PHA production, degradation and material properties

PHAs are a type of polyester, meaning that the ester functional group links chains of hydroxyalkanoate groups together (Raza et al., Reference Raza, Abid and Banat2018). PHAs are classified by the length of the carbon chain: short-chain length PHAs (scl-PHAs) have less than five carbon atoms, medium-chain length PHAs (mcl-PHAs) have between 5 and 14 carbon atoms and the less-studied long-chain length PHAs have more than 14 carbon atoms (Raza et al., Reference Raza, Abid and Banat2018). PHAs can be copolymerized, and there are over 150 different monomers that are known to be incorporated into PHA chains, which makes PHAs the largest group of natural polyesters (Kim et al., Reference Kim, Kim, Chung and Rhee2007). The first discovered, and the most common, PHA is the scl-PHA polyhydroxybutyrate (PHB) (Fig. 1) (Raza et al., Reference Raza, Abid and Banat2018).

Figure 1.

(a) General chemical structure of PHAs with (b) nomenclature and carbon number determined by the functional alkyl R group (adapted from Tan et al., Reference Tan, Chen, Li, Ge, Wang, Razaad, Li, Zhao, Mo and Wang2014a). (c) Pseudomonas putida CA-3 cells with PHA granules from a transmission electron micrograph (Ward Patrick et al., Reference Ward Patrick, De Roo and O'Connor Kevin2005).

Ongoing research efforts into PHAs have focused on both nanobiotechnology applications and using them to manufacture more environmentally safe alternatives to petroleum-derived plastics (Raza et al., Reference Raza, Abid and Banat2018). PHAs are hypothesized to be created and utilized as energy and carbon stores in times of stress and starvation in microorganisms (Urtuvia et al., Reference Urtuvia, Villegas, González and Seeger2014). PHAs are synthesized in both nutrient-rich and nutrient-poor environments by at least 92 genera of Gram-positive and Gram-negative bacteria (Alamgeer, Reference Alamgeer2019; Vicente et al., Reference Vicente, Proença and Morais2023) including both heterotrophic and autotrophic bacteria as well as cyanobacteria living under aerobic and anaerobic conditions (Palleroni and Palleroni, Reference Palleroni and Palleroni1978; Xiao and Jiao, Reference Xiao and Jiao2011), and in extreme environments of astrobiology relevance (Wang et al., Reference Wang, Dragone, Avard and Hynek2022a). PHAs are also synthesized by archaea and eukaryotes, including plants and recombinant yeast (Terentiev et al., Reference Terentiev, Breuer, Babel and Kunze2004; Xiao and Jiao, Reference Xiao and Jiao2011).

PHAs are synthesized and degraded in the PHA cycle (Fig. 2). The PHA cycle branches off from many well understood and common carbon metabolisms including glycolysis, the citric acid cycle, fatty acid biosynthesis, the β-oxidation pathway, the glyoxylate cycle, the ethylmalonyl–coenzyme A pathway, the Entner–Doudoroff pathway and methane oxidation (Urtuvia et al., Reference Urtuvia, Villegas, González and Seeger2014; Liu et al., Reference Liu, Xie, Xing, Liu, Ding and Ren2020; de Vogel et al., Reference de Vogel, Schlundt, Stote, Ratto and Amaral-Zettler2021; Wang et al., Reference Wang, Dragone, Avard and Hynek2022a).

Figure 2.

(a) Overview of the PHA cycle with chemical structures, connected metabolic pathways, and (b) enzyme-catalysing reactions. CoA, coenzyme A; ACP, acyl carrier protein. Adapted from de Vogel et al. (Reference de Vogel, Schlundt, Stote, Ratto and Amaral-Zettler2021).

PHAs form insoluble, complex and well-organized granule structures inside cells measuring from 0.2 to 0.5 μm in diameter intracellularly (Jendrossek et al., Reference Jendrossek, Selchow and Hoppert2007) and from ~100 nm to several micrometres extracellularly (Bäckström et al., Reference Bäckström, Brockelbank and Rehm2007). PHA granules (Fig. 3) are commonly surrounded by proteins bound to the core, with PHA granule-associated proteins (PhaP or ‘phasins’) being the most common. Phasins are a group of amphipathic proteins that bind specifically and hydrophobically with the PHA core, and they can coat up to 54% of the PHA granule (Tian et al., Reference Tian, He, Lawrence Adam, Liu, Watson, Sinskey Anthony and Stubbe2005; Bäckström et al., Reference Bäckström, Brockelbank and Rehm2007). A phospholipid membrane surrounding the PHA core is heavily debated (Maestro and Sanz, Reference Maestro and Sanz2017); while original studies detected phospholipids attached to the PHA granule, Wahl et al. (Reference Wahl, Schuth, Pfeiffer, Nussberger and Jendrossek2012) and Bresan et al. (Reference Bresan, Sznajder, Hauf, Forchhammer, Pfeiffer and Jendrossek2016) argue that prior findings of phospholipids were due to contamination in PHA extraction and preparation techniques.

Figure 3.

Schematic of a PHA granule structure from Maestro and Sanz (Reference Maestro and Sanz2017).

