*2.5. Precipitation of Potassium Dodecyl Sulfate (KDS) and Determination of Residual SDS Concentration*

SDS is a water-soluble molecule, whereas the salt potassium dodecyl sulfate (KDS) is water-insoluble. This is the main idea of how to get rid of SDS from wastewater after isolation of PHA from bacterial biomass. A simple addition of KCl to supernatant containing SDS results in the formation of an insoluble precipitate of KDS, which can conveniently be separated by sedimentation or centrifugation. For this purpose, we prepared 4 M aqueous KCl solution and after isolation and following centrifugation, we added it in a 1:1 ratio to the supernatant to obtain a precipitate.

Residual concentration of SDS after PHA isolation procedure and also the efficiency of KDS precipitation from SDS solutions with different initial amounts of the detergent were determined employing a spectroscopic assay based on the use of Stains-All dye (Sigma-Aldrich, Darmstadt, Germany) as described by Rupprech et al. [29]. The analysis was performed using a microplate reader (ELISA reader, ELx808, BioTek, Winooski, VT, USA).

#### **3. Results and Discussion**

Every isolation experiment was preceded by the cultivation of microorganisms *H. halophila* and *S. thermodepolymerans* in mineral media with the most suitable carbon sources (glucose for *H. halophila* and xylose for *S. thermodepolymerans*) to obtain biomass with high PHA content. Within individual cultivations, we tried to keep the cultures under the same conditions for gaining results of biomass and PHA content as similar as possible, despite we measured both characteristics (biomass content gravimetrically and PHA content by GD-FID) within each experiment for independent comparison among results of cultivations. For each experiment, amounts of PHA in biomass are stated under almost all tables for the comparison with the purity of isolated polymer using different treatments, because for individual experiments all the yields were related to individual cultivation results. Based on the data from experiments, for *H. halophila* average concentration of biomass was (5.5 ± 0.3) g/L with a PHA fraction in biomass of about (78 ± 7) wt.%. For *S. thermodepolymerans* average biomass concentration was (6.1 ± 0.1) g/L with (70 ± 4) wt.% PHA.

#### *3.1. Optimization of SDS Concentration*

Within the whole PHA isolation development process, the first step was focused on the optimal concentration of detergent SDS. The idea was to expose the extremophilic bacterial cells naturally containing compatible solutes as a part of their adaptation strategy to hypotonic conditions with low amounts of SDS to improve cell disruption and enable solubilization of hydrophobic cell components which would otherwise get attached to PHA granules reducing the purity of the materials. For both used strains, tested SDS concentrations were 1; 2.5; 5 and 10 g/L; results for individual strains are listed in Table 1. For *H. halophila* the process was carried out in a water bath duringa2h lasting process at 70 ◦C; for *S. thermodepolymerans*, the process lasting the same time was carried out at 90 ◦C.


**Table 1.** Effect of different SDS concentrations on PHA purity and yield during 2 h lasting isolation at 70 ◦C for *H. halophila* and 90 ◦C for *S. thermodepolymerans*.

<sup>1</sup> Yield is defined as PHA (g/L) for individual isolation way divided by PHA (g/L) obtained from biomass without previous isolation step. PHA content in the biomass: *H. halophila* (84.02 ± 0.61) wt.%, *S. thermodepolymerans* (64.43 ± 5.96) wt.%.

In the case of *H. halophila*, the most effective SDS concentration for isolation of PHA was 5 g/L, when the purity of the polymer was more than 97 wt.% together with the highest product yield considering purity and also amount of the isolated material. For this moderately halophilic strain with optimal salt concentration for growth 66 g/L, utilization of 5 g/L of SDS induces effective hypotonic cell lysis and results in high purity of the isolated material. Therefore, the most suitable concentration of SDS was used for all the following experiments employing the halophilic strain.

For *S. thermodepolymerans*, the highest purity was determined for a SDS concentration of 2.5 g/L. In comparison with the halophilic culture, the concentration is 2 times less, which might be caused by the lower content of the compatible solutes in the cells of thermophilic culture. It can be expected that thermophilic bacteria are less prone to hypotonic lysis as compared to halophiles. This expectation is also supported by the fact that purities of PHA materials obtained for thermophilic *S. thermodepolymerans* are substantially lower than those obtained in its halophilic counterpart *H. halophila*. Further, aside from the purity of the material, we have also considered the yield of the isolation process which was highest for 5 g/L of SDS leading to the establishment of the most suitable concentration of SDS of 5 g/L—the same as for *H. halophila*.

The team of Arikawa et al. (2017) focused on using SDS in combination with sonication for the isolation of PHA from biomass of the mesophilic bacterium *Cupriavidus necator*, when the optimal concentration of detergent was set on 33 g/L [30]. In our case, we used a more than 6-times lower amount of detergent due to the advantageous effect of hypotonic shock, which is applicable on strains producing compatible solutes (for instance halophiles and thermophiles). Further, our approach relying on hypotonic shock does not require any special equipment, on the contrary, sonification of the bacterial cells can be challenging especially in scales of industrial production of PHA.
