84% of CDW, containing a maximum molar fraction of 1.33 mol% 3HV. Similar results in the amount of 3HV monomers incorporated in P(3HB-co-3HV) were obtained with *Halomonas* sp. TD01 (0.83 mol% 3HV) and *Herbaspirillum seropedicae* Z96 (1.8 mol% 3HV) [51,78]. However, these values were lower than that reported in *Burkholderia* sp. IPT 101 (6.2 mol% 3HV) under similar culture conditions [79]. When propionic acid is added to the culture medium, it is transformed into propionyl-CoA by the cell [80,81]. Part of the propionyl-CoA can be metabolized to generate energy, and part can be used to synthesize 3HV monomers in a Claisen-condensation with acetyl-CoA catalyzed by a 3-ketothiolase [40,79]. Given the very low efficiency in converting propionic acid to 3HV monomers (about 3%), strain HG01 utilizes mainly propionic acid as a carbon and energy source [79]. Valeric acid also promoted the synthesis of the P(3HB-co-3HV) copolymer by *Halomonas* HG01 (Table 3). Valeric acid demonstrated a clear advantage over propionic acid in supporting both cell growth and PHA accumulation. According to Tukey's analysis, CDW levels in cultures supplemented with valeric acid (4.45 g/L) were statistically equivalent to the control (4.40 g/L, p > 0.05) and significantly higher than those achieved with propionic acid (4.15 g/L). Similarly, PHA accumulation was maintained at high levels (69.40 wt.%) without significant differences from the control (71.23 wt.%, p > 0.05), whereas both conditions outperformed the other tested precursors. Interestingly, unlike propionic acid, valeric acid allowed the synthesis of PHA presenting a higher 3HV molar fraction (about 20-25 mol%) at high efficiency (about 80-90%). The comparison of 3HV incorporation between valeric and propionic acids
further highlighted the superior performance of valeric acid. Student's t-test indicated that this difference was highly significant (t = 48.39, p < 0.05), with valeric acid exhibiting approximately a 21-fold higher incorporation efficiency of 3HV into the copolymer. Valeric acid is activated to valeryl-CoA and then metabolized in the β-oxidation of fatty acids. C5 intermediates from β-oxidation can be channeled to 3HV synthesis via enoil-CoA hydratase or 3-ketoacyl-CoA reductase [43,82]. The higher efficiency observed with valeric acid can be explained by differences in its metabolic conversion. Valeric acid showed a yield coefficient ($Y_{3HV/Val}$) of approximately 0.81–0.89 g/g, corresponding to 83–91% of the maximum theoretical yield, whereas propionic acid presented a much lower efficiency ($Y_{3HV/Prop}$ ≈ 0.04 g/g, about 3%). This difference is mainly due to the direct conversion of valeric acid to 3-hydroxyvaleryl-CoA through β-ketothiolase, which acts as an immediate substrate for PHA synthase. By contrast, propionic acid is metabolized through more complex and less efficient pathways, leading to low 3HV incorporation into the copolymer. Previous reports involving other bacterial strains such as *Yangia* sp. ND199 (8.1 mol% 3HV) and *Halomonas marina* HMA (11.8 mol% 3HV) have also shown the relevance of valeric acid as a 3HV monomer precursor [72,77]. The highest 3HV molar fraction (95 mol% 3HV) incorporated in the polymer was reported with *Caldimonas taiwanensis* grown with soluble starch and valeric acid [45].

Even-chain organic acids were evaluated mainly as potential precursors of 3HHx. Similarly to the 3HV synthesis from propionic acid, 3HHx synthesis from butyric acid would depend on a 3-ketothiolase action. Metabolic intermediates from the β-oxidation of hexanoic and octanoic acids would be channeled for 3HHx synthesis. In the conditions tested in this study, *Halomonas* sp. HG01 was unable to synthesize PHA containing 3HHx as constituent.