Bacteria have been able to produce up to 90% (w/w) PHAs of their cell dry weight (Tan et al., Reference Tan, Wu, Chen and Chen2014b). PHAs are water insoluble (resistant to hydrolytic degradation), and they sink in aqueous environments (Noreen et al., Reference Noreen, Sultana, Sultana, Tabasum, Zia, Muzammil, Jabeen, Lodhi, Sultana, Mahmood Zia, Jabeen, Anjum and Ikram2020), which could help with biopreservation through burial. As a material, PHAs are easily attacked by acids and alkalis and dissolve in chlorinated solvents; nevertheless, PHAs have strong ultraviolet (UV) resistance (Bugnicourt et al., Reference Bugnicourt, Cinelli, Lazzeri and Alvarez2014). PHAs have exhibited melting points between 50 and 180°C and crystallinity between 30 and 70% (Bäckström et al., Reference Bäckström, Brockelbank and Rehm2007; Rehm, Reference Rehm2010). Zhang et al. (Reference Zhang, Liu, Yang, Chen, Lin, Han, Cao, Zeng and Ye2022) reported thermal and mechanical properties for multiple scl-PHAs and mcl-PHAs.

Photolysis of biodegradable plastics, including PHAs, has been shown in experiments to result in photodegradation and certain structural changes (Kaing et al., Reference Kaing, Guo, Sok, Kodikara, Breider and Yoshimura2024). For example, UV photodegradation has been shown to cause some chemical and structural changes to PHBs in experiments, leading to decreased biodegradability from increased crystallinity (Sadi et al., Reference Sadi, Fechine and Demarquette2010). Experiments investigating abiotic hydrolysis of PHAs discovered that longer side-chain lengths and higher crystallinity (>40%) interfere with abiotic degradation under alkaline conditions, providing a higher stability against hydrolysis (Tarazona et al., Reference Tarazona, Machatschek and Lendlein2020).

Since most research into PHAs has been focused on enhancing biodegradability in industrial settings, there is limited knowledge on PHA's preservation potential extracellularly and its long-term stability without microbial degraders in various environments, particularly for geologic timescales when searching for relict life. PHA's weak resistance to solvents would make biopreservation a challenge in aqueous environments with non-neutral pHs, however it is possible that the cell membrane and protein layer surrounding PHA granules could help improve preservation, especially if the cell membrane is composed of certain phospholipids from extremophiles that are temperature and pH resistant (Sollich et al., Reference Sollich, Yoshinaga, Häusler, Price, Hinrichs and Bühring2017). Encapsulation from extremophile phospholipids also would inherently assist biopreservation of any other biomolecule. It should be noted that extremophiles producing PHAs have been found in many extremophile environments (Gambarini et al., Reference Gambarini, Pantos, Kingsbury, Weaver, Handley and Lear2022), though the literature is scarce on PHA biosignature detection in these environments and the material's stability extracellularly.

The strong UV resistance of PHAs makes them an attractive candidate when looking for biosignatures in planetary environments with high radiation, like Mars and Europa, where radiation can degrade molecules (Merino et al., Reference Merino, Aronson, Bojanova, Feyhl-Buska, Wong, Zhang and Giovannelli2019). While PHAs may not be as well preserved as some other biosignatures (e.g. lipids, pigments, biomembranes), the high crystallinity, melting temperature, water insolubility and binding with proteins (and potentially lipids) may make PHAs a stronger biosignature than other labile compounds (e.g. amino acids, sugars, DNA/RNA). While long-term stability is challenged until further experimentation, the material properties of PHAs combined with the diversity of the polymer and the many ways in which they can bind and be preserved by other biomolecules make them a candidate biosignature for astrobiological purposes, especially in the search for extant life.

PHA-producing microbes in extreme environments

PHAs are not only found in all three branches of life (bacteria, archaea and eukarya), but they are found in many extremophiles living in astrobiological-relevant environments. The adaptability of some microorganisms to their extreme environments has even been attributed to PHAs and their material properties, as summarized in Table 1.

Table 1.

Summary of various PHA-producing microorganisms from varying environments

Acidic environments are astrobiologically relevant, as life is hypothesized to have been harboured in hydrothermal environments on Mars (Wang et al., Reference Wang, Dragone, Avard and Hynek2022a) and perhaps currently on the moons of Europa and Enceladus where hydrothermal activity could provide heat and nutrients for life (Deamer and Damer, Reference Deamer and Damer2017). Sulphur discovered via remote sensing on Europa could be associated with sulphur-metabolizing microorganisms (Carlson et al., Reference Carlson, Johnson and Anderson1999; Trivedi Christopher et al., Reference Trivedi Christopher, Stamps Blake, Lau Graham, Grasby Stephen, Templeton Alexis and Spear John2020) and life on Venus is even hypothesized to live in the sulphuric acid clouds where Earth temperatures and pressures are found (Izenberg et al., Reference Izenberg, Gentry, Smith, Gilmore, Grinspoon, Bullock, Boston and Słowik2021). Thus, biosignatures generated by acidophiles may be of importance in future astrobiology space missions.

