Halomonas alkaliantarctica as a platform for poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) production from biodiesel‐derived glycerol

Abstract Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by a wide range of microorganisms, including extremophiles. These unique microorganisms have gained interest in PHA production due to their ability to utilise low‐cost carbon sources under extreme conditions. In this study, Halomonas alkaliantarctica was examined with regards to its potential to produce PHAs using crude glycerol from biodiesel industry as the only carbon source. We found that cell dry mass concentration was not dependent on the applying substrate concentration. Furthermore, our data confirmed that the analysed halophile was capable of metabolising crude glycerol into poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) copolymer within 24 h of the cultivation without addition of any precursors. Moreover, crude glycerol concentration affects the repeat units content in the purified PHAs copolymers and their thermal properties. Nevertheless, a differential scanning calorimetric and thermogravimetric analysis showed that the analysed biopolyesters have properties suitable for various applications. Overall, this study described a promising approach for the valorisation of crude glycerol as a future strategy of industrial waste management to produce high value microbial biopolymers.


INTRODUCTION
Industrial biotechnology continues to drive innovation, improve bioprocess efficiency and contribute to a more sustainable and circular bioeconomy.In the last few decades, microbial processes are attracting interests of the scientific community as the sources of value-added bio-based products.Among them, polyhydroxyalkanoates (PHAs) are especially attractive as ecological alternative to conventional polymers derived from fossil fuels.They are a group of biopolymers that are produced by certain microorganisms in the form of intracellular granules.For many years, PHAs were thought to be a way to store carbon and energy (Policastro et al., 2021).However, recent studies demonstrated that the biological role of PHAs is more complex.The data suggested that the accumulation of PHAs provide stress robustness of bacteria, helping them maintain their structural integrity and function in challenging conditions (Obruca et al., 2020).PHAs possess unique properties such as biodegradability, non-toxicity, biocompatibility; therefore, they are considered as innovative biopolymers that can be used in many novel medical or agricultural applications (Ashori et al., 2019).
The specific details of PHA production can vary depending on the chosen microorganism, carbon source and desired PHA properties.The industrial production of PHA is still scarce, due to the high production costs (Koller & Mukherjee, 2022).Therefore, researchers continue to screen microorganisms that efficiently synthesise PHAs and explore ways to reduce their production costs.Extremophiles are of great interest to scientists because of their numerous applications in the fields of biotechnology (Kaur et al., 2019).The study of extremophiles for PHA production is still relatively in its early stages, but it holds promise for sustainable biopolymers production.Saline environments are widely distributed around the world.They are a source of halophilic bacteria that have a great potential to produce biomolecules such as PHAs.Moreover, in order to develop sustainable and economically viable microbial processes of PHA production, the requirement of the usage of cheap and renewable carbon source is essential.One of industrial feedstocks that can be applied as a substrate for microorganisms, is crude glycerol generated during biodiesel production.Due to the impurities, this residue has limited direct applications and the purification process is too expensive.For this reason, it has become very important to find an alternative method of its utilisation.Moreover, crude glycerol is one of the few waste sources that can be used directly as the carbon source for PHA production (Ray et al., 2016).Therefore, it is considered a promising alternative substrate in reducing production cost (Vicente et al., 2023).
Moreover, based on the concept of next-generation industrial biotechnology, extremophilic production strains have potential to transform the current PHA production into more competitive bioprocess (Yu et al., 2019).In this context, halophiles are currently considered as candidates for PHA production due to several advantages, including the utilisation of low-cost carbon sources, reduced risk of contamination and potential for producing tailor-made PHA compositions with unique properties (Yin et al., 2015).To the best of our knowledge, only in two publications the PHA biosynthesis process using Halomonas spp.grown on biodiesel byproducts were investigated.Shrivastav et al. (2010) analysed Halomonas hydrothermalis towards poly(3-hydroxybutyrate) [P(3HB)] production using Jatropha biodiesel byproduct.Also, algal biodiesel waste residue was investigated for P(3HB) synthesis by Halomonas daqingensis (Dubey & Mishra, 2021).The above-mentioned reports revealed that Halomonas spp.are capable of producing P(3HB) homopolymer.However, none of the study evaluated in details properties of the extracted biopolyesters.Moreover, there is still a lack of studies that showed the potential of Halomonas spp. to synthesise PHA copolymers from waste feedstock.In general, P(3HB) homopolymer is stiff and brittle due to its high crystallinity that limits its commercialization.Whereas, poly(3hydroxybutyrate-co-3-hydrovalerate) copolymer [P (3HB-co-3HV)] was reported to be more desirable than P(3HB) because their melting point is much lower, and they are less crystalline (Możejko-Ciesielska & Kiewisz, 2016).
Therefore, the aim of the present study was to evaluate the capability of Halomonas alkaliantarctica to produce P(3HB-co-3HV) copolymer using biodieselderived glycerol without additional precursors.Furthermore, we investigated the effect of the waste substrate concentration on biomass rate and PHA productivity.In addition, the extracted PHA copolymers were comprehensively studied based on their physico-thermal properties.