### 3.4. Production of P3HB in fed-batch bioreactor
To evaluate kinetics parameters of growth and P(3HB) production by *Halomonas* sp. HG01, a fed-batch bioreactor cultivation was conducted using glucose as the sole carbon source. The profiles of cell growth and P(3HB) accumulation are shown in Figure 2. Maximum cell concentration, P(3HB) concentration and P(3HB) content of 12.2 g/L, 7.7 g/L and 63%, respectively, were obtained in 72h, while the maximum productivity reached in 55 h was 0.12 g P(3HB)/L.h. As depicted in Figure 2, the maximum value of residual biomass during growth (cell biomass excluding the PHA mass) was 4.5 g/L in the first 24 h, and then remained constant due to nitrogen depletion (Figure 2). At this stage, the specific growth rate (μ=0.16±0.01 h⁻¹) calculated was found to be identical to that obtained in shaken flask cultures (data not reported). During the active exponential phase, a slight accumulation of P(3HB) was observed (Figure 2), which then increased greatly after nitrogen depletion (Figure 3). This behavior is in accordance with that observed in other *Halomonas* spp. [75,83,84]. Despite the high P(3HB) content obtained in the fed-batch bioreactor, with adequate air flow and controlled pH, the amount of accumulated P(3HB) was lower than that obtained in the shaken flask cultures. Cultivations in shaken flasks are characterized by oxygen-limited conditions. Several studies have shown that oxygen may play an important role in P(3HB) synthesis [85,86]. For example, reports demonstrated that mimicking the shaken flask conditions in a bioreactor, with a low air inflow, P(3HB) accumulation obtained is higher than when under high oxygen supply conditions [16]. Meanwhile, authors [87,88] found that, maintaining the oxygen level at 1 to 5% air saturation during the PHA accumulation phase, the polymer accumulation is higher than under an oxygen concentration of 10 to 15%. Such effect might be attributed to the influence of oxygen on the intracellular concentrations of the P(3HB) biosynthetic enzymes (3-ketothiolase and acetoacetyl-CoA reductase) and also to the carbon flow through the central catabolism, as observed in early studies by Blunt and collaborators in 2018 and 2019 [80,86]. Therefore, the reduction not only on the P(3HB) content but also on the efficiency in converting glucose into P(3HB)
(about 0.075 g/g, 25% of maximum theoretical value) could be a result of the high airflow in the fed-batch culture. The results obtained suggest that, in addition to nitrogen, oxygen may be another important factor to be considered to improve the P(3HB) production in *Halomonas* sp. HG01.

In the future, cultures on non-sterile conditions could be carried out to confirm that this more economical condition could be efficiently applied. Besides this, a better knowledge of the NaCl concentration more appropriated for the growth and PHA accumulation by *Halomonas* sp. HG01 will be useful for further bioprocess improvement.

<figure><img src="image_3.png" /><figcaption>**Figure 2.** Cellular growth, glucose consumption and P(3HB) production by *Halomonas* sp. HG01 during cultivation in mineral salts medium with glucose in fed-batch culture. CDW: cell dry weight. Residual biomass: cells without P(3HB).</figcaption></figure>
<figure><img src="image_4.png" /></figure>

**Figure 3.** Effect of nitrogen source limitation on P(3HB) production by *Halomonas* sp. HG01 during cultivation in mineral salt medium with glucose in fed-batch culture.

Another important point to be developed in the future is the characterization of the polymer produced by *Halomonas* sp. HG01. Molar mass, thermal and mechanical properties will be very useful in characterizing the PHA and defining its potential for different applications.