PHA metabolism is a key feature of microbes in the Acidiphilium genus, an aerobic acidophile capable of a wide array of metabolisms and adaptations including sulphur oxidation and metal detoxification (Li et al., Reference Li, Liu, Zhang, Meng, Liu, Wang, Li, Jiang, Zhong, Jiang and Yin2020). Wang et al. (Reference Wang, Dragone, Avard and Hynek2022a) noted that genes allowing for PHB metabolism, a type of PHA, were present in their Acidiphilium-dominated metagenome of microorganisms from the crater lake of the Poás Volcano. The Poás Volcano is an extreme Martian acid-sulphate hydrothermal analogue exhibiting frequent phreatomagmatic eruptions, with pH measurements as low as −0.87, and temperatures as high as boiling (Hynek et al., Reference Hynek, Rogers, Antunovich, Avard and Alvarado2018). While low pH fluids easily react with PHAs making them unstable in these kinds of environments, Wang et al. (Reference Wang, Dragone, Avard and Hynek2022a) suggested PHB metabolism may actually be one of the key factors allowing for the persistence of Acidiphilium spp. in this extremely acidic and dynamic environment. The authors hypothesized that the presence of PHB metabolism genes in the Acidiphilium spp. bacteria survived the frequent phreatomagmatic eruptions between their multiple field campaigns using PHB metabolism to manage periods of carbon starvation (in either the crater lake, connected groundwater or the surrounding soils) thus allowing future recolonization of a locale if it was sterilized. Note that the authors did not find nor search for PHB's in their field samples, so even if PHAs may be an integral feature of acidophilic microorganisms in extraterrestrial environments, further studies into PHA preservation in low pH would need to be conducted to assess their functionality as a biosignature in acidic environments.

Hydrothermal environments undergoing serpentinization reactions are also of astrobiological relevance as they are hypothesized to have been important for the origin of life on Earth (e.g. Russell et al., Reference Russell, Hall and Martin2010) and potentially extraterrestrial life in environments on Europa, Enceladus, Titan and Mars (Vance and Melwani Daswani, Reference Vance and Melwani Daswani2020), including Jezero crater (Simon et al., Reference Simon, Hickman-Lewis, Cohen, Mayhew, Shuster, Debaille, Hausrath, Weiss, Bosak, Zorzano, Amundsen, Beegle, Bell, Benison, Berger, Beyssac, Brown, Calef, Casademont, Clark, Clavé, Crumpler, Czaja, Fairén, Farley, Flannery, Fornaro, Forni, Gómez, Goreva, Gorin, Hand, Hamran, Henneke, Herd, Horgan, Johnson, Joseph, Kronyak, Madariaga, Maki, Mandon, Mccubbin, Mclennan, Moeller, Newman, Núñez, Pascuzzo, Pedersen, Poggiali, Pinet, Quantin-Nataf, Rice, Rice, Royer, Schmidt, Sephton, Sharma, Siljeström, Stack, Steele, Sun, Udry, Vanbommel, Wadhwa, Wiens, Williams and Williford2023), Perseverance's main target. Serpentinization is a water-driven reaction of mafic and ultramafic minerals (i.e. olivine and pyroxene) which generates serpentine, hydrogen and methane amongst other products. This can occur at temperatures between ambient and 400°C and at high pH values (Ehlmann et al., Reference Ehlmann, Mustard and Murchie2010); moreover, the alkalinity of Enceladus' Ocean, pH ~11–12, as inferred from Cassini spacecraft data, is attributed to serpentinization processes (Glein et al., Reference Glein, Baross and Waite2015). In a study performed by Bird et al. (Reference Bird, Kuenen, Osburn, Tomioka, Ishii, Barr, Nealson and Suzuki2021), PHA accumulations were produced by three cultures of facultative autotrophic, highly alkaliphilic bacterial strains (pH ~9.0–12.5) that were isolated from terrestrial serpentinizing springs. The cultures were dominated by members of the Comamonadaceae family, which have also been characterized for their poly(3-hydroxybutyrate-co-3-hydroxyvalerate)-degrading denitrifying activity (Khan Shams et al., Reference Khan Shams, Horiba, Yamamoto and Hiraishi2002; Bird et al., Reference Bird, Kuenen, Osburn, Tomioka, Ishii, Barr, Nealson and Suzuki2021). Thus, it is conceivable that microbial life in extraterrestrial environments dominated by serpentinization processes may also metabolize PHAs.

In addition to acidity and alkalinity, hydrothermal environments frequently exhibit high temperatures. Many thermophiles are capable of producing PHAs, and they have been reported to do so in temperatures of up to 80°C (Obulisamy and Mehariya, Reference Obulisamy and Mehariya2021). For example, Geobacillus stearothermophilus strain K4E3_SPR_NPP was shown to produce a PHA copolymer containing poly(3-hydroxybutyrate)-poly(3-hydroxyvalerate)-poly(3-hydroxyhexanoate) (Rodge et al., Reference Rodge, Shende and Patil2023), and Cupriavidus cauae PHS1 was able to produce a 47% cell dry weight of PHB at a temperature of 47°C (An et al., Reference An, Ha and Lee2023). Considering that the last universal common ancestor of life on Earth is hypothesized to be a thermophile or hyperthermophile (Nisbet and Sleep, Reference Nisbet and Sleep2001; Giulio, Reference Giulio2003; Gaucher et al., Reference Gaucher, Govindarajan and Ganesh2008), high-temperature compatibility is crucial when assessing PHA's astrobiological potential.

