Exploring nutrient limitation for polyhydroxyalkanoates synthesis by newly isolated strains of Aeromonas sp. using biodiesel-derived glycerol as a substrate

Microbial isolation and selection of PHAs producers

Activated sludge samples were collected in spring from an aeration tank at the municipal wastewater treatment plant in Olsztyn (Poland) and placed into 100-ml sterile glass bottles. To collect the samples, the oral permission from Mr. Ireneusz Jankowski, who was the Vice-Chief of the treatment plant, was received. The sludge samples were diluted with sterile saline solution (0.8% NaCl), then a volume of 0.1 ml was spread onto nutrient agar (Oxoid, Hampshire, United Kingdom). The plates were incubated at 30 °C for up to 72 h in the light. Single bacterial colonies were then picked and cultured in lysogeny broth (1% w/v tryptone, 0.5% w/v yeast extract, 1% NaCl) in 15-ml screw-capped tubes at 30 °C with 220 rpm shaking for 24 h.

The isolated strains were selected as potential PHA producers using polymerase chain reaction-based detection of the PHA polymerase gene. One milliliter of each bacterial strain culture was utilized for DNA isolation using the commercial Genomic Mini kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s protocol. The PCR mixture consisted of 50 ng of extracted DNA, 100 μmol of deoxynucleoside triphosphate (Promega, Madison, WI, USA), 1.5 μM MgCl₂, 0.5 μM of each primer, 1 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA), five μl of reaction buffer (500 mM potassium chloride, pH = 8.5; Triton X-100). To detect potential PHA producers, two primer pairs were used (Table 1). The first one, recognizing both short chain length- and medium chain length- PHAs synthase genes was elaborated by Romo et al. (2007) and the second primer pair was specific for both genes responsible for mcl-PHA synthesis (Solaiman, Ashby & Foglia, 2000) (Table 1). PCR was conducted using an Eppendorf® Mastercycler Gradient (Eppendorf, Wesseling-Berzdorf, Germany). The presence of PCR product was confirmed by analysing five μl of the PCR product on a 1.0% agarose gel stained with ethidium bromide.

Identification of bacteria by 16S rRNA analysis

Genomic DNA was extracted from a single PHA producing colony using the Genomic Mini kit (A&A Biotechnology, Poland) according to the manufacturer’s instructions. Pure bacterial isolates of Aeromonas sp. strains were identified by PCR amplification in a mixture containing 50 ng of DNA, 2.5 mM each of deoxynucleoside triphosphate (Promega, Madison, WI, USA), 400 ng of each primer, 10× PCR buffer (500 mM KCl pH 8.5; Triton X-100), 1.5 mM MgCl₂, 1 U of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA). The 16S rRNA gene was amplified using the primers described by Muyzer, De Waal & Uitterlinden (1993) and Rossau et al. (1991) (Table 1). PCR was performed in Eppendorf® Mastercycler Gradient (Eppendorf, Germany). PCR products were purified using a Clean-up kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer’s instruction. The sequence data were compared with those from the GenBank database using the BLAST function available on the National Center for Biotechnology Information database. Sequences were aligned using the ClustalW program (Thompson, Higgins & Gibson, 1994). A phylogenetic tree was constructed by the neighbor-joining method (Saitou & Nei, 1987) implemented by MEGA version 6.0 with the uncorrected p-distance model using nucleotide sequences of the 16S rRNA gene (Tamura et al., 2013). To determine the degree of statistical support for branches in the phylogeny, 1,000 bootstrap replicates of data were analyzed. The 16S rRNA gene sequences were deposited in GenBank under accession numbers: MH270335, MH270336, MH270337 for the Aeromonas spp. AC_01, Aeromonas spp. AC_02, and Aeromonas spp. AC_03, respectively.

Culture media and carbon sources

Seed cultures were grown in lysogeny broth (1% w/v tryptone, 0.5% w/v yeast extract, 1% NaCl) at 30 °C with shaking (200 rpm) for 16 h before inoculation. Bacteria were cultivated in three different mineral salt media (MSM): (1) non-limited medium (7 g/l KH₂PO₄; 3.5 g/l Na₂HPO₄·12H₂O; 5 g/l (NH₄)₂SO₄); (2) nitrogen-limited medium (7 g/l KH₂PO₄; 3.5 g/l Na₂HPO₄·12H₂O; 0.5 g/l (NH₄)₂SO₄); (3) phosphorus-limited medium (0.7 g/l KH₂PO₄; 0.35 g/l Na₂HPO₄·12H₂O; 5 g/l (NH₄)₂SO₄). Each medium was supplemented with MgSO₄·7H₂O (1 g/l) and trace element solution (2.5 ml/l). Each liter of trace element solution contained: 20 g FeCl₃·6H₂O, 10 g CaCl₂·H₂O, 0.03 g CuSO₄·5H₂O, 0.05 g MnCl₂·4H₂O, 0.1 g ZnSO₄·7H₂O dissolved in 0.5N HCl. All experiments were conducted using crude glycerol from biodiesel industry and pure glycerol (99% purity; Sigma Aldrich, St. Louis, MO, USA) at the concentration of 1% (w/v) (Kachrimanidou et al., 2014; Phukon et al., 2014). Crude glycerol was derived from non-GMO vegetable oil and was kindly provided by Biofuels S.A. Company (Malbork, Poland). This fraction was composed of 85% glycerol, 7% NaCl, 5.5% water, 2% organic matter-of-non-glycerin, and 0.5% methanol.