**3.5. Genome sequencing analysis *Halomonas* sp. HG01**

The first draft sequence of *Halomonas* sp. HG01 was obtained a few years ago with a 180-fold coverage [37]. Based on this draft, the complete *Halomonas* sp. HG01 genome sequence was accomplished in this work. This can be observed in the cumulative diagram profile (GC-Skew) shown in Figure 4. The profile of V/inverted V-shape for bi-directional replication between singular ori and ter indicates that the scaffolds were assembled into one single chromosome [89], exhibiting 68% of GC content.
<figure><img src="image_5.png" /><figcaption>**Figure 4.** Cumulative diagram profile (GC-Skew) obtained from the *Halomonas* sp. HG01 genome.</figcaption></figure>

The automatic annotation of the genes from *Halomonas* sp. HG01 whole genome sequence using RAST and KBase was performed and subsequently used to reconstruct the GEM. The HG01 genome is organized in one circular chromosome with 3,666,044 bp, which is graphically represented in Figure 5, indicating the GC distribution (68%) and GC skew. Blast Ring Image Generator (BRIG-0.95) [90] was used to compare the genome identity of the HG01 fully-genome sequenced with five different *Halomonas* species: *Halomonas* sp. SF2003 (4.4 Mb, 62.5% GC), *H. bluephagenesis* TD01 (4.1 Mb, 52.5% GC), *H. elongata* DSM 2581 (4.1 Mb, 63.5% GC), *H. halophila* CCM 3662 (3.6 Mb, 68% GC) and *H. boliviensis* LC1 (or *Vreelandella boliviensis* LC1 - 4.2 Mb, 54.7% GC). The location of the *Halomonas* sp. HG01 genes presented in Figure 5 are depicted in black. Additional genes from the PHA production pathway from Tables SM3 and SM4 are presented in red and those from the ectoine biosynthesis and regulation are given in blue.

A total of 3,366 protein-coding genes and 80 genes coding tRNAs or rRNAs were found. The genes were grouped in 26 subsystems (Figure SM1), each one representing a set of relational functional roles that, together, implement a specific biological process in *Halomonas* sp. HG01. Some important
genes, enzymes and metabolic pathways involved in the carbohydrate metabolism, PHA production and stress resistance have been refined in this analysis and evaluated using the GEM reconstructed in this work.

<figure><img src="image_6.png" /><figcaption>**Figure 5.** Graphical representation of the 3.66 Mb chromosome of *Halomonas* sp. HG01 indicating from the innermost ring: distribution of the GC content (black), GC skew (purple/green), homology with self</figcaption></figure>
(solid purple), *Halomonas* sp. SF2003 (pink), *H. bluephagenesis* TD01 (light blue), *H. elongata* DSM 2581 (dark blue), *H. halophila* CCM 3662 (purple) and *H. boliviensis* LC1 (yellow). The outermost red blocks indicate areas where *Halomonas* sp. HG01 has low homology with other species, and are annotated with possible genes of interest. The approximate position and locus tag of genes involved in ectoine biosynthesis are marked in blue, those producing PHA are in magenta color.

## 3.6 Stress regulation and resistance related genes

*Halomonas*, halotolerants chemoorganotrophic bacteria, are considered a versatile group which can adapt and colonize different environments, they need specialized enzymes or metabolic pathways to survive and improve their ecological fitness. The manual curation in the *Halomonas* sp. HG01 genome and in its GEM reconstructed (Table SM3) showed the presence of genes involved in many types of stress responses such as: osmoadaptation (osmotic stress), protection of cells and its components from freezing, cold shock, or heat shock, at high or low temperatures, respectively, and reactive oxygen species (oxidative stress and detoxification) [91,92].