One of Mars Perseverance's top-level goals is to search for biosignatures in a palaeo-fluviolacustrine environment by searching for biomarkers at or near the surface (Farley et al., Reference Farley, Williford, Stack, Bhartia, Chen, De la Torre, Hand, Goreva, Herd, Hueso, Liu, Maki, Martinez, Moeller, Nelessen, Newman, Nunes, Ponce, Spanovich, Willis, Beegle, Bell, Brown, Hamran, Hurowitz, Maurice, Paige, Rodriguez-Manfredi, Schulte and Wiens2020). PHAs are found in a variety of soil environments utilizing either aerobic metabolism (e.g. Alcaligenes eutrophus, Alcaligenes latus, Pseudomonas oleovorans) or anaerobic metabolism (e.g. Clostridium botulinum, Clostridium acetireducens) (Santhanam and Sasidharan, Reference Santhanam and Sasidharan2010; Flüchter et al., Reference Flüchter, Follonier, Schiel-Bengelsdorf, Bengelsdorf, Zinn and Dürre2019). Since the majority of extraterrestrial astrobiology targets contain low concentrations of oxygen gas or are completely oxygen-free (Cockell et al., Reference Cockell, Schwendner, Perras, Rettberg, Beblo-Vranesevic, Bohmeier, Rabbow, Moissl-Eichinger, Wink, Marteinsson, Vannier, Gomez, Garcia-Descalzo, Ehrenfreund, Monaghan, Westall, Gaboyer, Amils, Malki, Pukall, Cabezas and Walter2018), the ability for PHA metabolism to be compatible with both aerobic and anaerobic respiration better supports its use as a biosignature for a variety of environments. Note that the aerobic soil bacteria A. latus was studied extensively by Palleroni and Palleroni (Reference Palleroni and Palleroni1978) and was found to have optimal growth at low oxygen levels (atmospheric O₂ at ~8%) and could utilize molecular hydrogen for autotrophic growth.

PHA metabolism has also been found in Methylosinus trichosporium and Methylocystis hirsute, methanotrophic bacterial strains that convert methane to PHB when nutrient supplies are ‘imbalanced’ (Liu et al., Reference Farley, Williford, Stack, Bhartia, Chen, De la Torre, Hand, Goreva, Herd, Hueso, Liu, Maki, Martinez, Moeller, Nelessen, Newman, Nunes, Ponce, Spanovich, Willis, Beegle, Bell, Brown, Hamran, Hurowitz, Maurice, Paige, Rodriguez-Manfredi, Schulte and Wiens2020). Methanotrophs and methanogens have been postulated on Mars as a source of seasonal variation in atmospheric methane (Webster et al., Reference Webster, Mahaffy, Atreya, Moores, Flesch, Malespin, McKay, Martinez, Smith, Martin-Torres, Gomez-Elvira, Zorzano, Wong, Trainer, Steele, Archer, Sutter, Coll, Freissinet, Meslin, Gough, House, Pavlov, Eigenbrode, Glavin, Pearson, Keymeulen, Christensen, Schwenzer, Navarro-Gonzalez, Pla-García, Rafkin, Vicente-Retortillo, Kahanpää, Viudez-Moreiras, Smith, Harri, Genzer, Hassler, Lemmon, Crisp, Sander, Zurek and Vasavada2018; Seto et al., Reference Seto, Noguchi and Cappellen2019). Methanogens generate methane, often live in symbiosis with methanotrophs (Wang et al., Reference Wang, Ge, Feng, Liu, Li, Wang, Xiao and Zhang2022b), and have been postulated to live on the surface of Saturn's moon Titan (McKay and Smith, Reference McKay and Smith2005). While Titan's ultra-low temperatures allowing for liquid nitrogen and frozen water on the surface would likely make life on the moon very different for any terrestrial organisms (McKay, Reference McKay2016), any discovery of PHAs on Titan would warrant an astrobiological interpretation.

While low temperatures resembling that of Titan are unfathomable on Earth, the survival mechanisms of PHA-producing psychrophiles are very well studied. PHAs in Pseudomonas sp. 14-3 were shown to protect the cells from oxidative stress (Ayub et al., Reference Ayub, Pettinari, Méndez and López2007), and an increased motility in biofilms developed by Pseudomonas extremaustralis was attributed to PHA accumulations (Tribelli and López, Reference Tribelli and López2011). In another study, Tribelli et al. (Reference Tribelli, Pezzoni, Brito, Montesinos, Costa and López2020) reported the bacterial diversity of cultivation-dependent freshwater samples from the Ecology Glacier foreland in King George Island, Antarctica. They noted that genes for PHA synthesis were present in all five sampling sites (temperatures ranging from 2.8 (±1.8) to 6.1 (±2.1)°C) in more than half of their isolated strains. In particular, PHA synthesis genes were found in six strains of Pseudomonas and four strains of Janthinobacterium. In their natural environments, these organisms were subject to very cold and variable temperatures, low nutrient availability and low water quality; however, it was hypothesized that PHAs helped the bacteria survive longer by providing a reserve source for when there were low nutrient and carbon supplies (Tribelli et al., Reference Tribelli, Pezzoni, Brito, Montesinos, Costa and López2020). Similarly, Ciesielski et al. (Reference Ciesielski, Górniak, Możejko, Świątecki, Grzesiak and Zdanowski2014) isolated the psychrophile P. extremaustralis, a producer of PHBs and mcl-PHAs, from a temporary water pond in Antarctica and subjected it to extreme cold and radiation. The authors found that higher PHB accumulation conferred with colder temperature tolerances and higher radiation resistance after a lethal ultraviolet-A (UVA) exposure (Ciesielski et al., Reference Ciesielski, Górniak, Możejko, Świątecki, Grzesiak and Zdanowski2014). These studies suggest that PHAs are not only present in these psychrophilic and radiation-resistant microorganisms, but that PHAs confer better survival in both cold and high radiation environments. PHAs would then be beneficial to any microorganisms living in cold and/or high radiation environments (e.g. on Mars, Europa and Enceladus).