PHA extraction
The cell dry mass (CDM) and PHA concentration was determined at three time-points (24, 48 and 72 h).H. alkaliantarctica cells were centrifuged (11,200 Â g for 10 min) and after removing the supernatant, the bacterial pellet was lyophilized for 24 h by Lyovac GT2 System (SRK Systemtechnik GmbH, Riedstadt, Germany).PHA extraction was conducted by shaking the freeze-dried cells in chloroform (purity ≥ 99.8%; Sigma-Aldrich; USA) for 5 h at 50 C.The obtained mixture was further filtered through No. 1 Whatman filter paper and washed with cold 70% methanol.The collected pellet was allowed to evaporate at the room temperature.PHA content (% of CDM) was determined in the lyophilized biomass and was defined as the percentage of the ratio of PHAs concentration to CDM concentration.

Analysis of monomeric composition
The PHA composition was evaluated using Gas Chromatography coupled with Mass Spectrometry (GC-MS QP2010 PLUS, Shimadzu, Japan) as previously reported by Możejko-Ciesielska and Pokoj (2018).The lyophilized bacterial cells were treated with acidified methanol containing 3% v/v H 2 SO 4 and chloroform.Obtained methyl esters were injected into a BPX70 (25 m Â 0.22 mm Â 0.25 mm) capillary column (SGE Analytical Science, Victoria, Australia).Helium was used as a carrier gas at 1.38 mL/min.The initial temperature was programmed as 80 C, followed by an increase to 240 C at 10 C/min.The temperature of the ion source was maintained at 240 C. The quantitative analysis was used P(3HB-co-3HV) copolymer (Sigma Aldrich, USA) as a standard.
The attenuated total reflectance mode (ATR-FTIR) of a Fourier Transform Spectrophotometer (FTIR) Nicolet iS10 (ThermoScientific, USA) was used to record infrared spectra of the PHAs samples.The spectral were recorded in the range from 4000 to 650 cm À1 .Each spectrum analysed was the average of 16 recorded measurements.

Analysis of thermal properties
The phase transitions of extracted polymers were determined using a Q200 differential scanning calorimeter (DSC) (TA Instruments, USA).Samples of about 1 mg of the thin PHAs films were first heated to 210 C and kept isothermal for 1 min.to combine several layers of film.Then a cooling run to À30 C was performed at a temperature change rate of 10 C/min.In the last stage, the biopolymers samples were heated to 210 C at a heating rate of 10 C/min.The glass transition temperature (T g ), melting point (T m ), the enthalpy of melting process (ΔH cc ) and degree of crystallinity (X c ) were evaluated from the heating DSC curve.The X c values were calculated from the formula: where ΔH m100% -change in melting enthalpy of a 100% crystalline sample-109 J/g (Jost et al., 2017).The thermal resistance of the extracted copolymers was tested using the Q500 thermobalance (TA Instru ments, USA).Samples of about 1 mg were evaluated in a nitrogen atmosphere in the temperature range from 25 to 600 C with a temperature change rate of 10 C/min.

