Substrate Concentration and Thermal Effects During Polyhydroxyalkanoate Bioproduction

Polyhydroxyalkanoates (PHAs) have long been thought to have the potential to replace petrochemical-based polymers because their production cost has been a challenging factor hindering their production processes. Previously characterized quantities of Micrococcus


Introduction
Biodegradable polymers, which occur naturally, belong to the family of bio-polyesters, and their physical properties depend upon the type of monomer in the polymers (Sneha et al., 2017). Polyhydroxyalkanoates (PHA) are polyesters synthesized in nature by various microorganisms, including fermentation of lipids and sugar by bacteria (Songyuan et al.,2019). Hydroxy fatty acids are components of PHAs, representing an intricate class of intracellular storage polymers produced by different bacteria and archaea. PHAs are generated in the presence of excess carbon sources, while growth is inhibited due to limited nutrient availability (Jimoh et al., 2018). The usage of plastic materials, which seems to be very important to human life, is now causing severe environmental problems due to their nonbiodegradability thus an alternative polymers attracting considerable interest is the PHAs, which may be utilized in similar applications as the existing synthetic polymers (Muhammad et al., 2015).

Processing of corn cobs
Corn cobs obtained during the harvesting period from Osogbo metropolis, Osun state, Nigeria, were air-dried at 30°C for two weeks and processed into smooth powdery form. The corn cobs powder was stored in an air-tight container and kept at 4°C in a refrigerator for further analysis.

Proximate Analysis of Corn Cobs Powder
The proximate analysis was determined using the standard analysis methods of the Association of Official Analytical Chemists (2019).

Estimation of cell dry weight
Estimating dry cell weight was done according to the modified procedure of Mascarenhas and Aruna (2019). The culture (10 ml) was transferred onto a filter paper of known weight; air-dried, and reweighed at 24-hour intervals until a constant cell dry weight was obtained using the formulae below: Cell dry weight (g/l) = weight of the dried cell in a petri dish -the weight of the empty petri dish.

Biomass Yield
The cultured medium (2 ml) was withdrawn at 24-hours intervals to determine its biomass yield using UV-vis spectrophotometer at 650 nm. (Jimoh et al., 2018).

Estimation of Reducing Sugar Concentration
Cell free-culture medium (1 mL) withdrawn at 24-hour intervals was boiled in a water bath at 100 ºC for 5 minutes. Subsequently, 10 ml of distilled water was added, and the absorbance of each sample was determined at 540 nm using JENWAY 6300 spectrophotometer. The corresponding reducing sugar concentration values were extrapolated from the glucose standard curve (Jimoh et al., 2013).

Extraction and Preliminary Quantification of PHAs
PHAs were extracted according to Mascarenhas and Aruna's modified method (2019). The culture medium (10 ml) was centrifuged after 72 hours at 10,000 rpm for 20 minutes. Cell pellets were washed with sterile phosphate-buffered saline (pH 7.2) and recentrifuged at 8000 rpm for 15 minutes. The supernatant was disposed of, and the pellet was suspended in 10 ml of chloroform, vortexed and incubated at 37°C for 24 hours; then further centrifuged at 8000 rpm for 12 minutes (Jimoh et al., 2018). After centrifugation, the supernatant was transferred onto sterile Petri dishes at 30°C for evaporation of chloroform and to obtain polymer granule composition. The polymer granule obtained was dissolved in concentrated H2SO4 and heated in a water bath at 100°C for 10 minutes; thus, depolymerization of PHA when heated with concentrated H2SO4 leads to the formation of brownish-coloured crotonic acid. After cooling, the absorbance was read at 235 nm against a concentrated H2SO4 blank using a spectrophotometer. A standard curve was busked with Pure PHA (20-100μg/ml) (Sigma, Aldrich); thus, PHA concentration and percentage yield were determined using the formulae below: Cell dry weight (g/l) = weight of the dried cell in a petri dish -the weight of the empty petri dish.

Results and Discussion Proximate Analysis of Corn cobs
The proximate composition of corn cobs utilized in this research work was determined during PHA production. The corn cobs' high moisture content (0.037 ± 0.014 %) shows its support for microbial growth, which eventually increases microbial activity (Aletan and Kwazo, 2019). Ash content (3.550 ± 0.054%) expresses the mineral content of the corn cobs, which indicates the availability of low mineral concentration (especially the macro elements) and vice versa (Otunola and Afolayan, 2019). The crude lipid content (45.995 ± 1.068%) indicates that corn cobs are an excellent source of fat-soluble vitamins and contribute significantly to the energy content of the PHA product that is synthesized from such agricultural waste (Der et al., 2012). Corn cobs contain the essential component of the diet required for microbial survival through the availability of crude protein (0.085 ± 0.007%). The carbohydrate content of 13.740 ± 0.651% shows that corn cobs can serve as a good energy and carbon source during microbial metabolic activity (Lu-Ji et al., 2011). The two-way ANOVA results reveal a significant difference between corn cobs' mean proximate composition (Table 1). where the recorded values were significantly different at p˂ 0.05. Data are mean ± SD (standard deviation), n= 2. The statistical level of significance analyzed by two-way ANOVA is followed by the Tukey post hoc pairwise multiple comparisons test.

Osmotolerance and Thermotolerance Effect during PHA production
The biomass yield, reducing sugar concentration, and PHA yield obtained during PHA production varied due to varying environmental conditions (substrate concentration and temperature).

Biomass yield and Reducing sugar concentration
Maximum biomass yield was obtained at 8%, as shown in (Figure 1). The reducing sugar concentration increases as the substrate concentration increases, indicating the utilization of glucose by the organisms (Figure 2). The highest biomass yield and reducing sugar concentration were also obtained at 37°C, thus, supporting Micrococcus flavus growth and activity (Figures 4 and 5). Based on the results acquired in this research, corn cobs are economically, and environmentally valuable raw materials for industrial-scale PHA production complemented with metabolically active Micrococcus flavus SS21B strain.

PHA concentration and PHA yield
In this study, PHA yield obtained using corn cobs as carbon sources made it easier for Micrococcus flavus to absorb nutrients (Rodrigues and Druzian, 2018). Variations in PHA yield obtained using varying substrate concentrations and temperature (figure 3) indicated the potential of Micrococcus flavus SS21B to synthesize and accumulate PHA as carbon and energy storage materials or as storage for redundant reducing power under the condition of limiting nutrients in the presence of excess carbon source (Nwinyi and Owolabi 2019). The accumulated PHA can be degraded by intracellular depolymerases and metabolized as a carbon and energy source instantly the supply of the limiting nutrient is reestablished. The effect of temperature (37°C) during the synthesis of PHA is significant, and the percentage yield and concentration of PHA increase as the substrate concentration increases (Figure 3). These environmental conditions support Micrococcus flavus SS21B strain activity during PHA production ( Figure 6). Thus, the availability of PHA through microbial synthesis using corn cobs would replace petrochemicallysynthesized materials (plastic materials, packages, absorbents, biomedical materials, tissue engineering materials) due to the availability of agricultural residues.

Conclusion
Microbial synthesis of PHA at 8% and 37°C will increase the quantitative and qualitative characteristics of PHA-synthesized products since PHAs are produced from biodegradable renewable sources. Its utilization would also improve the state of the environment by solving the problems of solid waste management that result from the accumulation of corn cobs in the environment, thus preventing air pollution and flooding caused by the recycling of corn cobs (Jimoh et al., 2018). Bacillus Megaterium Strain Jha using Inexpensive Agro-industrial Wastes.