Genetic engineering of low-temperature polyhydroxyalkanoate production by Acidovorax sp. A1169, a psychrophile isolated from a subglacial outflow

Vol.:(0123456789) 1 3 Extremophiles (2023) 27:25 https://doi.org/10.1007/s00792-023-01311-5 ORIGINAL PAPER Genetic engineering oflow‑temperature polyhydroxyalkanoate production byAcidovorax sp. A1169, apsychrophile isolated fromasubglacial outflow JakubGrzesiak1· JanGawor1· MałgorzataMartaRogala1· XenieKouřilová2· StanislavObruča2 Received: 24 July 2023 / Accepted: 24 August 2023 © The Author(s) 2023 Abstract In recent years, extremophilic microorganisms have been employed as producers of the microbial bioplastics polyhydroxyalkanoates (PHA), which are of great biotechnological value. Nevertheless, cold-loving or psychrophilic (cryophilic) bacteria have been neglected in this regard. Here, we present an investigation of the Arctic glacier-derived PHA producer Acidovorax sp. A1169. Biolog GEN III Microplates were used as a screening tool to identify the most suitable carbon substrate concerning PHA synthesis. The strain produced homopolymer poly(3-hydroxybutyrate) (PHB) most efficiently (2g/L) at a temperature of 15°C when supplied with fructose or mannitol as carbon sources with a substantial decrease of PHB biosynthesis at 17.5°C. The PHB yield did not increase considerably or even decreased when carbon source concentration exceeded 10g/L hinting that the strain is oligotrophic in nature. The strain was also capable of introducing 3-hydroxyvalerate (3HV) into the polymer structure, which is known to improve PHA thermoplastic properties. This is the first investigation providing insight into a PHA biosynthesis process by means of a true psychrophile, offering guidelines on polar-region bacteria cultivation, production of PHA and also on the methodology for genetic engineering of psychrophiles. Keywords Extremophile· Arctic· Bioplastics· Low-temperature biotechnology· Oligotrophy Introduction Bacterial intracellular storage materials provide its producers with an immense advantage in their native habitats by enabling proliferation at nutrient-deficit circumstances and thus allowing the out competition of species lacking this ability (Moradali and Rehm 2020). One such compound are polyhydroxyalkanoates (PHA), microbial polyesters of hydroxyacids that are stored intracellularly as insoluble granules when carbon source is present in excess to be subsequently depolymerized and catabolized under carbon source limitation conditions. Enzymes responsible for the polymerization process are the PHA synthases encoded by the phaC gene. So far, four classes of this enzyme were discovered and described. PHAs can be divided into shortchain length (scl-PHA, C3-C5 per monomer) PHAs and medium-chain length (mcl-PHA, C6-C14) PHA (Koller 2018). Generally, scl-PHA are produced with the participation of enzymes encoded by phaA and phaB genes and by the action of class I, III and IV PHA synthases, mclPHA are synthesized by class II synthases with precursors being delivered by lipid de-novo synthesis or catabolism of long-chain fatty acids (Reddy etal. 2003). The chemical and physical properties of the resulting polymers were what drew the attention of the industry as they resemble those of plastics made from petroleum or its derivatives. Biodegradability and biocompatibility were the defining factors that led to the large-scale production of the PHA polymer (Tan etal. 2021). The most basic and common member of the PHA family is a homopolymer poly(3-hydroxybutyrate) (PHB) produced by Bacteria and Archaea alike (Müller-Santos etal. 2021). Its thermoplastic properties are greatly improved when various copolymers are produced, for instance by the introduction of Communicated by Oren. * Jakub Grzesiak [email protected]w.pl 1 Institute ofBiochemistry andBiophysics, Polish