Two archaeal halophilic strains, Natrinema altunense and Haloterrigena jeotgali, found in the extremely salty lake, Chott El Jerid (salt concentration up to 330 g l⁻¹), are producers of PHB and polyhydroxyoctanoate (Abdallah et al., Reference Abdallah, Karray, Mhiri, Mei, Quéméneur, Cayol, Erauso, Tholozan, Alazard and Sayadi2016, Reference Abdallah, Karray and Sayadi2020). Briny hydrothermal activity has been postulated on Mars (e.g. Schmidt et al., Reference Schmidt, Ruff, Mccoy, Farrand, Johnson, Gellert, Ming, Morris, Cabrol, Lewis and Schroeder2008) making halophilic compatibility an interesting consideration for PHAs astrobiological potential. A leading hypothesis for the recurring slope lineae (RSLs) on Mars is the presence of briny water, and thus RSLs have been given special consideration under many space agencies' planetary protection protocols as life could potentially live in this kind of environment (McEwen et al., Reference McEwen, Schaefer, Dundas, Sutton, Tamppari and Chojnacki2021). Moreover, halopsychrophiles, living in both cold and briny environments, have been proposed to possibly be capable of inhabiting hypothesized saline subsurface oceans of the dwarf planet Ceres, those of the moons Titan and Europa, and in Cl-rich subsurface brines on Mars (Merino et al., Reference Merino, Aronson, Bojanova, Feyhl-Buska, Wong, Zhang and Giovannelli2019).

While PHAs are not ubiquitous in all living organisms, they are found in all three branches of life, in both heterotrophs and autotrophs, in aerobes and anaerobes and in a wide array of environments (i.e. cold, hot, acidic, basic, briny, high radiation). Not only are PHAs (or PHA genes) found in these extreme environments, but the adaptability of these microorganisms to these extremes are also attributed to the presence and utilization of PHAs in the case of nutrient imbalance (Tribelli et al., Reference Tribelli, Pezzoni, Brito, Montesinos, Costa and López2020), cold temperatures and lethal UVA radiation (Ciesielski et al., Reference Ciesielski, Górniak, Możejko, Świątecki, Grzesiak and Zdanowski2014), oxidative stress (Ayub et al., Reference Ayub, Pettinari, Méndez and López2007) and survival in an acidic environment exhibiting frequent phreatomagmatic eruptions (Wang et al., Reference Wang, Dragone, Avard and Hynek2022a). Thus, it is conceivable that microorganisms on other worlds may exploit PHAs, perhaps utilizing it as a primary metabolic and survival strategy.

Astrobiology detection and instrumentation

While the use of PHAs would be beneficial to microorganisms living in many extreme environments and potentially extraterrestrial worlds, the ability to detect them using current and upcoming techniques searching for biosignatures comes with limitations. The two primary technical limitations to detecting PHAs on other worlds we have identified are that: (i) the samples taken for analysis may contain impurities so that they interfere with or mask the PHA signal, and (ii) there may not be high enough PHA mass in samples to reach minimum detection limits. However, these limitations are problems for all molecular biosignatures, not just for PHAs.

To the knowledge of the authors, literature on detecting PHAs in situ is extremely limited, and this is the first paper that discusses using PHAs as an astrobiological biosignature. To determine whether certain instruments and techniques could be utilized to detect PHAs for astrobiology purposes, we review and report numerous methods that are utilized to measure PHAs in modern laboratories that are analogous to current or upcoming astrobiological instrumentation. Samrot et al. (Reference Samrot, Samanvitha, Shobana, Renitta, Senthilkumar, Kumar, Abirami, Dhiva, Bavanilatha, Prakash, Saigeetha, Shree and Thirumurugan2021) also discuss many characterization techniques for analysing PHAs beyond those of astrobiological relevance. Note from the section ‘PHA production, degradation and material properties’ that PHAs are diverse and can range from short-chain to long-chain lengths. This inherently results in varying detection signals for different kinds of PHAs. In this section, we review the most cited and relevant techniques for studying varying PHAs (Table 2) and then later provide future recommendations for furthering PHA detection technology for astrobiological purposes in the section ‘Conclusions and future directions’.

