Bacterial production of polyhydroxyalkanoates (PHAs) using various waste carbon sources

Microorganisms and chemicals

Four bacterial strains Bacillus subtilis (accession number KM282517), Bacillus cereus (accession number KM282518), Pseudomonas aeruginosa (Maqbool et al., 2016), Alcaligenes sp. used in this study were collected from Industrial Biotechnology Laboratory Government College University Faisalabad, Pakistan. These strains were reactivated and fresh cultures were prepared on nutrient agar plates. The inoculum was prepared by adding the bacterial cells to the nutrient broth. This inoculum was used for further studies. All the chemicals used in this study were of analytical grade, purchased from RDH (Germany), CarlRoth Gmbh (Germany), Sigma-Aldrich (USA), and Europroxima (The Netherlands).

Bacterial screening for PHA production

Sudan Black-B staining was used for the screening of bacterial strains to confirm the production of PHA. Alcoholic Sudan Black-B (0.3%) was used for the bacterial staining, with some modifications as described by Mohanrasu et al. (2020). Two methods were used for the bacterial screening with Sudan Black-B. In the first method, the agar plates with the PHA accumulating strains and control strains were flooded with the prepared Sudan Black B solution for 30–45 min (Alshehrei, 2019). The second method was the smear slide method, in which the slides were observed under a microscope after staining with alcoholic Sudan Black-B solution (Muneer et al., 2022).

Carbon sources

This study used rice bran and sugarcane molasses as cost-effective carbon sources. Rice bran was collected from the local rice milling point at Sheikhupura Road, Faisalabad, Pakistan. The sugarcane molasses was arranged from the Tandlianwala and Kanjwani Sugar Mills Limited, Faisalabad, Pakistan. Raw sugarcane molasses contains variable content of different sugars like glucose, fructose, and sucrose, which aid in PHA production. Some inhibitory metal ions are also found in cane molasses, which are unsuitable for PHA production; therefore, they were pretreated before its use.

Pre-treatment of sugarcane molasses

Raw sugarcane molasses contains variable content of various carbohydrates like glucose, fructose, and sucrose, which aid in PHA production. Some inhibitory metal ions are also found in cane molasses, which are unsuitable for PHA production. Deionized distilled water was added to the concentrated sugarcane molasses to set the required sugar content. Activated charcoal was added to the diluted molasses, stirred for 1–2 h using a magnetic stirrer, and centrifuged at 6,000 rpm for 10 min. The pH of the filtered supernatant was set at 7–7.5 and autoclaved (Jo et al., 2021).

PHA production media & culture conditions

The selected bacterial strains were cultured in shaking flasks containing minimal solution medium (MSM). Before inoculating into MSM, these bacterial strains were cultured in the nutrient broth (NB) medium containing: Peptone; 5 g/L, Yeast extract; 1.5 g/L, Beef extract; 1.5 g/L, NaCl; 5 g/L, glucose; 10 g/L, pH; 7, incubated at 37 °C, 150 rpm for 24 h. 10ml of each inoculum was added in autoclaved MSM. The composition of MSM was derived with some modifications by (Gomaa, 2014). MSM consisted of different salts in the content ratio of (g/L): Na₂HPO₄.2H₂O; 2.0, NaHCO₃; 0.5, MgSO₄.7H₂O; 0.5, NH₄Cl; 1.0, CaCl₂.2H₂O; 0.01, KH₂PO₄; 2.0, and some trace elements including H₃BO₃; 0.3, ZnSO₄.7H₂O; 0.08, CoCl₂.6H₂O; 0.2, NiCl₂.6H₂O; 0.02, MnCl₂.4H₂O; 0.03, CuCl₂.2H₂O; 0.01. Five different concentrations of rice bran (0.5%, 1.0%, 1.5%, 2.0%, and 2.5%) and pretreated sugarcane molasses (2.0%, 4.0%, 6.0%, 8.0%, and 10.0%) were added to the MSM medium in different flasks as a carbon source for PHA production. Three replicates were used during the experiment using each carbon source and for their various concentrations. The data obtained is given in Tables 1 and 2.