Academy ofSciences, Pawińskiego 5A, 02-106Warsaw, Poland 2 Department ofFood Chemistry andBiotechnology, Faculty ofChemistry, Brno University ofTechnology, Purkynova 118, 61200Brno, CzechRepublic Extremophiles (2023) 27:25 1 3 25 Page 2 of 14 hydroxyacid with > 4 carbon atoms (such as 3-hydroxyvalerate) (Tan etal. 2021). Being totally biologically produced, PHA synthesis costs far exceed that of oil-based plastics, so its application is now largely restricted to high-value applications e.g. in the medical field (implants, drug carriers). Therefore, lowering the cost of PHA production is key to increasing PHA competitiveness in the global market (Możejko-Ciesielska and Kiewisz 2016). Several factors of the production process can be optimized in order to make PHA more affordable. Choosing the right PHA producer strain is of pivotal importance. Many strategies involving selection in permissive conditions were applied, leading to the isolation of bacterial and archaeal species with a promising PHA-producing potential. Secondly, the identification of the appropriate carbon source is usually one of the first steps in PHA biosynthesis optimization. Other conditions such as medium composition and pH, and incubation temperature, are also subjects of optimization (Tripathi etal. 2013). A plethora of environmental strains was tested in such a manner, with two being frequently used in science and industry: Cupriavidus necator (scl-PHA producer) and Pseudomonas putida (mcl-PHA producer) (Zhang etal. 2022; Weimer etal. 2020). Genetic manipulation can be employed to further increase PHA yields in model producer strains, mainly by enhancing carbon flow into the PHA biosynthesis pathways or preventing intracellular PHA degradation by inactivating PHA depolymerases encoded by the phaZ gene (Wang etal. 2023). In recent years, several “outside the box” microbes have been tested for their ability and efficiency to produce PHAs (Koller 2017). Thermophiles, such as Schlegelella thermodepolymerans or Aneurinibacillus sp. as well as halophiles (Halomonas spp.) were employed to convert waste products into PHAs of varying kinds, with the advantage of unusual cultivation conditions providing the needed robustness of the process against contamination by common mesophilic microflora which substantially reduces the cost associated with sterility demands and opens avenues for establishment of effective continuous or semicontinuous cultivation scenarios (Kourilova etal. 2020; Rehakova etal. 2023; Wang etal. 2022). Therefore, the employment of extremophiles may grant the industry new, surprising benefits as was recently formulated in the concept of Next-Generation Industrial Biotechnology by professor Chen (Chen and Jiang 2018). Nevertheless, psychrophiles have been pretty much neglected in this respect. They grow best below the temperatures usually applied in industrial microbial biotechnology (T < 20°C) (Madigan etal. 2019). Psychrophiles display several advantages when cultured at low temperatures, inter alia: decreased energy input, especially practical in colder climates or around the winter season (Margesin etal. 2008). Low temperature also has a stabilizing effect on the substrate and the products of bacterial metabolism, especially important if one of those is thermolabile, like many sugars (Wolfenden and Yuan 2008). Furthermore, the solubility of atmospheric oxygen is greatly improved in lower temperatures, which substantially prevents problems with the aeration of the process and reduces associated costs (Georlette etal. 2004). Psychrophiles are widespread, occupying niches and habitats across the globe, mostly polar and alpine environments as well as the vast expanses of the deep ocean, providing a great opportunity for bioprospecting for PHA producers (Margesin etal. 2008; Rogala etal. 