**1. Introduction**

Polyhydroxyalkanoates (PHAs) are microbial storage polyesters, which are accumulated by various prokaryotic microorganisms. Apart from their primary storage function for carbon, energy, and reduction equivalents, PHAs also significantly enhance the stress robustness of microorganisms and accumulation of them is probably a part of adaptation to some extreme conditions such as high salinity [1]. PHAs are, therefore, widespread metabolites among extremophilic prokaryotes [2]. Moreover, they represent a very promising ecologically friendly alternative to traditional petrochemical polymers–PHAs are completely biodegradable and compostable materials, which can be produced from totally renewable resources including also waste products stemming from food production and other industries. Their properties and application fields are very similar to petrochemical plastics such as polypropylene [3].

**Citation:** Novackova, I.; Kourilova, X.; Mrazova, K.; Sedlacek, P.; Kalina, M.; Krzyzanek, V.; Koller, M.; Obruca, S. Combination of Hypotonic Lysis and Application of Detergent for Isolation of Polyhydroxyalkanoates from Extremophiles. *Polymers* **2022**, *14*, 1761. https://doi.org/ 10.3390/polym14091761

Academic Editor: Shashi Kant Bhatia

Received: 21 March 2022 Accepted: 23 April 2022 Published: 26 April 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Nevertheless, the main problem prohibitive for large-scale production of PHAs is their high production cost. There are several strategies how to overcome this limitation. One of them is the employment of extremophiles as PHA-producing chassis. The main advantage of extremophiles is their capability of growth and production of various metabolites including but not limited to PHA under conditions, which reduce or even eliminate the risk of contamination of the process by common mesophilic microflora, e.g., high or low temperature, high salinity, extreme pH-values, etc. Due to their robustness against unfavorable microbial contamination, which can destroy whole fermentation batches, biotechnological processes using extremophilic microorganisms can be operated under reduced requirements for sterility, or eventually even completely without sterilization of the cultivation media or equipment. Consequently, it has a significantly positive impact on the energy and economic balance of these processes. Moreover, high tolerance of the process against contamination allows running the cultivation process in highly productive continuous or semi-continuous mode. Therefore, employing extremophilic microorganisms in biotechnology is nowadays a modern trend, which is called the concept of industrial biotechnology of the next-generation ("Next-Generation Industrial Biotechnology", NGIB) [4].

In this context, the employment of extremophilic microorganisms can significantly reduce the price of PHA. It can take advantage of the ability of numerous extremophiles to produce PHA natively. Predominantly many halophilic microorganisms (microorganisms adapted to the high salinity of environments) belong among promising PHA producers– PHA production ability was described for several representatives of genus *Halomonas* such as *Halomonas boliviensis* [5], *Halomonas neptunia* and *Halomonas hydrothermalis* [6], *Halomonas bluephagenesis* [7], *Halomonas halophila* [8], or extremely halophilic representatives of the Archaea domain, such as *Haloferax mediterranei* [9]. As mentioned above, the accumulation of PHA is a part of the adaptation strategy of halophiles to hyperosmotic environment; however, the main adaptation strategy is usually the accumulation of either organic (osmolytes) or inorganic (K+ ions) substances, which compensate for the osmotic pressure of the surrounding, as observed especially for extreme haloarchea [10].

Instead of halophiles, the production of PHAs was also observed in a restricted number of thermophilic bacteria, which are adapted to high temperature condions [2]. The most studied strains belong to the genera *Caldimonas* [11], *Tepidimonas* [12], in addition to the very promising PHA producer *Schlegelella thermodepolymerans*, which showed expedient potential for PHA production using lignocellulose-based substrates with high xylose content [13]. For thermophiles, PHA most likely does not directly take the role in the adaptation to high temperature, since the adaptation is usually connected with changes of protein structure, enhancement of the activity of chaperones and other heat-shock proteins, and also accumulation of organic compatible molecules similarly to halophilic prokaryotes [14].

