Optimizing Polyhydroxyalkanoate production using a novel Bacillus paranthracis isolate: A response surface methodology approach

Abstract

Microorganisms have emerged as promising resources for producing economical and sustainable bioproducts like Polyhydroxyalkanoate (PHA), a biodegradable polymer that can replace synthetic plastics. In this study, we screened a novel isolate, Bacillus paranthracis RSKS-3 strain, to produce PHA from sewage water, identifying it using Whole Genome Sequence. This study represents the first report on optimizing PHA production using B. paranthracis RSKS-3, employing Design Expert 12.0 software. Our findings reveal that four factors (temperature, inoculum size, potassium dihydrogen phosphate, and magnesium sulfate) significantly affect PHA production in the Plackett-Burman design experiment. Through Response Surface Methodology, we optimized PHA production to 0.647 g/L with specific values for potassium dihydrogen phosphate (0.55 %), inoculum size (3 %), magnesium sulfate (0.055 %), and a temperature of 35 °C, in agreement with the predicted value of 0.630 g/L. This optimization resulted in a substantial 13.29-fold increase in PHA production from 0.34 g/L to 4.52 g/L, underscoring the promising role of B. paranthracis RSKS-3 in eco-friendly PHA production and advancing sustainable bioproduct development.

Keywords: Bacillus paranthracis RSKS-3; Design expert 12.0; Optimization; Plackett-burman design; Polyhydroxyalkanoate (PHA); Response surface methodology; Sustainable development; Whole genome sequence.

Fig. 1

Phylogenetic analysis based on 16s rRNA of RSKS-3 (B. paranthracis RSKS-3) having SAMN39897631 accession number in NCBI database.

Fig. 2

Pareto chart of standardized effect of fermentative parameters for PHA produced by B. paranthracis RSKS-3. Four factors (KH₂PO₄, inoculum size, temperature, and MgSO₄) out of nine shows significant effect as they crossed above t-value limit.

Fig. 3

The predicted Vs experimental values PHA concentration of Plackett-Burman design for 12 runs of nine factors.

Fig. 4

Box-Cox plot of PB design for nine factors. The Box-cox plot here represents the data is normalized and do not require any transformation.

Fig. 5

2D Contour graphs representing 2-way interaction among A, B, C, and D (KH₂PO₄, inoculum size, temperature, and MgSO₄ respectively) on PHA production by B. paranthracis RSKS-3. The shape of contour plots signifies how factors influence the response (PHA in this case). Interaction between AD, AC, BC, BD, and CD shows a positively shaped elliptical contour plot which positively enhances the PHA production, whereas, AB interaction shows a negative elliptical contour plot that limits the PHA production.

Fig. 5

2D Contour graphs representing 2-way interaction among A, B, C, and D (KH₂PO₄, inoculum size, temperature, and MgSO₄ respectively) on PHA production by B. paranthracis RSKS-3. The shape of contour plots signifies how factors influence the response (PHA in this case). Interaction between AD, AC, BC, BD, and CD shows a positively shaped elliptical contour plot which positively enhances the PHA production, whereas, AB interaction shows a negative elliptical contour plot that limits the PHA production.

Fig. 6

A PHA film produced by B. paranthracis RSKS-3 under optimized synthetic media. A chloroform-dissolved PHA was poured into a watch glass and air-dried using a hot air oven at 60 °C temperatures.

Fig. 7

UV spectra of PHA film produced by B. paranthracis RSKS-3 and standard crotonic acid. The PHA film produced was treated with concentrate H₂SO₄ and converted to crotonic acid. The produced crotonic acid was analyzed as spectra from 800 to 190 nm against standard crotonic acid using a UV spectrophotometer.

Fig. 8

FTIR of PHA film produced by B. paranthracis RSKS-3 under statistically design-optimized conditions. The peaks at 2923, 1751, 1457, 1155, and 779 cm⁻¹ correspond to CH, C=O, CH₂, C–O, and C–O–C functional groups.

Fig. 9

XRD of PHA film with 3 intensities peaks at 27.289, 31.639, and 45.354°.

Fig. 10

Scanning Electron Microscopy showing surface morphology of (A) standard PHA and (B) PHA of B. paranthracis RSKS-3.

Fig. 11

NMR analysis, where, (A) C′ NMR (B) H′ NMR of extracted PHA from B. paranthracis RSKS-3.

Fig. 12

Thermo-Gravimetric Analysis of extracted PHA from B. paranthracis RSKS-3.