Halophilic microorganisms evolved mechanisms to adapt to high-salinity environments. One such mechanisms is the maintenance of a high cytoplasmic potassium chloride concentration (such as $K^+$) within the cells to balance the extracellular osmotic pressure of NaCl [93,94]. A second strategy, identified in several bacterial species, is the intracellular accumulation of low molecular weight polar organic compounds highly soluble in water in high concentrations, called compatible solutes or Osmoprotectants, such as, ectoine, choline, betaine and glucans [91]. Such compounds can be synthesized de novo or uptaken directly from the environment by the bacterial cells to promote their survival [95]. Ectoine is also produced by cultivation of *H. boliviensis* and *H. elongata* [88]. These solutes also operate stabilizing enzymes, DNA, cytoplasmic membranes, and whole cells against other different stress conditions, as for example, heating, drying and freezing [91,92]. A total of 35 genes involved in osmotic
stress resistance were identified in HG01 genome (Table SM3), including those involved in the
metabolic pathways of biosynthesis of the osmoprotectants ectoine, hydroxyectoine, glycine-betaine,
choline and osmoregulated periplasmic glucans. Ectoine and its derivatives hydroxyectoine are polar and
highly water-soluble compounds with low-molecular weight accumulated by bacterial cells to survive or
resist under hypersaline environments - osmoregulatory function - due to their salt-in-cytoplasm
mechanism [92,95,97]. Ectoine was first identified by [98] in the extremely halophilic phototrophic
bacterium *Ectothiorhodospira halochloris*. In the *Halomonas* sp. HG01 genome (Table SM3), the genes
involved in ectoine production are organized in an operon *ectABC*, which comprises an *ectD* gene that is
responsible for the hydroxyectoine synthesis from ectoine. This operon structure was also reported in
*Halomonas elongata* DSM 2581, *Halomonas salina* BCRC 17875, *Halobacillus halophilus* and *Bacillus
pasteurii* [75,99]. In the ectoine synthesis pathway, a L-2,4-diaminobutyratetransaminase, enzyme
encoded by *ectB* gene, catalyses the reaction from L-aspartate-β-semialdehyde (ASA) to L-2,4-
diaminobutyrate (DABA). DABA is then turned into N-acetyl-2,4-diaminobutyrate (ADABA) by the
2,4-diaminobutyrate acetyltransferase (encoded by *ectA*). Finally, ADABA is converted to ectoine by an
ectoine synthase (encoded by *ectC*) [100,101]. Expression of the operon *ectABC* is usually induced in
response to upped osmolarity or extreme growth temperatures [102,103]. Besides ectoine, betaine
(glycine betaine), one of the most powerful osmoprotectant compound, despite not being synthesized by
most bacteria [104], seems to be produced by *Halomonas* sp. HG01 strain, demonstrating the strain
versatility. Genes involved in the metabolic pathways of betaine and choline biosynthesis were
identified in strain HG01 (Table SM3) and previously described in *Halomonas elongata* DSM
3043[100]. Betaine is synthesized from its precursor choline in two steps: (1) In the first step, the
production of choline from choline-O-sulfate is catalysed by choline-O-sulfatase (encoded by *betC*
gene) or/and its uptake into the cell by a high-affinity choline uptake protein BetT (encoded by *betT*
gene). After that, choline is converted into betaine aldehyde by a membrane-bound choline dehydrogenase (encoded by *betA* gene); (2) Then, in the second step, betainealdehyde dehydrogenase (encoded by *betB* gene) catalyses the conversion of betaine aldehyde to betaine / glycine betaine. The reactions seem to be controlled by the HTH-type transcriptional regulator BetI, encoded by *betI* gene that also was identified in the HG01 genome (Table SM2). In addition to this, *Halomonas* sp. HG01 also has glycine betaine/choline ABC transport systems and permeases to uptake these compounds into the cells (Table SM3).

Furthermore, heavy metals tolerance genes were found in the HG01 genome related to copper, cobalt-zinc-cadmium, mercury, arsenic, chromium composts, or iron. Multidrug resistance genes - fluoroquinolones, beta-lactamases and efflux pumps - were also identified. Many of these were assigned as being of extrachromosomal origin, as for example, genes normally found in plasmids (colicin V production gene, heavy metals or antibiotic resistance genes), bacteriophages and phage (genes involved in phage tail proteins, replication, packaging machinery, tail fiber proteins, capsid proteins, DNA synthesis and integrons), which has been promoted by a horizontal transfer suffered by HG01 strain, demonstrating the plasticity of its genome.

### 3.7. Carbon source uptake and catabolism in *Halomonas* sp. HG01

The interesting adaptability of *Halomonas* species to diverse growth conditions makes them good candidates for biotechnological applications. Therefore, a search was carried out on genes, enzymes and metabolic pathways involved in catabolism of different carbohydrates.