RESULTS AND DISCUSSION
Effect of crude glycerol concentration on growth and PHA production Extremophiles have been recently discovered to be capable of utilising renewable feedstocks into high value products (Joulak et al., 2022).The study of halophiles has attracted scientific interest due to their ability to survive in extreme environments and their potential applications in the production of biomolecules.There are no reports that evaluated the potential of H. alkaliantarctica to grow and produce PHAs during fermentation process with biodiesel-derived glycerol.Moreover, any Halomonas spp.has not been investigated for production of PHA copolymers using crude glycerol derived from vegetable oil so far.Therefore, the aim of this study was to evaluate whether H. alkaliantarctica is capable of converting crude glycerol into PHA copolymers and to characterise them taking into consideration their physico-chemical properties.
Our results confirmed that the crude glycerol did not hamper the growth of the analysed bacterial species (Figure 1).We found that CDM values were not dependent on the applying substrate concentration.Shake flasks yielded comparable biomass value at all applied crude glycerol concentrations.Nevertheless, the CDM values were higher at 48 and 72 h in comparison to 24 h of all experimental variants.The biomass concentration reached the maximum value in the cultivation with 80 g/L of crude glycerol in 72 h (1.7 g/L).Lower CDM rates (below 0.5 g/L) were determined by Dubey and Mishra (2021)  fermentor supplemented with algal biodiesel waste residue.Lower bacterial cell density was also reported by Shrivastav et al. (2010) in the cultivation of H. hydrothermalis with Jatropha biodiesel byproduct (about 0.4 g/L).As shown on Figure 2A,B, H. alkaliantarctica was able to produce PHAs in all conducted experiments within 24 h of the cultivation.PHA content was the highest in bacterial cells cultivated in BMB medium supplemented with 50 g/L of biodiesel-derived glycerol (about 17% of CDM).At higher level of this feedstock, the PHA content in CDM decreased (Figure 2A).However, in all experimental variants the PHA content was higher compared to the values reported in shake-flasks culture of H. halophila, H. salina or H. meridiana grown on waste frying oil which synthesised only 0.38%, 0.66% and 2.96% of CDM, respectively (Pernicova et al., 2019).Moreover, our results showed that the highest PHA concentration (0.27 g/L) and PHA productivity (5.63 mg/(LÁh)) was reached in the cultivation with 50 g/L of crude glycerol in 48 h.We also observed that the further increase of the waste substrate concentration (above 50 g/L) resulted in a decrease of PHAs productivity at all measured time-points (Figure 2B).However, Liu et al. (2022) suggested that NaCl concentration can play a role in PHA production.The authors showed that pathways involved in salt tolerance can be blocked or weakened in H. cupida J9 cells when using glycerol as a feedstock.In a consequence, these bacterial cells can respond to a high salt concentration by increased production of PHA as an effective protectant against salt stress.Also, Kucera et al. (2018) reported that the concentration of NaCl used during cultivation of H. halophila influenced PHA productivity.The highest P (3HB) yield was observed in the cultivation supplemented with 60 g/L of NaCl, whereas at lower and higher salt concentration (20, 40, 80 and 100 g/L), the homopolymer productivity decreased.The same observations were made by Rodríguez-Contreras et al. (2016) in the culture of halophilic bacterium Bacillus megaterium uyuni S29.The authors proved that NaCl concentration is an essential factor influencing PHA productivity when employing halophilic bacteria.