Table 2.

Summary of techniques to identify PHA's in situ for astrobiology missions

Gas chromatography-mass spectrometry (GC-MS) is a popular method for identifying molecular compounds, and instruments have flown on various spacecraft. In GC-MS, a mass spectrometer uses an ionized beam of electrons to break the GC output to calculate a mass to charge ratio (m/z) with a relative abundance. Four GC-MS experiments have been sent to Mars: one on each Viking lander, the Sample Analysis at Mars (SAM) instrument on Mars Science Laboratory and the Mars Organic Molecule Organizer (MOMA) instrument on ExoMars (Klein, Reference Klein1979a; Mahaffy et al., Reference Mahaffy, Webster, Cabane, Conrad, Coll, Atreya, Arvey, Barciniak, Benna, Bleacher, Brinckerhoff, Eigenbrode, Carignan, Cascia, Chalmers, Dworkin, Errigo, Everson, Franz, Farley, Feng, Frazier, Freissinet, Glavin, Harpold, Hawk, Holmes, Johnson, Jones, Jordan, Kellogg, Lewis, Lyness, Malespin, Martin, Maurer, Mcadam, Mclennan, Nolan, Noriega, Pavlov, Prats, Raaen, Sheinman, Sheppard, Smith, Stern, Tan, Trainer, Ming, Morris, Jones, Gundersen, Steele, Wray, Botta, Leshin, Owen, Battel, Jakosky, Manning, Squyres, Navarro-González, McKay, Raulin, Sternberg, Buch, Sorensen, Kline-Schoder, Coscia, Szopa, Teinturier, Baffes, Feldman, Flesch, Forouhar, Garcia, Keymeulen, Woodward, Block, Arnett, Miller, Edmonson, Gorevan and Mumm2012; Arevalo et al., Reference Arevalo, Brinckerhoff, Amerom, Danell, Pinnick, Xiang, Getty, Hovmand, Grubisic, Mahaffy, Goesmann and Steininger2015). As PHAs are composed of multiple polyester units and do not have volatile tendencies, the PHA biopolyester must be broken down into volatile (or semi-volatile), low molecular weight and thermally stable compounds prior to being analysed by any GC system (Tan et al., Reference Tan, Ge, Pan, Ngin Tan, Wang and Koller2016). Of the ways to prepare PHAs for GC-MS analysis, methanolysis, pyrolysis using tetramethylammonium hydroxide (TMAH) and derivatization using N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) are techniques utilized by both SAM and MOMA. Both SAM and MOMA can perform MTBSTFA derivatization prior to GC-MS and TMAH pyrolysis prior to GC-MS (i.e. pyr-gc-ms), and SAM can also perform methanolysis (Mahaffy et al., Reference Mahaffy, Webster, Cabane, Conrad, Coll, Atreya, Arvey, Barciniak, Benna, Bleacher, Brinckerhoff, Eigenbrode, Carignan, Cascia, Chalmers, Dworkin, Errigo, Everson, Franz, Farley, Feng, Frazier, Freissinet, Glavin, Harpold, Hawk, Holmes, Johnson, Jones, Jordan, Kellogg, Lewis, Lyness, Malespin, Martin, Maurer, Mcadam, Mclennan, Nolan, Noriega, Pavlov, Prats, Raaen, Sheinman, Sheppard, Smith, Stern, Tan, Trainer, Ming, Morris, Jones, Gundersen, Steele, Wray, Botta, Leshin, Owen, Battel, Jakosky, Manning, Squyres, Navarro-González, McKay, Raulin, Sternberg, Buch, Sorensen, Kline-Schoder, Coscia, Szopa, Teinturier, Baffes, Feldman, Flesch, Forouhar, Garcia, Keymeulen, Woodward, Block, Arnett, Miller, Edmonson, Gorevan and Mumm2012; Arevalo et al., Reference Arevalo, Brinckerhoff, Amerom, Danell, Pinnick, Xiang, Getty, Hovmand, Grubisic, Mahaffy, Goesmann and Steininger2015). The mass to charge ratio for PHAs depends on the type of PHA, the method of preparation and the ionic source, but m/z values have been reported to range from 40 to 600 (Tan et al., Reference Tan, Chen, Ge, Li, Wang, Zhao, Mo, Tan and Wang2014c), which overlaps well with the detectable ranges of both SAM and MOMA. In addition to these Martian rovers, NASA's Dragonfly mission, which is a rotorcraft poised to arrive on Titan in 2034, will carry a Dragonfly Mass Spectrometer (DraMS). DraMS uses heritage hardware from SAM and MOMA and is capable of performing pyrolysis and derivatization GC-MS (Trainer et al., Reference Trainer, Brinckerhoff, Freissinet, Lawrence, Peplowski, Parson, Zacny, Turtle, Barnes, Lorenz, Hörst and Stickle2018; Barnes et al., Reference Barnes, Turtle, Trainer, Lorenz, Mackenzie, Brinckerhoff, Cable, Ernst, Freissinet, Hand, Hayes, Hörst, Johnson, Karkoschka, Lawrence, Le Gall, Lora, McKay, Miller, Murchie, Neish, Newman, Núñez, Panning, Parsons, Peplowski, Quick, Radebaugh, Rafkin, Shiraishi, Soderblom, Sotzen, Stickle, Stofan, Szopa, Tokano, Wagner, Wilson, Yingst, Zacny and Stähler2021). It must be noted that while GC-MS can identify and quantify PHAs in samples, the retention time and m/z values will depend on the individual instrument, sample preparation and internal standards. GC-MS was one of the first astrobiological relevant techniques used to search for life when it was utilized in the Viking landers; it is currently being utilized by spacecraft, and it will be exploited in future astrobiological missions. With access to and testing with SAM-, MOMA- and DraMS-analogues, it would be beneficial to determine the signals for various PHAs for each of these instruments.