Apart from the microbial biosynthesis of PHA, also the expenses associated with the isolation of PHA, an intracellular product, from microbial biomass contribute to the high cost of PHA. Extraction of the polymer from the microbial cells is often performed by organic solvents; however, considering limited polymer solubility, the extraction is limited to selected, predominantly chlorinated, solvents such as chloroform or dichloromethane. Alternatively, compounds typically described as "PHA anti-solvents", such as acetone, can be employed for PHA extraction under conditions of highly elevated temperature (120 ◦C) and pressure (7 bar) [15]. Nevertheless, the approaches based on organic solvents are technologically difficult, and the use of toxic and hazardous solvents eliminates the positive ecological impact of final materials [16]. The other possibility is the utilization of supercritical CO2 for PHA extraction. Despite a very positive ecological connotation, due to the low solubility of PHA in supercritical CO2 and the need for additional compounds acting as solubility mediators ("modifiers"), such as methanol, the process shows low efficiency [17,18]. Also, an alternative PHA isolation approach based on the removal of other components of biomass can be used, when PHA granules remain in the intact, solid state and can be further separated by centrifugation or filtration. Form an economical viewpoint, this approach makes definitely sense: Considering the fact that the PHA fraction

in biomass can exceed 90 wt.%, it appears cumbersome to recover these 90 wt.% instead of simply removing the minor (10%) fraction of biomass. Indeed, many reagents and strategies can be used to remove other cell components instead of PHA. For instance, in literature the use of hydrolytic enzymes (predominantly commercially available proteases– Protease L330, Esparase, Alcalase, Neutrase, Allprotease, etc.) is described [19], but in practice, these approaches are limited by the high cost of enzymes. An alternative way is the digestion of biomass using various chemical reagents such as sodium hydroxide [20], sodium hypochlorite [21], EDTA, and detergents such as SDS [22] or ammonium laurate [23]. Individual approaches can be combined as was mentioned in Kathiraser et al., who used enzymes together with detergents and EDTA for PHA isolation [24].

Hypotonic lysis is an additional tool that can be used for the simple disruption of microbial cells. This approach is based on the exposition of cells to surrounding with significantly lower osmotic strength than osmolarity of cultivation medium. During sudden exposition to a hypotonic environment, water begins to penetrate into cells, which leads to disruption of their integrity, rupture of cells, and release of the intracellular cell content. This hypoosmotic cell lysis approach was described predominantly for extreme halophilic prokaryotes such as *Hfx. mediterrenei*, which are cultivated in media with salt contents of more than 200 g/L and are, therefore, extremely sensitive to hypotonic treatment. During exposure to distilled water, cells disruption and release of PHA granules into the solution occur immediately. After centrifugation, there are two phases generated—the bottom pinkish phase represents cell mass residues (colorized by carotenoid pigments), while the upper white phase consists of PHA granules [25]. However, the purity of PHA isolated in the described way is not too high due to the PHA granules being covered by a proteinaceous membrane; therefore, the following purification using for instance hypochlorite, H2O2, or other reagents is usually necessary [8,26].

In this work, we decided to investigate the possibility of utilization of hypotonic lysis to isolate PHA from moderately extremophilic microorganisms—halophilic *Halomonas halophila* and also thermophilic *Schlegelella thermodepolymerans.* We hypothesized that both microorganisms could be partially sensitive to hypotonic lysis due to the accumulation of compatible solutes as a strategy of their adaptation to extreme conditions. To further support the process of cell disruption and increase the purity of the isolated materials, we have also introduced a low concentration of 10 g/L of sodium dodecyl sulfate (SDS). Since SDS might reduce the positive environmental impact of the process by serious contamination of wastewater, we introduced a step in which SDS leftovers after PHA isolation are precipitated with KCl and can be easily removed from wastewater and potentially even regenerated for repeated use.