Effect of crude glycerol concentration on PHA composition
The results from gas chromatography coupled with mass spectrometry analysis showed that H. alkaliantarctica is capable of producing copolymers contained higher content of 3HB monomer and lower content of 3HV fraction (Table 1).We observed that the repeat units content in the purified PHA copolymers depended on the concentration of biodiesel-derived glycerol.Moreover, the content of the monomers varied in different cultivation time.The concentration of 3HV fraction  was higher in 48 h than in 24 h in all experimental variants.Halomonas spp.are known to be able to produce P(3HB) (Dubey & Mishra, 2022;Kawata & Aiba, 2010).Nevertheless, PHA copolymers are of high interest due to their favourable thermomechanical properties compared to PHA homopolymers.Incorporation of 3HV monomer to the polymer chain lowers melting temperature, reduces crystallinity, improves flexibility that are important parameters for their future application (Hammami et al., 2022).Most bacterial species required precursors to produce P(3HB-co-3HV) such as Cupriavidus necator (Grousseau et al., 2014), Bacillus aryabhattai (Balakrishna Pillai et al., 2020) or Halomonas sp.YLGW01 (Kim et al., 2023).To our knowledge, the analysed H. alkaliantarctica is the only halophilic bacterial strain reported to be able to synthesise P (3HB-co-3HV) copolymers using biodiesel-derived glycerol without any co-substrates.
The chemical structure of the extracted biopolymers was also confirmed by Fourier transform infrared spectroscopic spectra (FTIR spectra) (Figure 3).The observed bands were characteristic for P(3HB-co-3HV) copolymer.Similar signals were reported in earlier studies suggesting the chemical structure of the above-mentioned PHA copolymer (Volova et al., 2013).The band in the range of 3100-2700 cm À1 was associated with the presence of -CH 3 and -CH 2 groups in macromolecules.A single peak band with a maximum of $1722 cm À1 was derived from the C=O carbonyl groups found in macromolecules.Also, Abd El-malek et al. (2020) showed the strongest band at 1720 cm À1 for PHA extracted from Halomonas pacifica ASL10 and Halomonas salifodiane ASL11.Additionally, our results confirmed that in the spectrum range of 1500-800 cm À1 , which is the fingerprint region characteristic for PHAs, several bands with numerous peaks are characteristic for the type of the produced copolymers.Peaks with maxima of $1460, $1379, $980, $900 and $825 cm À1 were associated with the presence of -CH 3 and -CH 2 groups in macromolecules, as well as C-C bonds.It has been previously proved that a strong vibration at 1379 cm À1 is characteristic for PHA (Sathiyanarayanan et al., 2017).Moreover, in the obtained FTIR spectra a series of peaks appeared at $1278, $1228, $1180, $1132, $1101 and $1053 cm À1 were associated with bonds in the C-O-C region.