Infrared (IR) spectroscopy is used to characterize absorptions in particular wavelengths that can be mapped to specific molecules based on characteristic vibrations released from the molecular bonds or angles shifting in space (e.g. Stuart, Reference Stuart2015). Poly(3-hydroxybutyrate) (P(3HB)) has a notably strong carbonyl absorption peak at 1728 cm⁻¹ in the IR spectrum and a sharp absorption band at 5.80 μm (Godbole, Reference Godbole2016). Compared to current instrumentation, Infrared Spectrometer for ExoMars (ISEM) has a spectral range between 1.15 and 3.30 μm with a spectral resolution varying from 3.3 nm at 1.15 μm to 28 nm at 3.30 μm (Korablev et al., Reference Korablev, Dobrolensky, Evdokimova, Fedorova, Kuzmin, Mantsevich, Cloutis, Carter, Poulet, Flahaut, Griffiths, Gunn, Schmitz, Martín-Torres, Zorzano, Rodionov, Vago, Stepanov, Titov, Vyazovetsky, Trokhimovskiy, Sapgir, Kalinnikov, Ivanov, Shapkin and Ivanov2017), which is beyond the range of what can detect P(3HB). Yet there are no technical issues in sending mid-to-thermal IR instruments to Mars, which have already been demonstrated on orbiters including Viking, Mars Global Surveyor and Mars Odyssey.

Fourier-transform infrared (FTIR) spectroscopy instruments, however, have the appropriate spectral ranges to detect PHAs. FTIR employs a mathematical process known as Fourier transformation to interpret the interference of two beams of radiation to generate an interferogram which achieves high-resolution spectral ranges (e.g. Stuart, Reference Stuart2015). The FTIR spectra of a pure PHA containing short-chain length and medium-chain length monomers such as hydroxybutyrate, hydroxyoctaoate and hydroxydecanoate have shown strong detection bands at 1728, 1740 and 1732 cm⁻¹, respectively (Hong et al., Reference Hong, Sun, Tian, Chen and Huang1999). Additionally, the intensity of the methylene band near 2925 cm⁻¹ is very strong which helps confirm PHA detection, and accompanying bands near 1280 and 1165 cm⁻¹ help identify the type of PHA (Hong et al., Reference Hong, Sun, Tian, Chen and Huang1999). When analysing PHAs within bacterial cells, the strong marker bands were 1732, 1744 and 1739 cm⁻¹ corresponding to intracellular PHB, mcl-PHA and P (HB + mcl-PHA), respectively (Hong et al., Reference Hong, Sun, Tian, Chen and Huang1999). While no FTIR instruments have been used or developed for an upcoming space mission, Anderson et al. (Reference Anderson, Andringa, Carlson, Conrad, Hartford, Shafer, Soto, Tsapin, Dybwad, Wadsworth and Hand2005) proposed an FTIR for Mars research instrument with a resolution between 700 and 5000 cm⁻¹, which covers the appropriate spectral range for PHA detection. Note that in addition to the limitations as discussed in the beginning of this section, the IR and FTIR detection bands for PHAs are non-specific to PHA and can also be interpreted as another compound. Thus, we propose if any type of IR analysis is used for PHA detection that it be followed up with another technique more sensitive to PHAs for biosignature confirmation.

