Journal of Energy Bioscience 2024, Vol.15, No.5, 289-300 http://bioscipublisher.com/index.php/jeb 293 pre-treatment has been found to be more effective than acid and alkaline treatments in increasing biohydrogen generation from kitchen waste (Jais et al., 2023). Similarly, the combination of physical and chemical methods, such as ultrasonication with hydrogen peroxide, has demonstrated superior results in enhancing hydrogen production from organic wastes (Salem et al., 2018). The optimization process also involves adjusting the volatile solid concentrations and mixing ratios of different waste types to achieve the highest hydrogen production rates (Nam, 2023). 4.3 Challenges in scaling pre-treatment processes While pre-treatment methods have shown promising results at the laboratory scale, several challenges remain in scaling these processes for industrial applications. One of the primary challenges is the economic feasibility of the pre-treatment methods. For instance, although freeze-thaw pre-treatment has been identified as the most profitable process, the costs associated with maintaining low temperatures can be prohibitive (Ma et al., 2011). Additionally, the energy requirements for thermal and chemical pre-treatments can be substantial, potentially offsetting the benefits of increased biohydrogen yields (Jeyakumar et al., 2020). Another challenge is the variability in the composition of kitchen waste, which can affect the consistency and efficiency of the pre-treatment process (Pagliaccia et al., 2019). Addressing these challenges requires further research into cost-effective and energy-efficient pre-treatment methods, as well as the development of robust systems that can handle the heterogeneity of kitchen waste. 5 Lipid Accumulation by Microorganisms 5.1 Microbial strains used for lipid accumulation (bacteria, yeast, fungi) Various microbial strains, including bacteria, yeast, and fungi, have been explored for their lipid accumulation capabilities. For instance, the oleaginous yeast Yarrowia lipolytica has been extensively engineered to enhance lipid production, achieving lipid contents as high as 61.7% in bioreactor fermentations (Tai and Stephanopoulos, 2013). Similarly, the yeast Rhodosporidium toruloides has shown significant lipid accumulation when cultivated in bioethanol wastewater, reaching a lipid content of 34.9% (Zhou et al., 2013). Bacterial strains such as Bacillus cereus have also been used in co-culture systems with yeast to optimize lipid production and wastewater treatment (Karim et al., 2021). 5.2 Mechanisms of lipid biosynthesis Lipid biosynthesis in microorganisms involves several key enzymatic steps. In Yarrowia lipolytica, overexpression of diacylglycerol acyltransferase (DGA1) and acetyl-CoA carboxylase (ACC1) has been shown to significantly enhance lipid production by driving the synthesis of triglycerides (TAGs) and fatty acids (Tai and Stephanopoulos, 2013; Qiao et al., 2015). The process involves the conversion of acetyl-CoA to malonyl-CoA by ACC1, followed by a series of reactions leading to the formation of fatty acids, which are then esterified to glycerol to form TAGs. In Schizochytriumsp., adaptive laboratory evolution (ALE) has been used to enhance lipid biosynthesis under stress conditions, improving both lipid yield and antioxidant defenses (Sun et al., 2018). 5.3 Process parameters affecting lipid yield (temperature, pH, etc.) Several process parameters significantly influence lipid yield in microbial cultures. Temperature and pH are critical factors; for example, optimal lipid accumulation in a co-culture of Lipomyces starkeyi and Bacillus cereus was achieved at a pH of 6.5 and a temperature of 32.5 ℃ (Karim et al., 2021). In Schizochytrium sp., low temperature and high salinity were used to enhance DHA content and lipid accumulation while preventing lipid peroxidation (Sun et al., 2018). Additionally, nutrient availability, such as carbon and nitrogen sources, plays a crucial role. Feeding strategies, such as the addition of glucose during the cultivation of Rhodosporidium toruloides, have been shown to significantly increase both biomass and lipid production (Zhou et al., 2013). 5.4 Case studies of high lipid-producing microbes Several case studies highlight the potential of high lipid-producing microbes. In one study, Yarrowia lipolytica was engineered to overexpress key enzymes in the lipid biosynthesis pathway, resulting in a lipid content of 41.4% in a 2-L bioreactor fermentation (Tai and Stephanopoulos, 2013). Another study demonstrated that
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