Metabolic pathways involved in the catabolism of sucrose, glucose and fructose were identified as expected, since both carbon sources were efficiently used by *Halomonas* sp. HG01 (Figure 6 and Table 1). Sucrose is an important disaccharide applied as substrate in many industrial applications due to the
high amount available in nature and its low cost [105]. However, only a limited number of
microorganisms can utilize it, primarily due to the absence of the necessary transporters and enzymes for
its hydrolysis [48,105,106]. The HG01 genome annotation revealed genes putatively encoding a sucrose
hydrolysis enzyme, a sucrose catabolic repressor (LacI family), and a sucrose phosphotransferase/PTS
permease (EC 2.7.1.211) related to sucrose uptake into the cells (Table SM3 and Figure 6). In relation to
glucose and fructose catabolism, glucose seems to be phosphorylated by a glucokinase (Table SM4),
however the system responsible for glucose uptake remains to be identified in strain HG01. With respect
to fructose uptake and phosphorylation, the operon *fruBKA* was identified in *Halomonas* sp. HG01
genome, being regulated by a fructose catabolic repressor FruR from the LacI family, encoded by a *fruR*
gene located downstream of the operon. It involves the uptake of fructose through a sugar-specific
phosphotransferase system - PTS<sup>Fru</sup>, fructose-specific IIB and IIC components (EC 2.7.1.69) encoded by
the *fruA* and *fruB* genes, respectively, which phosphorylates this sugar in fructose 1-phosphate (F1P). A
gene encoding a fructokinase (EC 2.7.1.4) was identified when its amino acid sequences from
*Halomonas* sp. HL-48 and *H. elongata* DSM 2581 were used to carry out a Blast against the *Halomonas*
sp. HG01 genome. This enzyme is responsible for the conversion of fructose into fructose 6-phosphate
(F6P). Both F1P and F6P are phosphorylated to fructose 1,6-bisphosphate (F16BP) by
phosphofructokinase (EC 2.7.1.56) encoded by the gene *fruK*. After that, F16BP can be catabolised by
the Embden-Meyerhoff-Parnas (EMP) or the Entner-Doudoroff (ED) pathways identified in the GEM of
strain HG01 (Table SM03). Previous studies have demonstrated that some *Halomonas* species have used
ED followed by the Pentose Phosphate pathway (PPP) for degradation of different carbohydrates
(glucose, sucrose, xylose, maltose, starch hydrolysate, etc.) as described to *H. boliviensis* [21], *H.
bluephagenesis* TD01[43,81] and *H. elongate* [53,107]. Many bacteria rely on the ED pathway for the
oxidation of glucose, since the *pfk* gene encoding the 6-phosphofructokinase, a main enzyme from the
EMP pathway, is missing, as for example in *Pseudomonas* sp. [108,109], however, this enzyme is present in *Halomonas* sp. HG01 (Table SM4). In *E. coli*, both ED and EMP pathways are present, but ED is activated only when gluconate is present as carbon source. According to [107], in *Halomonas elongata* the metabolic pathways involved in glucose utilization are the EMP for energy generation; gluconate pathway, PPP and ED pathway for reducing power (NADPH) supply, fatty acids and nucleotide biosynthesis. However, in the strain *H. elongata* 1H9T, the glucose metabolism has been suggested to be conducted through the ED [53]. Revealing the conditional relevance of these two glycolytic pathways in *Halomonas* spp. will be of great importance to the curation of the reconstructed GEM and to establish proper *in silico* experiments for metabolic engineering. Figure 6 illustrates the metabolism pathways utilized by *Halomonas* sp. HG01 for growth and P(3HB) synthesis from different carbon sources.