Extraction and purification of PHA

PHA extraction was done using the previously reported methodology with some modifications (Mohandas et al., 2018; Aljuraifani, Berekaa & Ghazwani, 2019b). After the incubation period, the cell culture was collected and harvested. The cells of the culture containing rice bran and cane molasses were harvested differently. The biomass of the cell culture containing the rice bran was collected. An equal volume of sodium hypochlorite and chloroform was added to the biomass and incubated at 37 °C for 1 h. After incubation, the mixture was at 8,000 rpm for 15 min, discarding the upper phase containing cellular digestions. The lower chloroform phase was collected using a sterile syringe to collect biopolymers by adding 10 volumes of ice-cold methanol. Then, the precipitated PHA was dried at 65 °C for 24 h. The biomass of the sugarcane molasses cell culture was collected, washed with distilled water, and lyophilized by adding 40% sodium hypochlorite, vortexed vigorously, and incubated at 37 °C for 1 h. After incubation, centrifuged the mixture at 8,000 rpm for 15 min, discarded the supernatant, washed twice with distilled water, and then splashed the pellet with acetone and methanol. Centrifuged at 10,000 rpm for 5 min, suspended the pellet in boiling chloroform, and dried at 65 °C for 24 h. The formula in Eq. (1) is used to calculate the PHA content of the extracted biopolymer. (1)${\displaystyle \mathrm{PHA}\left(\text{\%},\mathrm{w}/\mathrm{w}\right)=\frac{\text{Weight of PHA}\left(\mathrm{g}/\mathrm{L}\right)}{\mathrm{CDM}\left(\mathrm{g}/\mathrm{L}\right)}\times 100.}$

Optimization of the process parameters

To optimize the different parameters for PHA hyperproduction, other culture conditions such as concentration of carbon source, incubation period, pH conditions, and temperature were selected, which helped to determine the optimum conditions for PHA production by B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa respectively. The carbon sources of different concentrations were used to optimize the production of PHA. In the case of rice bran as a carbon source, five concentrations of rice bran 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% were added in five separate Erlenmeyer flasks containing MSM media. In the case of sugarcane molasses as a carbon source, five concentrations of pretreated sugarcane molasses 2.0%, 4.0%, 6.0%, 8.0%, and 10.0% were added in five separate Erlenmeyer flasks containing MSM media. Alcaligenes sp. and P. aeruginosa were incubated for 120 h, and B. subtilis and B. cereus were incubated for 96 h in the shaking incubator. Cell dry mass (CDM) and PHA yield were determined at 24, 48, 72, 96, and 120 h along with the percentage yield of PHA derived from bacterial strains with both carbon sources. The pH of the bacterial growth medium (MSM) was optimized to determine the effect of pH on PHA production. The pH of the medium was varied from pH 4.0−9.0 for each bacterial strain by adding NaOH & HCl.

Analytical characterization

Fourier transform infrared spectrophotometry (FTIR) was used to analyze the chemical structure of the extracted polyhydroxyalkanoates produced from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa in the form of granular powder (Morya et al., 2021). FTIR analysis was done by dissolving the PHA powder in the chloroform and KBr (potassium bromide) pellet. The spectra were recorded in the 600 to 4,000 cm⁻¹ range using FTIR spectrophotometer. The extracted PHA samples were further thermally characterized by a differential scanning calorimeter (TA instruments Trios V4.1) to determine the extracted PHA’s melting temperature (Tm). The heating temperature was adjusted from 25−350 °C, to check the melting temperature (T_m) of the extracted samples under the nitrogen atmosphere at the heating rate of 10 °C per minute.

Statistical analysis

These experiments were performed in triplicate and the obtained data was statistically analyzed with one-way ANOVA. The p-values were calculated. Significant, and non-significant values were noted.

Bacterial screening for PHA production

B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa strains were grown on freshly prepared nutrient agar plates from the stock cultures. All the strains showed good growth (Fig. 1A) on the plates and were found satisfactory for further study. These bacterial strains were stained with Sudan Black-B. The results (Fig. 1B) indicated the production of PHA in all four bacterial strains (Shah, 2014). Under the compound microscope, the black granules (Fig. 1C) represented the Sudan Black-B positive isolates for PHA production.

Extraction and purification of PHA biopolymer

PHA polymer was extracted by using sodium hypochlorite and chloroform digestion from cellular biomass (Fig. 2). PHA production from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa was analyzed after different time intervals, with rice bran and sugarcane molasses as carbon source respectively. Under optimized conditions of rice bran, P. aeruginosa effectively utilized 0.5% rice bran and produced the maximum PHA yield, which was 93.7%, 3.2 g/L CDM, 3.0 g/L PHA content, after 72 h at 37 °C. According to previously reported studies, Pseudomonas sp. P (16) produced a 90.9% PHA yield (Aljuraifani, Berekaa & Ghazwani, 2019b), which has little difference from the research results presented in PHA production yield obtained from Alcaligenes sp. was 87.1%, 3.1 g/L CDM, and 2.7 g/L PHA content with 0.5% rice bran, after 72 h at 37 °C. PHA production yield obtained from B. subtilis was 69%, 2.9 g/L CDM, and 2.0 g/L PHA content with 0.5% rice bran after 24 h at 37 °C. PHA production yield obtained from B. cereus was 35.5%, 4.5 g/L CDM, and 1.6 g/L PHA content with 0.5% rice bran after 24 h of incubation at 37 °C. The maximum CDM and PHA content was obtained from P. aeruginosa after 72 h of incubation with 2 and 1.5% rice bran, respectively, 21.7 g/L and 3.8 g/L.