Culture conditions for PHA synthesis

The cultivations were carried out in an orbital shaker at 200 rpm, at 30 °C and pH-value adjusted to 7.0. Fermentation shake flasks were inoculated with 5% v/v of the seed and were subsampled for PHA following a one or two step cultivation. One-step PHAs synthesis was conducted in MSM medium for 48 h. In the case of the two-step production, cells were firstly cultivated in lysogeny broth containing glucose (10 g/l) for the first 8 h. Harvested and washed bacterial cells were then transferred into MSM medium supplemented with crude or pure glycerol and incubated for 40 h in the light. All cultivations were conducted in triplicate.

Analytical procedures and PHA extraction

Cell growth was monitored by measuring the absorbance at 595 nm (OD₅₉₅). Bacterial cells were harvested by centrifugation at 11,200×g for 10 min and washed once with ethanol and distilled water. Cell concentration, defined as cell dry weight (CDW) per liter of a culture medium, was determined by weighing lyophilized cells. Lyophilization was achieved by Lyovac GT2 System (SRK Systemtechnik GmbH, Riedstadt, Germany). PHAs were extracted by shaking lyophilized cells in chloroform (purity ≥99.8%; Sigma-Aldrich, St. Louis, MO, USA) for 5 h. The mixture was filtered through No. 1 Whatman filter paper to remove the cellular debris. After mixture filtration, the isolated biopolyesters were precipitated with cold methanol (purity ≥99.8%; Sigma-Aldrich, St. Louis, MO, USA) and then allowed to evaporate at the room temperature.

Gas chromatography analysis

To evaluate the PHAs composition, the lyophilized cells were suspended in acidified methanol containing 3% v/v H₂SO₄ and an equal volume of chloroform (purity ≥99.8%, Sigma-Aldrich, St. Louis, MO, USA). They were then incubated in the oven (Memmert Type SM 400, Memmert, Schwabach, Germany) for 20 h at 100 °C for esterification. The obtained methyl esters were quantified according to the method described by Furrer et al. (2007) using gas chromatography (Varian CP-3800; Varian, Santa Clara, CA, USA) equipped with a Varian VF-5 ms capillary column (30 m × 0.25 mm, film thickness 0.25 μm). Known quantities of pure 3-hydroxyacids (Larodan, Solna, Sweden) were used as standards. The flame ionization detector temperature was 270 °C with the injection port temperature of 250 °C. Initial column temperature was set to 80 °C and next raised with a rate of at 10 °C/min to 240 °C. Total time for analysis of one sample amounted to 16 min.

Phylogenetic analysis

The phylogenetic tree demonstrated that the bacterial isolates could be assigned into the Aeromonas genus (Fig. 1). The data revealed that isolate AC_01 exhibited the highest similarity with Aeromonas hydrophila. Strains AC_02 and AC_03 were determined to be potential members of A. aquatica and A. salmonicida, respectively.

Effects of pure and crude glycerol on growth of the isolates

The main goal of this study was to evaluate if the by-product from biodiesel production—crude glycerol—could be converted into PHAs. The results showed that all strains were able to utilize the pure and crude glycerol (Fig. 2). During the cultivations, an increase in the absorbance of the culture was observed indicating the bacteria grew. In all experimental treatments, the maximum specific growth rates (μ_max) were more than 0.2/h. However, two strains, AC_02 and AC_03, reached two and three times higher μ_max growing on crude glycerol than on pure glycerol under nitrogen and phosphorus limitation (Fig. 2). The results confirmed that by-product from biodiesel industry did not inhibit the growth of the analyzed Aeromonas strains and their growth rate did not depend on applying a macronutrient limitation strategy. CDW of bacteria cultured with crude glycerol was comparable with pure glycerol (Table 2).

In the two-step cultivation procedure, shake-flasks yielded biomass concentrations from 1.7 to 3.06 g/l after 40 h. Lowest biomass was for AC_02 grown on pure glycerol under phosphorus limited conditions and highest biomass was for AC_03 cultivated on crude glycerol under phosphorus deficiency, respectively. As predicted, the CDW value was higher at the end of the two-step cultivations compared with the one-step bioprocess (Table 2).

PHAs biosynthesis and their chemical characterization

Our results confirmed that the adequate supply of nitrogen and phosphorus during shake-flasks cultures of the analyzed strains did not stimulate the synthesis of PHAs. PHAs were not detected in bacterial cells under non-limiting conditions in the all conducted cultivations. Furthermore, during the two-stage cultivation, lysogeny medium was used as a rich nutrition to support cell growth in the first 8 h. As expected, no PHAs were synthesized during this time. It was observed that nutrient limitation is necessary for the synthesis of significant amounts of PHAs in the analyzed Aeromonas strains grown on pure and crude glycerol. Therefore, to estimate the effect of nutrient deficiency on the productivity of PHA synthesis, the one-step and two-step cultivations were conducted under nitrogen and phosphorus limitation. Our findings indicate that using pure and crude glycerol as carbon and energy sources, Aeromonas strains were able to synthesize biopolymers in all culture types and feeding regimes (Table 2). However, the PHA content was higher under a phosphorus-limiting environment, except for AC_03 strain grown on crude glycerol during one-stage cultivation, when three times more biopolymers were reached under nitrogen-limiting conditions. The highest PHA concentration was reached during the two-step bioprocess of Aeromonas spp. AC_03 cultured on pure glycerol (42% of CDW) under phosphorus limitation. The same strain synthesized two times less PHAs growing on crude glycerol at the same culture conditions and nutrient regime, however it was the highest PHA yield obtained in the cultures supplemented with by-product from biodiesel production.

Gas chromatography analysis provided information about the repeat-units composition of the purified biopolymers (Fig. 3). It revealed that generally the PHAs produced by the analyzed strains were polyhydroxybutyrate (P(3HB)) (Table 2). Our study confirmed that besides P(3HB), only Aeromonas spp. AC_02 was able to synthesize poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer P(3HB-co-3HV) under nitrogen limitation in the one-step cultivation supplemented with pure glycerol (Table 2).