<figure><img src="image_7.png" /><figcaption>**Figure 6.** Overview of carbohydrate and volatile fatty acid utilization pathways and PHA biosynthesis in *Halomonas* sp. HG01, deduced from a GEMs reconstructed using KBase and RAST based on its genome. The pathways include the Embden-Meyerhof-Parnas (EMP), Entner-Doudoroff (ED), and pentose phosphate</figcaption></figure>
(PPP) pathways, which converge in the production of acetyl-CoA, the precursor for P(3HB) and P(3HB-co-3HV). Key genes identified encode critical enzymes: *fba* (fructose-bisphosphate aldolase), *pgl* (6-phosphogluconolactonase), *edd* (6-phosphogluconate dehydratase), *eda* (2-keto-3-deoxy-6-phosphogluconate aldolase), *gnd* (6-phosphogluconate dehydrogenase), *fruK* (fructokinase), *glpK* (glycerol kinase), *gpd1* (glycerol-3-phosphate dehydrogenase), *prpE* (propionyl-CoA ligase), and *prpC* (2-methylcitrate synthase). PHA biosynthesis is mediated by *phaA* (3-ketothiolase), *phaB* (acetoacetyl-CoA reductase), *phaC* (PHA synthase), and *bktB* (3-ketothiolase). Propionate is primarily directed toward the methylcitrate cycle (MCC), with low incorporation of 3HV (dashed lines), whereas valerate promotes a continuous flux toward 3HV, enhancing its incorporation into the copolymer.

Growth and PHA accumulation by *Halomonas* sp. SF2003 using different carbon substrates (glucose, sucrose, fructose, galactose, mannose and maltose) or a mix of sugars and acids were reported by [52]. In relation to the conditions evaluated in this study of *Halomonas* sp. HG01, no significant growth was observed when cultured on lactose, xylose, or glycerol, as only carbon sources (Table SM2). The inability of this strain to use these substrates suggests a partial or total lack of a genetic machinery and metabolic pathways for degrading these substrates. The investigation for genes related to lactose and galactose utilization indicated the absence of them in the HG01 genome. However, genes involved in glycerol catabolism were identified (Table SM4 and Figure 6). All the genes needed for glycerol uptake (*ugp* genes - encode Glycerol-3-phosphate ABC transporter proteins), phosphorylation (*glpK* gene - encodes the glycerol kinase) and oxidation (*gpd* gene - encodes the glycerol-3-phosphate dehydrogenase) are present in the genome of *Halomonas* sp. HG01. Therefore, the inability of strain HG01 to use glycerol should be explained by the presence of pseudo-genes or improperly expressed genes. Glycerol can either be phosphorylated by the glycerol kinase to glycerol-3-phosphate(G3P) which is then oxidized by the G3P dehydrogenase (G3PDH) to dihydroxyacetone phosphate (DHAP), or glycerol can be oxidized by glycerol dehydrogenase to dihydroxyacetone (DHA) which is then phosphorylated by DHA kinase to DHAP. Both alternative pathways are referred to as G3P pathway and DHA pathway, respectively. Besides, four different pathways have been described for the utilization of
xylose, (i) xylose isomerase, (ii) xylose reductase and dehydrogenase, (iii) Weimberg and (iv) Dahms
[110], none of which were identified in the genome of *Halomonas* HG01.

**3.8 PHA production pathway in *Halomonas* sp. HG01**

P(3HB) was produced from glucose, fructose and sucrose by *Halomonas* sp. HG01 (Table 1). As reported by [111] Sedlacek *et al.* (2019), PHA granules protect the cells from adverse effects of osmolarity fluctuations; being the PHA biosynthesis a positive feature during adaptation to hypertonic conditions and osmotic fluctuations. The biosynthesis of P(3HB) from acetyl-CoA comprises the participation of three enzymes: 3-ketothiolase (*phaA*), NAD(P)H-dependent acetoacetyl-CoA reductase (*phaB*), and PHA synthase (*phaC*) [25,68]. Genes encoding the enzymes involved in PHA synthesis were also detected in the *Halomonas* sp. HG01 genome (Table SM4 and Figure 5). However, unlike other microbial genera where the genes are clustered and organized in one *phaCAB* operon, the genes responsible for PHA synthesis in *Halomonas* sp. HG01 are scattered in the genome as reported in other *Halomonas* spp. [18,52]. *Halomonas* sp. HG01 has a class I poly-(R)-hydroxyalkanoic acid synthase with a nucleotide sequence showing a similarity of >97% to enzymes from different *Halomonas* species and 88% to the *Pseudomonas aeruginosa* PAO1 *phaC*₂ (BlastN and Blastx analysis). A polyhydroxyalkanoate synthesis repressor (encoded by *phaR* gene) also is present. No P(3HB) depolymerase encoding gene (*phaZ*) was detected in this strain.

