1. Introduction
Polyhydroxyalkanoates (PHA) are biodegradable and biobased polymers produced by bacterial fermentation. PHA accumulate intracellularly as water insoluble inclusion bodies that function as carbon and energy storage [
1,
2,
3], The replacement of conventional oil-based plastics by PHA in various segments has been thoroughly discussed due to their similar thermo-physical properties, with PHA offering environmentally friendly advantages, such as biocompatibility, biodegradability and compostability [
1,
4]. PHA production costs, however, are currently higher than those for oil-derived plastics (EUR 1.18–6.12/kg vs. <EUR 1/kg), which limits their potential to niche-market high value applications [
1]. Continuous efforts have been made to reduce PHA production costs by applying mixed microbial cultures (MMC) and waste streams as carbon source for PHA production, as an alternative to pure cultures and the required sterile conditions and expensive substrates. Hence, PHA downstream processing is now the most economically impacting factor in the PHA production chain [
1,
4].
The best established and most commonly used methods for PHA recovery employ halogenated solvents, such as chloroform. Although these methods often generate superior recovery yields and product purity values, they are environmentally harmful, make use of large volumes of noxious solvents and require excessive energy input, making the recovery process unsustainable and economically unfeasible [
4,
5]. Thus, research has been leaning toward the development of more environmentally friendly approaches for the extraction and purification of PHA [
4].
The use of alkaline compounds, such as sodium hydroxide (NaOH), can be a valid, cost-effective and green alternative to chlorinated compounds for the extraction of PHA [
2]. Furthermore, alkaline treatment for PHA recovery has been considered more economically feasible when compared to an organic solvent-based process [
6]. Hydroxides cause saponification of the lipids present in the cell wall of the microorganism, leading to increased membrane permeability and release of proteins and non-PHA cell material [
7].
Mohammadi et al. (2012) [
8] used NaOH to recover PHA (poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), PHBHHx) from lyophilized recombinant
Cupriavidus necator (PHA content of ca. 38.3%), having tested different NaOH concentrations, digestion times and reaction temperatures. The authors obtained over 96% of PHA recovery and purity by treating lyophilized cells with 0.05 M NaOH at 4 °C for 3 h. The fact that this efficient, simple, non-toxic and environmentally friendly treatment resulted in negligible degradation of the polymer molecular weight (Mw) supports the utilization of alkaline-based methods as an alternative to organic solvents for PHA recovery. Jiang et al. (2015) [
3] assessed the feasibility of using NaOH for the extraction and purification of PHA (polyhydroxybutyrate, PHB) from MMC (PHA content of ca. 70%) fed with acetate and compared the results with those obtained from extraction with dichloromethane. These authors verified that the PHA purity in the final product increased when both NaOH concentration and treatment time increased, though PHA recovery decreased in both situations. Applying NaOH at 0.20 M to fresh biomass for 1 h, the authors obtained a PHA purity of 87% and a PHA recovery of 97%. When treatment time was increased to 3 h, PHA purity increased to 92% and PHA recovery was reduced to 94%. The comparison between the results of these alkaline-based methods and the ones obtained with extraction by dichloromethane, 98% of PHA purity and 56% of PHA recovery, suggests that PHA extraction with NaOH might be a viable alternative to extraction with organic solvents.
With the purpose of developing a low-cost extraction method with high PHA recovery to achieve a cost-effective polymer production, Heinrich et al. (2012) [
9] studied a simplified method for PHA extraction at large scale from cells of
Ralstonia eutropha H16 (now reclassified as
Cupriavidus necator), cultivated with a synthetic carbon/magnesium solution, using sodium hypochlorite (NaClO, 13%,
v/v). NaClO is a strong oxidizing chemical that dissolves non-PHA cellular mass (NPCM), while PHA granules remain in the solid form [
10]. Very high purities have been reported for PHA extraction with NaClO, but as PHA is not completely insoluble in NaClO, the often-associated decrease in polymer molecular weight after extraction has caused concern when dealing with this chemical [
9,
10,
11,
12]. In the study by Heinrich et al. (2012) [
9], PHA with an average purity of 93% was extracted with a maximum recovery of 87% when the largest extraction volume tested, 50 L, was used. Nevertheless, treatment with NaClO led to a 50–70% decrease in the polymer molecular weight and higher dispersity of the polymer. Villano et al. (2014) [
11] recovered PHA (poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV) from fresh MMC, produced using a synthetic mixture of acetic and propionic acids, by operating an extraction reactor using two digestion agents: NaOH (1 M) and NaClO (5% active Cl
2). In the study by Villano et al. (2014) [
11], PHA extraction displayed greater performance when NaClO was used for the digestion of NPCM, as opposed to NaOH, both in terms of polymer recovery (ca. 100%,
w/w) and purity (>90%,
w/w). The results showed, however, a wide polymer molecular weight distribution, which might be detrimental to the application of the process. Furthermore, treatments using NaClO may be hazardous and not viable for large-scale application due to the risk of formation of toxic halogenated compounds [
5].