2020). Numerous study indicates that PHA production ability is common among microbes adapted to cold environments (Goh and Tan 2012; Ciesielski etal. 2014; Kumar etal. 2018; Rogala etal. 2020), there are also indications that PHA prevents bacterial cells from the harmful effect of low temperature (Nowroth etal. 2016) and even repeated freezing and thawing (Obruca etal. 2016). Therefore, there were some attempts to employ polar-region bacteria as PHA producers, yet the optimal cultivation temperatures were outside the range of what can be considered psychrophilic, so true low-temperature PHA-production analysis was not published to date to the best of our knowledge (Kumar etal. 2020; Pacheco etal. 2019; Choi etal. 2021). The aim of the presented research was therefore to explore the capacity of a psychrophilic PHA producer to biosynthesize the polymer at low temperatures and to assess the dynamics of PHA production during submerged cultivation. After an elaborate screening of 200 polar-region bacterial isolates described in Rogala etal. (2020), a chosen few were provisionally analysed by gas chromatography for their PHA content and quality. Acidovorax sp. A1169 emerged as one of the more promising strains. It was isolated from the waters of a subglacial outflow stream emerging from beneth Hans Glacier, a tidewater glacier located at the shore of Hornsund fjord at Spitsbergen Island in the Arctic. We hypothesized that Acidovorax sp. A1169 could be an efficient PHA producer and given the right conditions it can accumulate PHAs at least to 50% of cell dry weight. Materials andmethods Bacterial strains, plasmids andculture conditions The bacterial strains and plasmids used in this study are listed in Table1. The Arctic isolate Acidovorax sp. A1169 was obtained from the Central Collection of Strains of the Institute of Biochemistry and Biophysics, Polish Academy of Sciences. It was previously recognized as a potent, lowtemperature PHA producer (Rogala etal. 2020). Escherichia coli strain DH10B was used for plasmid transformation and propagation, while E. coli strain S17-1 was used Extremophiles (2023) 27:25 1 3 Page 3 of 14 25 for mobilization of the suicide plasmid pAKE604 into Acidovorax sp. A1169. Wild-type of Acidovorax sp. A1169 strain and its gene knockout mutant (Δi-phaZ) were cultured in R3A medium (1g/L tryptone, 1g/L peptone, 1g/L beef extract, 1g/L yeast extract, 1g/L K2HPO4, 0.5g/L NaH2PO4, 0.5g/L Na-pyruvate, 0.1g/L MgSO4‧7H2O) at 15°C (if not otherwise indicated). All E. coli strains were cultured in LB broth on a shaker at 200rpm and 37°C or on LB agar at 37°C. Where required, kanamycin was added to a final concentration of 25 or 50µg/L to ensure plasmid maintenance and selection. DNA isolation andsequencing Genomic DNA was isolated by the CTAB method (Wilson 2001). Plasmid isolation was performed with the Plasmid Midi AX or the Plasmid Mini kits (A&A Biotechnology) while DNA purification was conducted with the Clean-up Concentrator kit (A&A Biotechnology) according to manufacturer instructions. The genome of Acidovorax sp. A1169 was sequenced using an Illumina MiSeq apparatus (Illumina Inc., USA). The Illumina paired-end sequencing library construction was performed with 1μg of post-nebulized DNA extract and the KAPA Library Preparation Kit reagents (KAPA Biosystems, USA), according to the manufacturer’s instructions. The library was pooled and sequenced on a MiSeq platform using the 600-cycle MiSeq reagent Kit v.3 (Illumina, USA). Sequence quality metrics were assessed using FASTQC (Andrews 2010). Genome assembly, annotation, primer design, PCR amplification andcloning Raw sequencing reads were trimmed for quality and residual library adaptors were removed using fastp software (Chen etal. 2018, https:// acade mic. oup. com/ bioin forma tics/ artic le/ 34/ 17/ i884/ 50932 