The synthesis of P(3HB) demands acetyl-CoA and reducing equivalents (NAD(P)H). These should be
supplied by the pathways involved in the carbon source catabolism. Frequently, bacteria producing
P(3HB) from carbohydrates relies on the ED pathway which supplies 2 moles of acetyl-CoA and 1 mol
of NADPH per mol of glucose oxidized perfectly meeting the demands for P(3HB) synthesis. A lower
P(3HB) yield from carbon source was observed from fructose than glucose (Table 1). The hypothesis to
justify this different performance could be based on the fact that *Halomonas* sp. HG01 catabolizes
fructose through the EMP pathway and glucose by the ED pathway, which supplies the proper
coenzyme for P(3HB) synthesis. However, this subject is more complex since, in *Halomonas* sp. GH01,
NADH is the cofactor used by its PhaB enzyme in the P(3HB) production - instead of NADPH [43].
Furthermore, the presence of a membrane transhydrogenases encoded by the *pntAB* genes, along with
NAD kinases encoded by the *nadK* gene in various *Halomonas* species and *Halomonas* sp. HG01 (Table
SM05), may facilitate the conversion of NADH to NADPH. This conversion could enhance the
flexibility of flux distribution within the metabolic network, although it would require a higher energy
input [68,112,113].

Recently, our group proposed that in the absence of the *gnd* gene, *Pseudomonas* sp. LFM046 adjusts a
cyclic operation of ED pathway to satisfy different demands of reducing equivalents and precursors for
growth or PHA production [108]. Although the *gnd* gene is absent in several species of *Halomonas*, it
was detected in *Halomonas* sp. HG01 (Table SM04).

In the context of copolymer production by *Halomonas* sp. HG01, the addition of 4-hydroxybutyric acid
to the culture medium resulted in the synthesis of a polymer containing 4HB. This suggests the presence
of a gene encoding an acyl-CoA synthetase in its genome; however, identifying the specific gene
responsible for this function is a challenging task due to the existence of multiple candidate genes.
Additionally, this finding indicates a degree of specificity of the PHA synthase of *Halomonas* sp. HG01
for this particular monomer. *Halomonas* sp. HG01 was also able to produce a PHA containing 3HV
monomers from propionic or valeric acids (Table 3). Interestingly [83] Kulkarni *et al.* (2011) reported
that *Halomonas campisalis* MCM B-1027 was able to produce P(3HB-*co*-3HV) supplied with only
maltose and calcium chloride via a propionate-independent pathway. It is noteworthy that the efficiency
of converting valerate to 3HV was much greater than the conversion of propionate to 3HV in *Halomonas* sp. HG01 (Table 3).

The synthesis of 3HV units from valeric acid can be made by channelling the intermediate of the β-oxidation cycle, trans-2-enoyl-CoA, by the action of a *R*-specific enoyl-CoA hydratase (EC 4.2.1.17), encoded by the *phaJ* gene identified in the genome of *Halomonas* sp. HG01 (Table SM04). To date, different types of PhaJ enzymes have been found, namely, a short chain-length (C4-C6) enoyl-CoA-specific type and a medium chain-length (C8-C12) enoyl-CoA-specific type [114]. Among PHA-producing bacteria, *Aeromonas caviae* was reported as the first bacterium identified as having a PHA biosynthesis gene cluster containing a *phaJ* gene [115], followed by *Haloferax mediterranei* [2]. However, *Halomonas* sp. HG01 has only one *phaJ* gene which was identified along its genome sequence and outside of the *pha* operon.