Until now, most PHA extraction studies have used either pure cultures or MMC produced with synthetic substrates [
1]. The present study focused on the optimization of PHA (PHBV) extraction from a MMC (ca. 70% PHA) produced at pilot scale with fruit pulp waste by NPCM digestion with either NaOH or NaClO. Optimization was performed using lyophilized biomass at a set concentration (20 g/L) through a central composite rotatable design (CCRD), a response surface methodology (RSM) design that efficiently seeks the optimum conditions for a multivariate system. RSM involves performing statistically designed experiments, coefficients estimation in mathematical models and response prediction and model accuracy testing [
13]. Central composite design, in particular, has been often used for the optimization of conditions from various processes, including chemical and biochemical reactions [
14]. In the current study, CCRD was used to determine different combinations of reagent concentration and digestion time to be tested and also to predict the conditions that maximize both PHA purity and recovery. This study also assessed the impact of intracellular PHA content, biomass pre-treatment and biomass concentration on PHA extraction performance and polymer characteristics, including molecular weight, thermal properties and infrared spectra.
2. Materials and Methods
2.1. Biomass Samples
Four different biomass samples containing the polymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were used in the PHA extraction trials, and all were produced in a three-stage process pilot unit plant operated at room temperature. This unit consisted of an acidogenic fermentation stage, performed in an upflow anaerobic sludge blanket (UASB) reactor with a working volume of 60 L; a culture selection stage, performed in a 100 L sequencing batch reactor (SBR) operating under a feast and famine regime; and a PHA accumulation stage, performed in a 60 L fed-batch reactor. The UASB reactor, the SBR and the fed-batch reactor were designed inhouse. Fruit pulp waste was used as feedstock for this production.
The PHA accumulation process was performed with biomass purged from the culture selection SBR and a volatile fatty acid (VFA)-rich stream derived from the UASB. The accumulation reactor worked in pulse feeding mode under nutrient limitation. After accumulation, the different PHA-enriched biomasses were acidified with sulfuric acid (Sigma-Aldrich, Sigma-Aldrich, Burlington, MA, USA), centrifuged and stored at 4 °C before lyophilization.
Biomass A was collected at the end of a PHA accumulation process and had an intracellular PHA content of ca. 70% (31% of 3-hydroxyvalerate, 3HV). This sample was used for the PHA extraction optimization tests and to study the impact of the biomass concentration on the PHA extraction performance. Biomasses B and C were collected at distinct times of a second PHA accumulation process, namely after the first and second feed pulses, and had PHA contents of ca. 35–41% (18% 3HV) and ca. 44–52% (18% 3HV), respectively. Biomass D was collected at the end of a third PHA accumulation and presented a PHA content of ca. 73% (21% 3HV). Biomasses B, C and D were used to study the effect of the intracellular PHA content on PHA extraction. Additionally, to study the effect of biomass pre-treatment on PHA extraction, trials were performed using biomass C, not only lyophilized but also in its fresh state (i.e., only subjected to acidification and centrifugation) and in its dry state (i.e., subjected to acidification, centrifugation and drying at 60 °C for 3.5 days).
2.2. PHA Extraction
For the determination of the optimal conditions for PHA extraction from lyophilized PHA-enriched biomass using NaOH (97%, Sigma-Aldrich, Burlington, MA, USA) or NaClO (10–15% active Cl
2, Acros Organic, Geel, Belgium), biomass was mixed at 20 g/L in 50 mL tubes with 20 mL of NaOH or NaClO solutions, according to the conditions determined by the experimental design (see
Section 2.4), and incubated at 200 rpm and 30 °C. After incubation with NaOH, the suspension was centrifuged at 9300×
g for 10 min at 20 °C, the resulting pellet was washed with 40 mL of water and then frozen and lyophilized for 48 h. Regarding digestion with NaClO, 20 mL of water were added to the suspension after incubation and before the first centrifugation to enhance the solid–liquid separation. Afterward, the processing of the pellet was the same as previously described for suspensions, including NaOH. For comparison purposes, conventional PHA extraction with chloroform was performed. In this method, 10 g of lyophilized biomass were subjected to Soxhlet extraction with chloroform (99.5% Sigma-Aldrich, Burlington, MA, USA) at 80 °C over 48 h, followed by precipitation in ice-cold ethanol (EtOH; 1:10
v/v, Fisher Chemical, Waltham, MA, USA), as described by Pereira et al. (2019) [
15].
