Studies on Phenol Biodegradation with Simultaneous Lipid Production by Rhodosporidium Toruloides 9564T for Potential Biodiesel Feedstock

Abstract

Waste management has become a significant concern in recent decades as a result of urbanization and the exponential growth of the global population. Industries such as paper and pulp, petroleum refining, coal processing, pharmaceuticals, and dyes discharge a wide range of hazardous organic and inorganic compounds. Organic compounds, including heavy metals and aromatic compounds, have detrimental impacts on the environment. Prominent environmental pollutants include heavy metals (Cr, Cd, Co, Pb, Ar, Hg, Zn, and Fe), the most prevalent aromatic compounds (phenol and its derivatives, catechol, 4 nitrophenol, and 4-chlorophenol). These substances are known to be genotoxic, carcinogenic, and mutagenic. All of these contaminants are discharged into the effluent during the pulping stage of paper manufacturing, resulting in its dark colour. These pollutants possess a potent odor, are toxic and carcinogenic to aquatic and terrestrial organisms, and are classified as aqueous pollutants due to their solubility in water. The EPA designates these pollutants as priority pollutants for removal from the environment based on their toxicity. Consequently, in order to safeguard the environment and organisms, it is imperative that these contaminants be eliminated from the effluents originating from the paper and pulp sectors. Oleaginous yeast is more valuable for the remediation of these contaminants in wastewater because it produces valuable products after utilizing the wastewater. Biofuel is one of the most frequently produced byproducts of oleaginous yeast. To thoroughly treat the organic debris in the wastewater effluent, the research investigation has been organized around five distinct objectives. The initial objective was to investigate phenol degradation and lipid production by using Rhodosporidium toruloides 9564T, an oleaginous yeast. It was found that R. toruloides completely degraded 0.75 g/L phenol with a lipid accumulation of 26.3% by following the Ortho-cleavage pathway. After completing the first objective, the second objective involved the degradation of phenol derivatives and their impact on cell morphology. The results obtained for this objective confirm that R. toruloides possesses the ability to fully degrade catechol (upto 1 g/L), 4-CP (upto 0.5 g/L), and 4-NP (upto 0.1 g/L). The maximal lipid yield achieved during this investigation was 36% (catechol). The impact of heavy metals on phenol degradation and lipid production was investigated in objective 3. The heavy metals Zn, and Fe have been observed to improve phenol degradation by reducing the degradation period. Conversely, Cr, Cd, and Co have been found to have an adverse impact on phenol degradation and lipid production. The SEM and confocal image confirmed the change in cell morphology, size, and accumulation of lipids within the cells confirms the toxicity level of the metals during phenol degradation. The maximum lipid content produced in the case of Zn and Fe containing phenol MSM media i.e. 43.39 and 40.56% respectively among all the heavy metal used. The obtained GC data confirmed that R. toruloides is capable of producing biodiesel that falls within the range specified by the ASTM standards, and the biodiesel properties are similar to vegetable oil. These results serve as evidence of the high quality of the biodiesel produced by R. toruloides. The optimization of phenol degradation and lipid production was then investigated using the Design Expert software in objective four. Using the Placket-Burman design, an initial screening of the most significant factors (pH, temperature, agitation speed, incubation period, and inoculum size) was conducted. Four factors are chosen for optimization by RSM (CCD experiment) out of a total of five. An optimized condition was obtained after the validation was incubation duration 92.145 h, temperature 29.46 ℃, inoculum size 14.68% v/v, and pH 6.07 and the 100% phenol was removed with an increase in lipid production 0.915 g/L. Again the RSM data was validated by ANN MOO-GA confirming the optimization of phenol degradation and lipid production was properly done. A study has been conducted in the 5 L reactor subsequent to the optimization study as the final objective. A batch mode reactor study was performed to treat synthetic paper pulp industry wastewater. The results of the study confirmed that R. toruloide exhibited phenol degradation (specifically, removal of 0.75 g/L from black liquor within 72 h of incubation), lignin degradation at a rate of 300 mg/L, and complete adsorption of heavy metals. The whole production process was analysed for the environmental impact and techno-economic feasibility and the obtained data confirmed that after 3 years the production system are economically viable with less environmental impact. Based on the comprehensive findings of the study, it can be concluded that R. toruloides is among the most effective oleaginous yeasts for biodiesel production and effluent valorization.