Climate change and other environmental problems caused by the extensive usage of fossil fuels have attracted great attention, therefore, it is urgent to develop alternative energy sources from renewable non-food biomass (Kim et al., 2019, Zhang et al., 2023). Biodiesel has received increasing research interest as a high energy density, biodegradable, nontoxic, and environmentally friendly energy (Lazar et al., 2018, Ma et al., 2018b). However, the industrialization of biodiesel has not been realized due to two main arising issues. One is the low economic benefit, as high as 75–90% of the biodiesel production cost is used for purchasing the required feedstocks (Nomanbhay et al., 2018, Pereira et al., 2021). The other issue is the waste glycerol problem; for every 100 kg of biodiesel production, approximately 10 kg of crude glycerol is produced as a byproduct (Garlapati et al., 2016). Based on the annual demand of 8 billion gallons of biodiesel production, an estimated 5.87 billion pounds of crude glycerol are generated (Kumar et al., 2021). The majority of crude glycerol was usually treated as wastewater or purified into pure glycerol. The method of refining crude glycerol to pure glycerol needed a large investment, high cost, and the price of pure glycerol was low (Gao et al., 2016). Therefore, it may not be economical for small- or medium-sized biodiesel industries (Luo et al., 2016). Even crude glycerol is discharged directly without any treatment, resulting in environmental pollution and waste of resources (Nomanbhay et al., 2018). A more effective way such as converting crude glycerol to a value-added product is of great significance for achieving more sustainable biodiesel production.

Methods used to convert crude glycerol to other products include chemical and biological conversions. Chemical methods could convert crude glycerol into other productions using catalysts, such as acrolein, 1,3-propanediol, polyglycerol, glycerol carbonate, polyurethanes, solketal, and ethers (Kaur et al., 2020, Luo et al., 2016). However, most of the studies remained in the lab phase due to the need for a high energy chemical reaction, the addition of toxic chemicals and non-biodegradable catalysts (Chilakamarry et al., 2021). Biological methods use microbes and enzymes for the conversion of glycerol by aerobic or anaerobic metabolism and could convert crude glycerol into microbial products such as citric acid, intracellular lipids, bioplastics, and bioethanol (Kaur et al., 2020, Kumar et al., 2019).

Microbial lipids were synthesized by oleaginous microorganisms (Maza et al., 2021). Oleaginous microorganisms are defined by the ability to accumulate more than 20% of their dry body mass as lipids, and the synthesized lipids might be employed as feedstock for biodiesel preparation because of their high carbon to heteroatom ratios (Alvarez et al., 2019, Lazar et al., 2018, Zhang et al., 2021). These microbes rarely depended on natural conditions, did not occupy arable land, and have a fast growth rate and high lipid productivity (Cho and Park, 2018). Therefore, the use of crude glycerol by oleaginous microorganisms for lipid production may solve not only the problem of crude glycerol waste but also the high cost of feedstock for biodiesel production (Vivek et al., 2017). Rhodotorula toruloides was a highly efficient oleaginous yeast that has shown good lipid production characteristics when using glucose as substrates (Saran et al., 2017). Our previous study has demonstrated that crude glycerol can be utilized as a low-cost carbon source for lipid preparation (Gao et al., 2016). However, the optimal conditions for the preparation of microbial lipids, acceptance for biodiesel production, and economic and environmental benefits have not been well studied.

This study aimed to provide a new strategy for a closed loop of biodiesel production and to ensure sustainable and stable development of the biodiesel industries. The component content of the raw crude glycerol was analyzed to prepare the synthetic crude glycerol based on this composition. Synthetic crude glycerol was utilized as carbon sources for R. toruloides to produce microbial lipids. The fermentation conditions for lipid production, such as the inoculation rate, nitrogen (N) source, C/N ratio, initial crude glycerol concentration, and fermentation time, were explored and optimized. The composition of the lipids under the optimal conditions was analyzed and estimated according to the Hoekman equation. An economic value and environmental implications analysis for a closed loop of crude glycerol to biodiesel was conducted.