Under optimized conditions of sugarcane molasses, P. aeruginosa produced the maximum PHA yield of 95%, 1.9 g/L PHA content, and 2.0 g/L CDM in the presence of 2% sugarcane molasses after 96 h, at 37 °C. Ali & Jamil (2017) reported a 45.74% production yield of PHA, with 1.40 g/L CDM in the presence of glucose as the carbon source, which is less than the presented results in PHA production yield obtained from Alcaligenes sp. was 90.9%, 1.0 g/L PHA content and 1.1 g/L CDM with 8% cane molasses, after 72 h at 37 °C. PHA production yield obtained from B. subtilis was 91.6%, 1.1 g/L PHA content, and 1.2 g/L CDM with 2% cane molasses after 24 h at 37 °C. PHA production yield obtained from B. cereus was 80%, 1.2 g/L PHA content, and 1.5 g/L CDM with 2% cane molasses after 24 h at 37 °C. The maximum CDM and PHA content was obtained from P. aeruginosa after 96 h with 10% cane molasses, respectively, 5.9 g/L and 4.3 g/L. To find the optimum pH, the PHA production was studied from pH 4–9 (Figs. 3 and 4). PHA production was maximum at pH 8 with sugarcane molasses; in the case of rice bran, maximum PHA was produced at pH 9. Hence, Alcaligenes sp. effectively utilized the cane molasses for PHA production.

FTIR spectroscopy

The functional groups of the extracted PHA from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa were analyzed by the FTIR spectrophotometer. Figure 5 indicated the presence of functional groups, which represented the more intense absorption band at 1,038.65 cm⁻¹, 1,056.51 cm⁻¹, 1,063.49 cm⁻¹ and 1,064.46 cm⁻¹ respectively, that described the -C-O- stretch of the ester group present in the structure of PHA. This absorption spectrum is similar to the previous studies in which the absorption band at 1,046 cm⁻¹, 1,053 cm⁻¹, and 1,043 cm⁻¹ indicates the presence of C-O carbonyl groups (Mohapatra et al., 2017; Liu et al., 2021; Tyagi & Sharma, 2021). Other absorbance peaks of specific characteristics are shown between the 1,500–2,000 (C =O stretch), 2,000–2,500 (-C ≡C- stretch), 2,500–3,000 (C-H stretch), 3,000-3,500 (O-H group), 600–1,000 (C-C stretch) which are similar to previous reports (Maaloul et al., 2013; Abid, Raza & Hussain, 2016). From the prior review of the literature and in the comparison to standard PHA, it can be concluded that the presence of these functional groups indicated the presence of poly-3-hydroxybutyrate (Ojha & Das, 2018; Evangeline & Sridharan, 2019). Hence the polymer extracted from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa was P(3HB), i.e., poly-3-hydroxybutyrate.

Differential scanning calorimetry (DSC) analysis

The PHA extracted from B. subtilis, B. cereus, Alcaligenes sp., and P. aeruginosa was thermally characterized by differential scanning calorimetry (DSC) to determine its melting temperature (T_m). DSC profiles of all four samples are shown in Fig. 6. PHA produced from Bacillus subtilis showed a sharp endothermic peak in the range of 160−170 °C, without significant change of overall heat absorption that represented its melting temperature (T_m). This data supported the previous reports (Jung et al., 2020; Lorini et al., 2021). In the case of B. cereus and Alcaligenes sp., no sharp peaks were observed, and the mild melting temperature curves were observed in the range of 130−150 °C and 230−240 °C. Recrystallization, crystal modification, and re-melting of thick crystals produce double endothermic peaks. This is similar to previous studies in which B. megaterium ATCC 14945 showed endothermic peaks at 133.15 and 145.42 °C (Vu et al., 2021; Tian et al., 2021). PHA produced from P. aeruginosa showed a sharp endothermic peak between the 230−240 °C range. Hence, the PHA produced during this study is thermally more stable at relatively high temperatures because melting temperature is the most important factor to describe the product quality and applications (Xu et al., 2021; Karagöz, 2021). The plastic for daily household applications, engineering, and high-temperature applications has a melting temperature of 100−300 °C, consistent with the PHA produced during this study and can be replaced with synthetic plastic (Feldmann, 2016).