34). Cleaned Illumina reads were assembled into contigs using SPAdes software (https:// github. com/ ablab/ spades). Draft genome was annotated using the BV-BRC platform (https:// www. bvbrc. org/). The SnapGene program SnapGene software (www. snapg ene. com) was used to design primers for the amplification of i-phaZ flanking regions (Table2). PCR amplifications were performed using PCR Mix Plus, PCR Mix Plus HGC and PCR Mix RAPID ready-to-use mixes for PCR (A&A Biotechnology). Appropriate flanking region pairs were cloned into pAKE604 (Kmr) vectors using the Anza Restriction Enzyme Cloning System (ThermoFisher) according to the manufacturer’s instructions and then transformed into E. coli DH10B chemically competent cells made using the Inoue method (Inoue etal. 1990). Transformants were checked by colony PCR using specific primers (Table2). Bacterial conjugation Recombinant plasmids were introduced into E. coli strain S17-1. Biparental mating with psychrophilic PHB producers was done as follows: saturated cultures of Acidovorax sp. A1169 and E. coli S17-1 were washed with PBS and combined in a 3:1 ratio. The resulting suspension was drop Table 1 Bacterial strains and plasmids used in this study Strain/plasmid Genotype/phenotype Source/references Acidovorax sp. A1169 Wild-type PHB producer Arctic glacier/Rogala etal. (2020) Δi-phaZ i-phaZ gene knockout mutant derived from A1169 This study E. coli DH10B F– mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara-leu)7697 galU galK λ– rpsL(StrR) nupG Thermo-Fisher Scientific S17-1 recA pro hsdR RP4-2-Tc::Mu-Km::Tn7,λ-pir; mobilizer strain Lab stock Plasmid pAKE604 oriMB1 oriTRK2 Apr Kmr lacZ sacB El-Sayed etal. (2001) Table 2 DNA primers used in this study Primer Primer sequence (5′–3′)Note 1169Z1F 1169Z1R TCAG GGA TCC ATA ACG GAG TTT CGA CCC CAT GCT (BamHI) TCAG GAA TTC GTA TCG TCC GAA AAG CGC TTGAA (EcoRI) For amplifying the 333-bp upstream The homologous sequence of i-phaZ 1169Z2F 1169Z2R TCAG GAA TTC CGG CAT CTT CAG CGG CCG GCG CTG (EcoRI) TCAG AAG CTT GTG CGG CGG GTG CGT GGT GCCGG (HindIII) For amplifying the 414-bp downstream The homologous sequence of i-phaZ 1169Z1F 1169rZ TCAG GGA TCC ATA ACG GAG TTT CGA CCC CAT GCT TTC ACC ACC GGT TTG CTG GCGA For the confirmation of the Δi-phaZ knock-out mutant Extremophiles (2023) 27:25 1 3 25 Page 4 of 14 plated onto Conjugation Agar containing: 1g/L tryptone, 1g/L peptone, 1g/L beef extract, 1g/L yeast extract, 1g/L K2HPO4, 0.5g/L NaH2PO4, 0.5g/L Na-pyruvate, 0.1g/L MgSO4‧7H2O, 3g/L HEPES, 3g/L NORIT® activated charcoal, 15g/L agar, pH was adjusted to 7.2 with 0.1M KOH, 0.1M HCl and a Hanna pH-meter. Plates were incubated at 15°C for 48h, after which the growth was scraped, serially diluted and plated onto R3A plates with Kanamycin (25mg/L) and incubated at 10°C until single colonies developed. Low temperature was used as a selecting factor for psychrophilic transconjugants as using the traditional method of generating Acidovorax sp. A1169 Rifampin resistant mutants, was unsuccessful (Smorawińska etal. 2012). Colonies of Km-resistant psychrophiles were picked, inoculated into R3A broth supplemented with 2.5% sucrose and incubated at 10°C with shaking until bacterial growth was apparent. The resulting suspension was diluted and plated onto R3A plates with 2.5% sucrose and incubated at 15°C until colony development. Obtained isolates were screened for the target sequence by PCR, using appropriate primers (Table2). Microarray metabolic fingerprinting Carbon source utilization abilities of Acidovorax sp. A1169 were assessed using GEN III Microplates (Biolog Inc., Hayward, CA, USA) as described in Gawor etal. (2016). After incubation in R3A broth on a rotary shaker (WL-972, JWElectronics) for 3days in 15°C the cells were harvested by centrifugation (9000rpm for 3min), washed twice, suspended