To assess the effect of biomass pre-treatment on PHA extraction performance, lyophilized, fresh and dry biomasses were mixed at 20 g/L in 50 mL tubes with 20 mL of NaOH or NaClO solutions, using the respective determined optimal conditions, and incubated at 200 rpm and 30 °C. The same experimental conditions were applied to lyophilized biomass with different intracellular PHA contents (biomasses B, C and D) and to lyophilized biomass with the same PHA content (biomass A) but at biomass concentrations of 20, 40, 60, 80 and 100 g/L to analyze the effect of intracellular PHA content and biomass concentration on PHA extraction performance. The processing of the resulting extraction products for both studies followed as described in the first paragraph of the present section.
2.3. PHA Content and Composition
PHA content and its composition in biomass and in extraction products were determined by gas chromatography (GC, Trace 1300, Thermo Scientific, Waltham, MA, USA), following a methanolysis method based on the one described by Cruz et al. (2016) [
16]. Summarily, the lyophilized samples (1 to 2 mg) were hydrolyzed with 1 mL of 20% (
v/v) sulfuric acid (Sigma-Aldrich, HPLC grade, Burlington, MA, USA) in methanol (Fisher Chemical, HPLC grade, Waltham, MA USA) and 1 mL of chloroform (Sigma-Aldrich, HPLC grade, Burlington, MA, USA), including heptadecanoate (HD) at 1 g/L, to function as internal standard. The reaction occurred at 100 °C over 3.5 h. The obtained methyl esters were analyzed in a Restek column (Crossbond, Stabilwax, Bellefonte, PA, USA) at a constant pressure of 14.50 Psi, using helium (Air Liquid, Paris, France) as the carrier gas. The oven temperature program was set as follows: 20 °C/min until 100 °C; 3 °C/min until 155 °C; 20 °C/min until 220 °C.
Commercial PHBV (Sigma-Aldrich, 88 mol% 3HB, 12 mol% 3HV, Burlington, MA, USA) was used as standard for the construction of calibration curves that allowed the determination of the mass of both 3HB and 3HV in each sample. The PHA content values were obtained by dividing the mass of PHA (mass of 3HB + mass of 3HV) in each sample by the mass of lyophilized total solids (TS) used for GC analysis, according to Equation (1):
To calculate the PHA recovery yield according to Equation (2), it was necessary to determine the PHA mass of both the final extraction product and of the biomass from which the extraction product was obtained (PHA
final and PHA
initial, respectively).
2.4. Experimental Design and Statistical Validation
RSM was used to assess the optimal conditions for PHA extraction using digestion with either NaOH or NaClO. CCRD was performed to analyze the impact and interaction between the experimental variables (X
i), reagent concentration (M for NaOH and % for NaClO) and digestion time (h), and the observed responses, PHA purity (%) and PHA recovery (%). The design applied consisted of nine experiments performed randomly: four factorial design points at levels ±1; four experiments of axial level α = ±1.414; and a central point with three replicates. The experimental values for the two independent variables were established according to the literature [
3,
6,
8,
9,
11,
12,
17,
18,
19,
20,
21]. The experimental tests for optimization of PHA extraction with NaOH and NaClO were performed as described in
Table 1.
The experimental data were fitted to the second-order model presented in Equation (3) to evaluate the system’s behavior.
In Equation (3), Yp corresponds to the predicted responses, and X1 and X2 are the coded values of the independent variables, namely reagent concentration and digestion time. b0, bi, bj, bij (i, j = 1, 2) are the coefficient estimates, b0 being the interception, b1 and b2 the linear terms, b11 and b22 the quadratic terms, and the b12 the interaction term. A statistical analysis was performed to evaluate the significance of each source of variation and select an appropriate quadratic model.