Effect of crude glycerol concentration on the properties of extracted PHAs
Besides chemical structure, the potential applicability of PHAs also depends on its thermal characteristic.Therefore, we investigated the effect of crude glycerol concentration on thermal properties of the extracted copolymers (Table 2).Properties studies on the biopolyesters extracted from Halomonas spp.cells cultured with biodiesel-derived glycerol as a sole carbon source have not been reported so far.To characterise comprehensively the PHAs biofilms, the copolymers extracted in 48 h of the cultivations were chosen for further analyses.
For all tested materials, three phase transitions are observed on the DSC curves: glass transition, cold crystallisation and melting (Figure 4).As can be seen in Table 1, the content of 3HV units in individual polymers is very similar.The changes in thermal properties must therefore result from another parameter or material characteristic.It was found that the recorded thermal parameters were strongly influenced by the crude glycerol concentration.We observed that the higher crude glycerol concentration the higher T g value.The glass transition temperature (T g ) of the tested materials ranged from À1.5 to 0.7 C. The increase in T g was probably due to the change in the degree of crystallinity of the copolymers which also increased with the increase in the concentration of crude glycerol.At a concentration of 10 g/L, the calculated degree of T A B L E 2 Thermal properties of P(3HB-co-3HV) copolymers extracted from the cultivation of Halomonas alkaliantarctica grown on crude glycerol in 48 h.
crystallinity was 1.3%, while at a concentration of 80 g/ L the degree of crystallinity increased to 13.6%.The melting temperature (T m ) of the analysed biopolymers was also affected by degree of crystallinity and reached about 165 C.This point value is similar to the P(3HBco-3HV) isolated from Halomonas campisalis cells in the cultivation with bagasse as sole substrate (Kulkarni et al., 2015).Higher T m value (178 C) was reported for P(3HB) produced by Halomonas sp.SF2003 cultured on agro-industrial effluents (Lemechko et al., 2019).It could be suggested that the concentration of crude glycerol influences the structure of polymer chains in a way that favours the crystallisation process.The increase in the degree of crystallinity influences the shift in the glass transition and melting temperatures.Furthermore, we observed that the degree of crystallinity increasing with the concentration of crude glycerol influenced on the thermal resistance of the extracted biopolymers (Figure 5).P(3HB-co-3HV) isolated from the H. alkaliantarctica cells grown on higher concentration of crude glycerol were characterised by higher decomposition temperatures (T d ), that is, the parameter adopted as the degree of thermal resistance and representing the temperature of loss of 5% of the sample weight.The highest thermal resistance was detected for the PHA copolymer produced at the highest carbon source concentration (T d = 213.1 C).Similar observation was made for maximum degradation temperature (T max ), that is, the parameter determining the temperature at which the thermal degradation process proceeds in the most intensive range.Kavitha et al. (2016) showed that P(3HB) homopolymer produced by Botryococcus braunii degraded at 240 C. Our results indicated that the carbon source concentration influenced also on the rate of the degradation process of the extracted copolymers.As the concentration of crude glycerol increased, the T max value of analysed PHA copolymers also increased.The T max value for PHA biofilm extracted from the cultivation supplemented with 80 g/L reached 288.3 C. Stanley et al. (2020) reported that the maximum degradation for P(3HB) synthesised by H. venusta occurred at 323.3 C.
The exploration of halophilic bacteria for PHA production has many benefits such as short fermentation time, lack of pathogenicity and ability to produce this biomolecule using renewable resources (Mitra et al., 2016).Our results proved that H. alkaliantarctica is able to utilise crude glycerol for the growth and P (3HB-co-3HV) production.Also, we found that PHA productivity, monomers content and thermal properties were dependent on the biodiesel-derived glycerol concentration.In addition, this bacterium produced the sclcopolymer without any 3HV precursors and make this biotechnological process a valuable strategy and industrially very important.Furthermore, the obtained data suggest that P(3HB-co-3HV) copolymers have properties that are important for industrial applications.Further work should include metabolic engineering along F I G U R E 5 TG and DTG curves of selected copolymers extracted from Halomonas alkaliantartica cells: (A) P(97.23%HB-co-2.77%3HV) from the cultivation with 10 g/L of crude glycerol; (B) P(98.18%HB-co-1.82%3HV) from the cultivation with 50 g/L of crude glycerol; (C) P(98.35%HB-co-1.65% 3HV) from the cultivation with 80 g/L of crude glycerol.
with genome editing to improve the ability of H. alkaliantarctica to metabolise broader range of waste feedstocks.A pressing need also arises to optimise the culture parameters in bioreactor for PHA production at industrial scale.

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who cultured H. daqingensis in the I G U R E 1 Growth of Halomonas alkaliantartica in the medium supplemented with crude glycerol.Mean values are calculated from triplicate measurements.

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I G U R E 2 Polyhydroxyalkanoate (PHA) production by Halomonas alkaliantartica in the medium supplemented with crude glycerol.(A) PHA content in cell dry mass (CDM), (B) PHA productivity in 24, 48 and 72 h of the cultivations.Mean values are calculated from triplicate measurements.T A B L E 1 Monomeric composition of extracted PHAs.