Raman spectroscopy analyses the lattice vibrations of molecules by measuring the shift in frequency by inelastically scattered light when incident photons hit the sample (Moon et al., Reference Moon, Li, Ramirez-Cuesta, Wu, Wachs and Bañares2023). Raman spectroscopy is similar to IR spectroscopy; however, instead of measuring infrared absorption lines, Raman spectroscopy focuses on the dipole moment that is induced in the molecule by the electric field of the photons which generates Raman emission lines (Moon et al., Reference Moon, Li, Ramirez-Cuesta, Wu, Wachs and Bañares2023). Samek et al. (Reference Samek, Obruča, Šiler, Sedláček, Benešová, Kučera, Márova, Ježek, Bernatová and Zemánek2016) identified the relevant spectral region for a PHB as 800–1800 cm⁻¹ utilizing a 785 nm laser. They calibrated their Raman emission lines for a PHB line at 1736 cm⁻¹ using two internal standards (DNA at 786 cm⁻¹ and amide I at 1662 cm⁻¹) by performing gas chromatography of PHB samples ranging in concentration from 12 to 90% (w/w) (Samek et al., Reference Samek, Obruča, Šiler, Sedláček, Benešová, Kučera, Márova, Ježek, Bernatová and Zemánek2016). This preparation resulted in several Raman peaks at 837, 1455 and 1736 cm⁻¹. In comparison, the Scanning Habitable Environments with Raman & Luminescence for Organics & Chemicals instrument (SHERLOC) on Perseverance has a Raman region between 800 and 4000 cm⁻¹ with a 248.6 nm laser (Bhartia et al., Reference Bhartia, Beegle, Deflores, Abbey, Razzell Hollis, Uckert, Monacelli, Edgett, Kennedy, Sylvia, Aldrich, Anderson, Asher, Bailey, Boyd, Burton, Caffrey, Calaway, Calvet, Cameron, Caplinger, Carrier, Chen, Chen, Clark, Clegg, Conrad, Cooper, Davis, Ehlmann, Facto, Fries, Garrison, Gasway, Ghaemi, Graff, Hand, Harris, Hein, Heinz, Herzog, Hochberg, Houck, Hug, Jensen, Kah, Kennedy, Krylo, Lam, Lindeman, Mcglown, Michel, Miller, Mills, Minitti, Mok, Moore, Nealson, Nelson, Newell, Nixon, Nordman, Nuding, Orellana, Pauken, Peterson, Pollock, Quinn, Quinto, Ravine, Reid, Riendeau, Ross, Sackos, Schaffner, Schwochert, O Shelton, Simon, Smith, Sobron, Steadman, Steele, Thiessen, Tran, Tsai, Tuite, Tung, Wehbe, Weinberg, Weiner, Wiens, Williford, Wollonciej, Wu, Yingst and Zan2021) and the Raman Laser Spectrometer (RLS) on ExoMars has a Raman region between 70 and 4200 cm⁻¹ with a spectral resolution of 6–10 cm⁻¹ with a 532 nm laser (Veneranda et al., Reference Veneranda, Lopez-Reyes, Manrique-Martinez, Sanz-Arranz, Lalla, Konstantinidis, Moral, Medina and Rull2020). Inherently, the purity of the sample in Samek et al. (Reference Samek, Obruča, Šiler, Sedláček, Benešová, Kučera, Márova, Ježek, Bernatová and Zemánek2016) allows for distinct Raman lines, and the calibration, internal standards and laser wavelength used by the authors resulted in the specific peaks for their PHB sample. For Raman spectroscopy to be used to detect PHAs on Mars or other planetary bodies, more studies working to identify PHA Raman lines using rover equivalent instrumentation and impure PHA samples should be conducted.

Another method, which we argue would be the most definitive, to detect PHAs in extraterrestrial worlds would be to exploit the binding between phasins and PHAs. Recall that phasins (PHA granule-associated proteins or PhaP) are a group of amphipathic proteins that bind specifically and hydrophobically with the PHA core. An instrument capable of detecting phasin–PHA associations in a sample would be a more definitive method to detect PHAs. The ‘Signs Of LIfe Detector’ 3rd generation (SOLID3) utilizes the LDChip300, a sandwich microarray immunoassay detection tool that analyses hundreds of different biosignatures from a small sample (Parro et al., Reference Parro, De Diego-Castilla, Rodríguez-Manfredi, Rivas, Blanco-López, Sebastián, Romeral, Compostizo, Herrero, García-Marín, Moreno-Paz, García-Villadangos, Cruz-Gil, Peinado, Martín-Soler, Pérez-Mercader and Gómez-Elvira2011). It employs fluorescent-based immunoassay antibodies (or antibody-like probes) to microscopically detect and report the presence of biosignatures. SOLID3 has been tested in multiple field campaigns and it was one of the instruments included in a proposed mission to Mars (Fairén et al., Reference Fairén, Gómez-Elvira, Briones, Prieto-Ballesteros, Rodríguez-Manfredi, López Heredero, Belenguer, Moral, Moreno-Paz and Parro2020). If a phasin could be added as an affinity tag, then SOLID3 may be able to detect PHAs on other worlds, in addition to the hundreds of different biomarkers it is already searching for.

There are many other methods to identify, quantify and analyse various types of PHAs including high-performance liquid chromatography, UV–visible spectroscopy, scanning and transmission electron microscopy, nanomagnetic resonance and other techniques as summarized by Koller and Rodríguez-Contreras (Reference Koller and Rodríguez-Contreras2015), Godbole (Reference Godbole2016) and Samrot et al. (Reference Samrot, Samanvitha, Shobana, Renitta, Senthilkumar, Kumar, Abirami, Dhiva, Bavanilatha, Prakash, Saigeetha, Shree and Thirumurugan2021). These methods may require either heavier machinery or more human input and customization that is not conducive to planetary mission instrumentation. Nevertheless, considering NASA's plans for a Mars 2020 sample return mission (Haltigin et al., Reference Haltigin, Hauber, Kminek, Meyer, Agee, Busemann, Carrier, Glavin, Hays, Marty, Pratt, Udry, Zorzano, Beaty, Cavalazzi, Cockell, Debaille, Grady, Hutzler, Mccubbin, Regberg, Smith, Smith, Summons, Swindle, Tait, Tosca, Usui, Velbel, Wadhwa and Westall2021) and plans for a human mission to Mars, these other techniques may soon be able to scrutinize returned samples.