Analysis of variance (ANOVA) was used to assess the fit of each model, which was considered an accurate prediction tool when it met the following criteria: a good correlation value (R
2 > 0.7, acceptable for biological samples [
22]) with statistical meaning (
p-value < 0.05, for a 95% confidence level) and with no lack of fit (
p-value > 0.05, for 95% confidence level) [
23]. Statistics and surface plots analysis provided information on the effect of reagent (NaOH or NaClO) concentration and digestion time on PHA purity and PHA recovery.
2.5. PHA Infrared Spectra
Fourier transform infrared (FTIR) spectra of the extracted PHA were collected between 400 and 4000 cm−1, at room temperature, using a Cary 630 FTIR spectrometer (Agilent Technologies, Santa Clara, CA, USA) with a thermoelectrically cooled dTGS detector and KBr standard beam splitter and equipped with a diamond attenuated total reflectance (ATR) accessory. All spectra were recorded via the ATR method, with a resolution of 1 cm−1 and 16 scans.
2.6. PHA Molecular Mass Distribution
The weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity index (Mw/Mn; PDI) of the extracted PHA were determined by size exclusion chromatography (SEC). For this analysis, 15 mg of each sample was first dissolved in 3 mL of chloroform at room temperature for 18 h. Then, the resultant solutions were filtered with glass fiber filters 47 mm (PALL, Port Washington, NY, USA) and analyzed by a Waters SEC system (Milford, MA, USA), with support SEC: PLgel 5 µm Guard, 50 × 7.5 mm; PLgel 5 µm 104 Å, 300 × 7.5 mm; PLgel 5 µm 500 Å, 300 × 7.5 mm (Polymer Laboratories, Church Stretton, UK). A temperature of equilibration of 30 °C was used, along with a flow rate of 1 mL/min, with degassing, and chloroform as the mobile phase. An amount of 100 µL of each sample was injected in the SEC circuit. The refractive index detector Waters 2410 was used for polymer detection, using the sensitivity 512 and a collect duration of 25 min. Relative molecular weights of the polymers were determined according to the universal calibration method adopting polystyrene standards with molecular weights between 800 Da and 504.5 kDa, and using Waters Millenium SEC software (Milford, MA, USA).
2.7. PHA Thermal Properties
Differential scanning calorimetry (DSC) analysis was used to probe the thermal properties of the extracted PHA. This analysis was performed using a differential scanning calorimeter DSC Q2000 (TA Instruments, New Castle, DE, USA). Each sample (approximately 4 mg) was placed in a sealed aluminum pan, perforated to allow water/solvents release. Thermograms were collected in a range of temperatures between −90 °C and 160 °C, with heating and cooling steps of 10 °C/min under a nitrogen atmosphere. Two cooling/heating cycles were performed for each sample. The glass transition temperature (Tg, °C) and the melting temperature (Tm, °C) were determined as the midpoint of the heat flux step and at the minimum of the endothermic peak, respectively, of the second heating run, due to water or solvent evaporation occurring during the first heating.
4. Discussion
In the present study, when the optimal conditions for digestion with NaOH (0.3 M, 4.8 h) were applied to lyophilized biomass (biomass A, PHA content of ca. 70%) produced at pilot scale using fermented fruit pulp as substrate, a PHA purity of 101.7 ± 7.6% and a PHA recovery of 102.9 ± 7.9% were obtained. These values are similar to the ones reported by Jiang et al. (2015) [
3], where lyophilized biomass with an intracellular PHA content of ca. 70%, produced with acetate as substrate, was digested using NaOH at 0.2 M for 1 h, resulting in the recovery of 95.5 ± 0.6% of the polymer with a purity of 95.9 ± 3.7%. However, in the current study, a higher NaOH concentration and a longer digestion time were necessary to obtain PHA purity and PHA recovery values similar to the ones reported by Jiang et al. (2015) [
3], possibly due to the fact that a real substrate with a more complex matrix was used for biomass production in the case at study. Furthermore, the results of the aforementioned authors suggest that sole NaOH treatment was unable to remove all NPCM from fresh biomass, the remaining impurities requiring the combined action of NaOH and sodium dodecyl sulfate (SDS) to be efficiently removed. In the present study, when testing the effect of different biomass pre-treatments on PHA extraction performance (
Figure 6), using biomass with a lower intracellular PHA content (ca. 44%, biomass C), it was also observed that NaOH digestion was less efficient when processing fresh biomass when compared to lyophilized and dry biomass. Hence, one possible approach for future work might be the addition of SDS to NaOH digestion for the extraction of PHA from fresh biomass.