in sterile 0.9% saline and added to a vial of MicroPlate IF C inoculation fluid until transmittance reached 90%. Biolog GEN III microplates (Biolog Inc., Hayward, CA, USA) were inoculated according to the manufacturer’s instructions. The plates were incubated in darkness at 15°C, the color development was read at 590nm (A590) in a Varioskan plate reader (Thermo Fisher Scientific, Waltham, MA, USA), and cellular respiration was measured kinetically by determining the colorimetric reduction of tetrazolium dye. Data were collected twice a week over a 15-day period. PHA production To assess which carbon source facilitates the most efficient granule synthesis in Acidovorax sp. A1169 several compounds, indicated by the Biolog system, were used as substrates for PHA production. PHA synthesis induction was done according to Kourilova etal. (2021a) with modifications. An active inoculum of wild-type strain was prepared (R3A broth, 15°C, 72h) and added (10% v/v) to the PHA production medium. The PHA production medium consisted of the following: 9g/L Na2HPO4·12H2O, 1.5g/L KH2PO4, 1g/L NH4Cl, 0.5g/L yeast extract, 0.2g/L MgSO4·7H2O, 0.02g/L CaCl2·2H2O and 1ml/L SL-11 trace element solution (5.2 g/L Na2-EDTA, 1.5g/L FeCl2·4H2O, 190.0g/L CoCl2·6H2O, 100.0mg/L MnCl2·4H2O, 70.0mg/L ZnCl2, 36.0mg/L Na2MoO4·H2O, 24.0mg/L NiCl2·6H2O, 6.0mg/L H3BO3, 2.0mg/L CuCl2·2H2O). After autoclaving a filter-sterilized solution of one of the following substrates was added to each flask of medium to a final conc. of 10g/L: mannitol, fructose, glycerol, glucose, lactate, sorbitol and mannose. After 96h of incubation at 15°C and 150rpm (INNOVA44 Incubator Shaker, New Brunswick Scientific), the biomass was harvested by centrifugation for further analysis. PHA production efficiency was further investigated at different temperatures (10.0, 12.5, 15.0 and 17.5°C) with fructose and mannitol as carbon sources (10g/L) after 96h at 15°C and 150rpm. To assess the optimum duration of the incubation period biomass and PHB content were measured daily for 6days at 15°C and 150rpm. The nitrogen source effect on the PHB accumulation was measured after substituting the NH4Cl in the production medium with urea (0.56g/L) or peptone (1.73g/L). The nitrogen source concentration was calculated to equate to that of 1g/L of NH4Cl assuming the peptone nitrogen content was 15%. The carbon source concentration effect was investigated by adding mannitol or fructose to the PHA production medium at a conc. of 10 and 20g/L. The strain’s ability of 3HV incorporation into the polymer structure when supplemented by proper structural precursors was tested by adding propanol, pentanol, propionate, valerate or levulinate to the PHA production medium at a conc. of 2g/L after 24h of cultivation with mannitol. To assess the i-phaZ deletion mutants ability to access intracellular PHA as carbon and energy source, it was subjected to PHA-accumulation conditions as described earlier. Therefore, prepared PHB-filled cells were washed with the PHA production medium without the primary carbon source and then introduced into the same medium at 10% v/v. Wild-type A1169 was used as a control strain. Cell growth was monitored during the 96h incubation period by colony forming unit count on R3A agar at 15°C. Biomass analysis Ten mL of bacterial suspension was collected after the incubation period on PHA production medium, centrifuged at 6000×g for 5min, washed with distilled water and dried at 80°C until constant mass was achieved. PHA composition and content of the dried biomass were determined by gas chromatography with a flame ionization detector (GC-FID) as described previously (Obruca etal. 2013). Extremophiles (2023) 27:25 1 3 Page 5 of 14 25 Data analysis Illumina reads were deposited in the NCBI Sequence Read Archive (SRA) as BioProject PRJNA991094. All results were compiled using Excel 2016 (MS Office) for Windows. Data visualization and statistical analysis have been performed using the R software (R v.4.2.3) and the following packages: ggplot2, ggpubr, (R Core Team 2002). Phylogenetic trees were made using the Mega-174 X software. Results anddiscussion Substrate preference ofthestrain Acidovorax sp. A1169 The genus Acidovorax (family Comamonadaceae, class Betaproteobacteria) currently comprises 20 validly published species (Du etal. 2023). 