The synthesis of 3HV units from propionic acid is dependent on a 3-ketothiolase since acetyl-CoA (C2) should be condensed to propionyl-CoA (C3) leading to the production of 3HV monomers [59]. This reaction is preferentially catalyzed by the 3-ketothiolase enzyme encoded by *bktB* genes [116] which was identified in the *Halomonas* sp. HG01 genome (Figure 5 and Table SM4). Therefore, the low efficiency in converting propionic acid into 3HV units should be justified by the presence of an efficient propionate catabolism. Several propionate catabolic pathways have been reported [117] but genetic evidence have been obtained just for two or three of these pathways. The efficient oxidation of propionate by the 2-methylcitrate cycle (MCC) was reported in previous works [79,117]. Genes for MCC organized in an operon were clearly identified in *Halomonas* HG01 (Table SM04). In addition, genes involved in the methylmalonyl-CoA pathway – another propionate catabolic pathway - were also identified in the *Halomonas* sp. HG01 genome (Table SM04). These genes were sometimes related to
the synthesis of propionyl-CoA instead of its catabolism [118]. Therefore, the synthesis of 3HV units
from unrelated carbon sources by *Halomonas* sp. HG01 should be investigated in the future.

**4. Conclusions**

*Halomonas* sp. HG01 can be considered as a potential cellular factory for P(3HB), P(3HB-*co*-3HV) and P(3HB-*co*-4HB) production from carbohydrates and organic acids under high salt concentration (100 g/L), allowing the conduction of less expensive PHA production processes. Genome analysis revealed important features and allowed us to make a preliminary reconstruction of its GEM, which will be essential to conduct *in silico* experiments evaluating the potential of this bacterium in different bioprocesses as well as the genetic modifications for its improvement. In conclusion, this study provided both genetic and metabolic engineering evidence of the great potential of *Halomonas* sp. HG01 as a relevant chassis for the biotechnological production of PHA and, in the future, other compounds.

**5. Conflict of interest**

The authors declare no financial or commercial conflict of interest.

**6. Acknowledgements**

We acknowledge the scholarship to Guzmán-Moreno, CW provided by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) and the research fellowship to Cardinali-Rezende, J from São Paulo Research Foundation (FAPESP) Grants nº 2014/08061-1 and 2016/24290-6. The research was financially supported partially by FAPESP (Grant nº 2010/51692-1) and partially by CAPES (Finance code 001). The National Council for Scientific and Technological Development CNPq 309086/2018-3 L.F. Silva and 311932/2022-3 J.G.C.Gomez productivity fellowships are also acknowledged
## 7. Author contributions

**Guzmán-Moreno, C.W:** Conceptualization; Investigation, Methodology, Visualization, Data curation, Formal analysis, Writing- Original draft preparation, Writing- Reviewing and Editing; **Cardinali-Rezende, J:** Conceptualization; Investigation, Methodology, Visualization, Data curation, Formal analysis; Resources; Software; Validation; Visualization; Writing- Original draft preparation, Writing- Reviewing and Editing; **Di Genova, A:** Methodology, Software, Data curation, Validation; Writing - Reviewing; **Ferrarini, M:** Software, Data curation, Validation; Writing - Reviewing; **Sagot, M.F.:** Methodology, Software, Data curation, Writing - Reviewing; **Rodrigues, K.L.:** Writing - Reviewing; **Taciro, M.K.:** Data curation, Formal analysis, Resources, Funding acquisition, Writing- Reviewing; **Silva, L.F.:** Data curation, Formal analysis, Resources, Writing- Reviewing; **Gomez JG.:** Supervision, Project administration, Resources, Visualization, Conceptualization, Data curation, Formal analysis; Funding acquisition; Writing- Original draft preparation, Writing- Reviewing and Editing.

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could
have appeared to influence the work reported in this paper.