In the present study, the application of the found optimal conditions for NaOH digestion (0.3 M, 4.8 h) to lyophilized biomass with an intracellular PHA content of ca. 41% (biomass B) (
Figure 5) resulted in an extraction product with a PHA purity of about 71% and a PHA recovery of ca. 99%. Mohammadi et al. (2012) [
8] performed experiments of PHA (PHBHHx) extraction from lyophilized recombinant
C. necator with a PHA content of ca. 38.3% at 4 °C and 30 °C. At 30 °C, the temperature used in the present study, the highest values of PHA purity and PHA recovery were ca. 95% and ca. 97%, respectively, obtained when biomass was subjected to digestion with NaOH at 0.1 M for 5 h. The lower PHA purity of the final product in the current study may be associated with the fact that MMC are reportedly more resistant to cell hydrolysis than pure cultures [
34].
In this study, using the optimal NaOH digestion conditions for the extraction of PHA from fresh biomass C with a PHA content of ca. 44% (
Figure 6) resulted in a PHA recovery and a PHA purity of around 88% and 57%, respectively. Villano et al. (2014) [
11] performed PHA extraction trials at room temperature using fresh biomass with an average PHA (PHBV) content of 46%, produced with a synthetic mixture of acetic and propionic acids. When subjecting the fresh biomass to NaOH at 1 M (the ratio between the volume of biomass and the chemical solution being 6:1) for 3 h, these authors reported a PHA recovery and a PHA purity of around 87% and 54%, respectively. Using a digestion time of 24 h, a PHA recovery and a PHA purity of about 80% and 56%, respectively, were obtained. These values are similar to the ones obtained in the present study. Regarding digestion with NaClO at 5% Cl
2 (5.25% NaClO; the ratio between the volume of biomass and the chemical solution being 6:1), the aforementioned authors reported complete recovery of PHA after 3 h and 24 h of digestion. Furthermore, PHA purities of ca. 90% and ca. 98% were obtained after 3 h and 24 h of digestion, respectively. In the present study, subjecting fresh biomass with a PHA content of ca. 44% (biomass C) to the optimal conditions for NaClO digestion (9.0% NaClO, 3.4 h) resulted in a PHA recovery of around 92% and a final product with a PHA purity of ca. 83% (
Figure 6). These values are lower than the ones obtained by Villano et al. (2014) [
11], which may be due to the fact that the biomass in the current study was produced using real waste as substrate, namely fermented fruit pulp with a complex matrix, while in the former, a synthetic mixture of acetic and propionic acids was used. The use of real waste as substrate may result in the presence of impurities that are harder to remove. On the other hand, the conditions used for PHA extraction from fresh biomass C (PHA content of ca. 44%) with NaClO in the present study were determined using lyophilized biomass with a PHA content of ca. 70%, biomass A (
Section 3.2). Thus, it is possible that applying the CCRD methodology to NaClO digestion for PHA extraction from fresh biomass C would determine a different set of optimal conditions that would result in increased PHA purity and PHA recovery. The same possibility can be posed for PHA extraction by NaOH digestion.
Considering Mw results, the polymer obtained from NaClO digestion (9.0%, 3.4 h) of lyophilized biomass with an intracellular PHA content of ca. 70% (sample II in
Table 7) presented an average Mw of 2.45 × 10
5 Da and PDI of 2.20. In the study by Villano et al. (2014) [
11], digestion using NaClO, in the conditions mentioned above resulted in a polymer with a Mw range between 3.4 × 10
5 and 5.4 × 10
5 Da, and a PDI between 4 and 10. It is possible that the considerable difference between the PDI values of the two studies is due to the process conditions, since in the present study, the polymer was extracted from lyophilized biomass with an intracellular PHA content of ca. 70%, while in the study by Villano et al. (2014) [
11], the polymer was extracted from fresh biomass containing ca. 46% of PHA.
Digestion of lyophilized MMC (biomass A, PHA content of ca. 70%) with NaClO, using the optimal conditions (9.0% NaClO, 3.4 h), resulted in a PHA recovery of ca. 90% and a PHA purity of around 99% (
Figure 4). Heinrich et al. (2012) [
9] extracted PHA (PHB) from lyophilized
R. eutropha H16 (intracellular PHA content of ca. 65.2%) by subjecting it to digestion with NaClO at 13% (
v/v) for 1 h at room temperature. In that study, PHA extraction was performed at 0.1 L and 50 L scales. At the scale of 0.1 L, a PHA purity of ca. 95.7% and a PHA recovery of about 91.3% were obtained, whereas at the scale of 50 L, an average of about 87% of the existing polymer was recovered, the final product presenting an average PHA purity of ca. 93%. The reported PHA purity and PHA recovery values using a pure culture are similar to the ones obtained in the current study with an MMC. Nevertheless, in the case at study, PHA extraction by NaClO resulted in a Mw reduction of about 7% when compared to the polymer recovered by chloroform (
Table 7), a value notably lower than the one observed by Heinrich et al. (2012) [
9], which ranged between 50% and 70%, similarly to previous studies [
18,
19].