16S rRNA gene sequence similarity with sequences of the aforementioned species deposited in the NCBI blastn database revealed that strain A1169 shared a 99.4% and 99.02% similarity with A. radicis and A. defluvii, respectively (Fig.1), not meeting the conditions for a new species (Kim etal. 2014). However, its placement on a separate branch of the phylogenetic tree and also its Arctic glacier origin suggest a new quality within the genus (Du etal. 2023). Acidovorax sp. A1169 genome annotation revealed the presence of one gene coding for a PHA synthase. The placement of its deduced amino acid sequence suggests it belongs to class I PHA synthases (Fig.2) capable of synthesizing scl-PHAs as was further confirmed by GC analysis of Acidovorax sp. A1169 biomass when various compounds were supplied as substrates (Fig.3c). Generally, isolates of the genus Acidovorax were obtained from different sources, both environmental (soil, rhizosphere, water, activated sludge) and clinical, hinting that phenotypic and genotypic features vary considerably between species/strains (Willems 2014). Therefore, the employment of phenotypic microarrays like the Biolog GENIII microplate can be justified as it provides an effective and reliable tool for screening the bacterium with respect to utilization of various carbon compounds and other cultivation conditions for environmental bacteria (Woźniak etal. 2019). However, the main purpose of this system is bacterial identification based on the carbon source utilization pattern and other metabolic features provided in the 96-well plates by the manufacturer (Sandle etal. 2013). On rare occasions, it has been used to ascertain a set of carbon compounds that support growth in microbiological media (DeAngelis etal. 2011). Seven compounds were revealed in the GENIII microplate assay as supporting high respiratory rates in Acidovorax sp. A1169 (Fig.3a). Highest values were noted for mannitol (A590 = 1.19 ± 0.19) and fructose (A590 = 1.19 ± 0.29), while the lowest within this group for glycerol (A590 = 0.91 ± 0.22). Biomass concentration analysis produced similar results when carbon sources were supplied into the PHA production medium at a concentration of 10g/L (Fig.3b). When A1169 was cultivated on mannitol and fructose the following biomass concentrations was achieved (g/L): 4.05 ± 0.21 and 4.22 ± 0.23, respectively, while on glycerol biomass concentration reached 2.82 ± 0.46. The highest PHB concentration was achieved on fructose (2.14 ± 0.22g/L) followed by mannitol (1.99 ± 0.11g/L) corresponding to 50.7% and 49.1% of PHB in cell dry mass, respectively (Fig.3c). There was a positive, significant correlation between values of the GENIII assay, dry biomass concentration and PHB content at p ≥ 0.05 (Fig.3d–f). Correlations between respiration rates and PHB concentration suggest Biolog GENIII microplates as a suitable guiding tool towards optimization of bacterial biomass and PHA accumulation. Hence, the highest PHB yield was achieved for the A1169 strain on fructose and mannitol. Fructose has proven to be the best carbon source for the flag PHB producer C. necator as well as the halophile Halomonas sp. YLGW01 (Nygaard etal. Fig. 1 Neighbor-joining phylogenetic tree based on 16S rRNA gene sequences showing the position of strain A1169 and related species in the genus Acidovorax (family Comamonadaceae). Numbers at nodes are bootstrap percentages based on the neighbor-joining algorithm. Simplicispira metamorpha DSM 1837T was used as an outgroup. Sequences were retrieved from the NCBI database. Bar shows substitutions per nucleotide position Extremophiles (2023) 27:25 1 3 25 Page 6 of 14 2021; Park etal. 2020). Besides the examined here Acidovorax sp., other members of the family Comamonadaceae were also able to turn fructose into PHB, however, other sources like glucose or xylose yielded higher concentrations of the biopolymer (Yamaguchi etal. 2019; Kourilova etal. 2020, 2021a). Nevertheless, even fructose can be considered as sustainable and renewable carbon substrate for PHA production when derived from properly chosen resources. For instance, Corrado etal. recently described PHA synthesis form hydrolysates of waste inulin rich in fructose content (Corrado etal. 2021). Mannitol was rarely considered as a substrate for PHB biosynthesis, although some mannitol-rich agro-industrial wastes such as celery waste (Apium graveolens) and ensiled grass press juice were recognized as potent substrates for PHA production employing Cobetia amphilecti and Burkholderia sacchari as producers (Cerrone etal. 2015; Gnaim etal. 2022). Effect oftemperature andcultivation time ongrowth andPHA synthesis inAcidovorax sp. A1169 Incubation temperature had a considerable effect on the biomass and also on the PHB concentration in Acidovorax sp. A1169 culture (Fig.4a, b). The highest PHA titers were achieved at 15°C on fructose [4.17 ± 0.02g/L CDW (cellular dry weight) and 1.99 ± 0.03g/L PHB] and mannitol (3.93 ± 0.02g/L CDW and 1.77 ± 0.05g/L PHB) as substrate. At 17.5°C biomass and PHB concentrations were the lowest, on fructose: 1.19 ± 0.06g/L CDW and 0.39 ± 0.02g/L PHB), while for mannitol: 1.24 ± 0.03g/L CDW and 0.49 ± 0.002g/L PHB. Such a low optimal temperature of PHB biosynthesis has not been observed before, even when polar-region or high-altitude strains Fig. 2 Neighbor-joining phylogenetic tree based on phaC (polyhydroxyalkanoic acid synthase) amino acid sequences showing the position of strains A1169 phaC sequence among other phaC sequences belonging to four synthase classes. Sequences were retrieved from the NCBI database. Bar shows substitutions per amino acid position. Numbers at nodes are bootstrap percentages based on the neighborjoining algorithm Extremophiles (2023) 27:25 1 3 Page 7 of 14 25 were tested (Kumar etal. 2020; Pacheco etal. 2019; Choi etal. 2021). Arctic and Antarctic Pseudomonas strains for example displayed the highest PHB production rates at 30°C, a temperature that is outside the range of what is considered psychrophilic (Pacheco etal. 2019; Choi etal. 2021; Madigan etal. 2019). In the case of Acidovorax sp. A1169, the increase in incubation temperature by 2.5°C caused not only a 77% drop in PHB yield, but also an approx. 66% decreases in the PHB-free biomass concentration, hinting that not only the enzymatic machinery behind PHB biosynthesis was affected but also essential cellular metabolism. The time frame of PHA synthesis is one of many factors that affect the efficiency of biopolymer production (Koller etal. 2010, 2017). The impact of Fig. 3 Carbon source utilization by Acidovorax sp. A1169 based on a the GENIII Microplate respiratory assay; b cellular dry mass (CDW) concentration; c PHB concentration. Correlation between variables: d GENIII microplate and cell biomass; e cellular dry mass (CDW) and PHB; f PHB and GENIII microplate Extremophiles (2023) 27:25 1 3 25 Page 8 of 14 incubation time on biomass production of strain A1169 varied between carbon sources (Fig.4c, d). The highest values of biomass and PHB concentrations were achieved for both, fructose and mannitol after 96h, yet for fructose these values dropped afterward, while for mannitol they remained relatively stable. C. necator achieved maximal PHB titers in flasks within 72h at 30°C while a variety of thermophiles display a similar PHB biosynthesis period at 50–55°C (Obruca etal. 2013; Aramvash etal. 2015; Nygaard etal. 2021; Kourilova etal. 2020, 2021a; b). Considering that the specific growth rate of mesoand thermophiles is approx. 