Regarding the effect of biomass concentration on PHA extraction, the results in
Figure 7 are in accordance with those obtained by Berger et al. (1989) [
19] and Choi and Lee (1999) [
6], who observed a decrease in polymer purity when cell concentration was increased in PHA extraction by NaClO and NaOH digestions, respectively. Additionally, Heinrich et al. (2012) [
9] also observed that in PHA extraction by digestion with NaClO at 13% (
v/v), a biomass concentration higher than 30 g/L led to the saturation of the NaClO solution.
Regarding the effect of biomass pre-treatment on PHA extraction,
Figure 6 shows that a higher PHA purity was obtained when NaOH digestion was applied to extract PHA from biomass C (intracellular PHA content of ca. 44%) that had been previously dried at 60 °C for 3.5 days, rather than fresh biomass C (ca. 77% of PHA purity in the former vs. ca. 57% in the latter). However, Mw results in
Table 7 show that drying biomass C prior to NaOH digestion (sample IV in
Table 7) resulted in a major decrease in Mw and increase in PDI when compared to the application of the same process to fresh biomass C (sample III in
Table 7). Sample III presented a Mw of 2.81 × 10
5 Da and a PDI of 2.23, whereas sample IV displayed a Mw of 0.87 × 10
5 Da and a PDI of 7.12. Lorini et al. (2021) [
35] also compared the Mw of polymers extracted from dried and fresh biomass and reported a 3-fold lower Mw for the one extracted from dried biomass. In the present study, it is possible that the drying process resulted in the physical association of the polymer in sample IV with low reversibility upon dissolution. This would justify the higher crystallinity of this sample when compared to sample III, as displayed in
Figure 9b, and would explain the decrease in Mw. Considering these results, it is possible to conclude that higher PHA extraction performance does not guarantee superior polymer quality.
DSC and FTIR results presented in
Figure 8 and
Figure 9b, respectively, suggest that the PHA extraction process influences the polymers’ crystallinity. These results are in agreement with the results obtained by Jiang et al. (2015) [
3], which show that chemical treatment and biomass pre-treatment have an impact on the polymer’s crystallinity.
5. Conclusions
PHA extraction conditions should be adjusted to the properties of each biomass that is to be processed. In the current study, NaOH and NaClO were tested as green alternatives to organic solvents for the extraction of PHA from MMC biomass produced at pilot scale with fruit pulp waste, and the optimal chemical digestion conditions were determined by a design of experiments methodology. This methodology can be transposed to determine the optimal conditions for PHA recovery from other types of biomasses, subjected to different or no pre-treatments, and with different intracellular PHA contents.
High PHA extraction performances were obtained using the optimal digestion conditions for either NaOH (0.3 M, 4.8 h) or NaClO (9.0%, 3.4 h). However, treatments using NaClO present the risk of formation of toxic halogenated compounds, hindering their application at a large scale. On the other hand, sole digestion with NaOH proved to be an efficient green alternative to chlorinated compounds.
NaClO was similarly efficient for PHA extraction from dry, lyophilized and fresh biomass, though displaying a slightly lower performance for dry biomass, while for NaOH, the greatest efficiency was attained for dry biomass and the lowest for fresh biomass. Digestion of lyophilized biomass with either NaOH or NaClO resulted in negligible loss of polymer molecular weight. However, biomass drying at 60 °C before PHA extraction resulted in a polymer with a broad molecular weight distribution and a decreased mean molecular weight. Furthermore, polymer characterization by DSC and FTIR suggested that its crystallinity is influenced by the applied extraction method.
Intracellular PHA content and biomass concentration were found to strongly influence the PHA extraction performance, irrespective of the digestion agent used. The higher the intracellular PHA content, the higher the PHA purity in the extraction products. On the other hand, when biomass concentration was increased above 20 g/L, the PHA purity of the extraction products tended to decrease. Thus, a compromise between the efficiency of polymer recovery/purity and the process productivity (related to the volume of biomass to be processed) should be considered according to the final polymer application.