2.5 × higher than psychrophiles, the additional 24h required by strain A1169 to achieve maximal PHB concentration can be considered efficient (Mohr and Krawiec 1980) and the bacterium Acidivorax so. A1169 can be considered as promising psychrophilic bacterium for PHA production aligning to the concept of Next-Generation Industrial Biotechnology (Chen and Jiang 2018). Identification ofsuitable nitrogen source andcarbon source concentration Replacing the ammonium chloride with peptone or urea greatly lowered the biomass and subsequently the PHB concentration in Acidovorax sp. A1169 culture (Fig.5a, b). Peptone stimulated higher PHB synthesis with lower cell dry weight (fructose: 0.82 ± 0.01g CDW/L and 0.23 ± 0.003g PHB/L; mannitol: 0.85 ± 0.13g CDW/L and 0.24 ± 0.002g PHB/L), whereas with urea the opposite was the case (fructose: 1.16 ± 0.01g CDW/L and 0.1 ± 0.0002g PHB/L; mannitol: 1.1 ± 0.14g CDW/L and 0.01 ± 0.001g PHB/L). As with the carbon source, nitrogen source effect seems to be bacteria species specific, with peptone and/or urea being preferable over ammonia salts by Vibrio proteolyticus, Pseudomonas aeruginosa and Erythrobacter aquimaris, while Rhizobium etli and Pseudomonas stutzeri preferred ammonia salts over peptone or even yeast extract (Hong etal. 2019; Tripathi etal. 2012; Mostafa etal. 2020; Belal 2013). Fig. 4 Effects of incubation temperature on biomass concentration using a fructose as carbon source; b mannitol as carbon source. Biomass concentration changes during cultivation when fructose (c) or mannitol (d) was the carbon source. Orange—cellular dry mass (CDW); blue—PHB Extremophiles (2023) 27:25 1 3 Page 9 of 14 25 The concentration of the carbon source—fructose or mannitol had an interesting effect on biomass concentrations (Fig.5c, d). In the case of fructose, biomass and PHB yield decreased with higher substrate concentration, whereas with mannitol it raised. However, the highest amounts of PHB per gram of supplied substrate were achieved for the conc. of 10g/L: fructose—0.19g PHB per g substrate, mannitol—0.16g PHB per g substrate, while at 20g/L: fructose—0.08g PHB per g substrate, mannitol—0.1g PHB per g substrate. This phenomenon of PHB biosynthesis suppression at relatively high substrate concentration has been observed before, albeit the optimal carbon source concentration being 20g/L or higher, with C. necator managing 40g/L without adverse effects (Wendy etal. 2022; Hong etal. 2019; Sriyapai etal. 2022; Nygaard etal. 2021). Growth and PHB production decrease in higher substrate concentration can be explained by osmotic and/or nutrient shock (Azevedo etal. 2012). The glacial origin of the strain may suggest both as glacier-hosted habitats are often poor in nutrients and solutes (Grzesiak etal. 2015). Characterization ofi‑phaZ deletion mutant Two PHB-depolymerase genes were recognized in the genome of Acidovorax sp. A1169. Analysis based on a phylogenetic neighbor-joining tree amended with amino acid sequences of confirmed intracellular and extracellular sclPHA depolymerases revealed that one of the recognized genes coding for an intracellular depolymerase (Fig.6a) (Knoll etal. 2009). As the inactivation of this enzyme Fig. 5 Effects of different nitrogen sources on biomass concentration using a fructose as carbon source; b mannitol as carbon source. Effects of carbon source concentration on the concentration of produced biomass (c, d). Orange—cellular dry mass (CDW); blue—PHB