(573ab) Fabricating an Economical and Sustainable Fermentation Medium to Produce Drop-in-Fuel and Value-Added Bioproducts from Alternative Feedstocks

Utilization of abundant renewable carbon present in lignocellulosic biomass can reduce the environmental concerns raised due to the use of fossil fuels. This can also be a driving force for scientific and technological advancement of biorefining facilities to efficiently produce drop-in fuels. Previously, a wide variety of non-transgenic bioenergy crops have shown promising results for the production of cellulosic fermentable sugars that can be conveniently converted to bioethanol and high value bioproducts. In the last few years, the energy density of bioenergy plants such as energycane, sugarcane, and sorghum, has been further increased by genetic engineering. These high biomass bioenergy crops are being metabolically engineered to sequestrate carbon in their vegetative tissues and direct the carbon flux for the synthesis and accumulation of energy-rich lipid molecules like triacylglyceride (TAG) molecules (Vanhercke et al. 2019; Parajuli et al. 2020; Luo et al. 2022). Therefore, transgenic bioenergy crops cater to both cellulosic sugars and vegetative lipids to produce drop-in fuel and value-added biochemicals (Maitra et al. 2022a; Maitra et al. 2022b). However, because the lipids in the vegetative tissues of transgenic crops are present in a complex form and lower amounts (Parajuli et al. 2020; Maitra et al. 2021), application of appropriate bioprocessing technologies is needed to efficiently deconstruct the recalcitrant lignocellulosic structure to recover vegetative lipids along with cellulosic sugars from the feedstock without degenerating lipids. Chemical-free hydrothermal pretreatment has been successfully established for the recovery of both cellulosic sugars and vegetative lipids that maintain the lipid profile during processing. During the processing, the biomass residues get enriched with lipids that were recovered at the end of the process (Maitra et al. 2022a; Maitra et al. 2022b).

Although the recovery of cellulosic sugars from bioenergy crops has been successfully established, the hydrolysate that is produced is not suitable for most microorganisms for growth and fermentation. The utilization of hydrolysate for fermentation still needs either an external supply of nutrients, additional detoxification steps, or both. There are no reports on the utilization of hydrolysate for fermentation without additional nutrients, salts, or detoxification. Detoxification steps and the addition of nutrients such as vitamins, yeast nitrogen base (YNB), yeast extract, and peptone (YP) increase the cost of the process. To alleviate the issue, the study presents the fabrication of a new sustainable and economic fermentation media using hydrolysate and juice derived from transgenic oil producing sugarcane.

Successful development of noble transgenic biomass has been achieved. Development of scalable processing technology is needed for sustainable biomanufacturing. The hydrolysate produced, especially from transgenic oilcane, had a detrimental effect on growth and fermentation. Unfortunately, both the engineered S. cerevisiae and commercial strains capable of utilizing both C6 and C5 carbons were unable to grow on 100% hydrolysate without any external nutrient. Hydrolysate was analyzed for the presence of compounds responsible for the inhibition. The hydrolysate was found to be rich in carbohydrates (~ 120 g/l of total fermentable cellulosic sugars), lipids (TAGs, DAGs, and phospholipids), and phenolics (various metabolites and compounds generated due to pretreatment) which could have an inhibitory effect on microorganisms. The amount of sugar degradation products such as HMF and furfurals were negligible. The first step was to add growth-supporting nutrients without any additional cost. To this end, juice from transgenic oilcane was not edible, rich in nitrogen, and had all the vitamins (LC-MS analysis) needed for growth. The media was designed with different ratios of hydrolysate to juice. All the combinations of hydrolysate to juice worked except ratios < 1:1. A 1:1 ratio of undetoxified hydrolysate and juice without any external nutrients was the optimized growth medium for both the lab and commercial strains. Once, the growth medium was optimized for the strains, operating conditions were optimized to improve the fermentation efficiencies.

It was demonstrated that pH control, microaerophilic conditions, and adaptation of strains in the same medium prior to inoculation enhance the fermentation efficiency. The specific and volumetric ethanol productivity of adapted engineered S. cerevisiae were 0.17 ± 0.001 and 1.04 ± 0.00, respectively, while that of commercial strain was 0.19 ± 0.02 and 1.06 ± 0.004, respectively, in the new sustainable media i.e., hydrolysate: juice (1:1). The adapted lab and commercial strains exhibited 1.7 times and 3.1 times higher specific ethanol productivity and 5.4 times and 5.0 times higher volumetric ethanol productivity, respectively. Adapted lab and commercial strains also demonstrated 1.1 times and 3.1 times higher specific consumption rates, respectively. The growth and fermentation kinetics of the strains suggest that uptake of substrates by the cells improved on adaptation and the improved carbon flux was directed towards the fermentation pathway. The production of byproducts such as glycerol and acetic acids were negligible by the strains. The growth and fermentation kinetics on the constructed media demonstrated that the proposed combination of hydrolysate and juice can be used as a noble economic and sustainable fermentation medium. The requirement of expensive nutrients such as vitamins, minerals, nitrogen, and buffer salts which are crucial for cell growth, metabolism and fermentation can be furnished without extra cost. Moreover, since the hydrolysate and juice would be derived from transgenic bioenergy crops which would be grown in marginal land there would be no competition for limited cultivable land.

References

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Maitra S, Dien B, Long SP, Singh V (2021) Development and validation of time‐domain 1 H‐NMR relaxometry correlation for high‐throughput phenotyping method for lipid contents of lignocellulosic feedstocks. GCB Bioenergy. https://doi.org/10.1111/gcbb.12841

Maitra S, Long SP, Singh V (2022a) Optimizing Chemical-Free Pretreatment for Maximizing Oil/Lipid Recovery From Transgenic Bioenergy Crops and Its Rapid Analysis Using Time Domain-NMR. Front Energy Res 10:1–13 . https://doi.org/10.3389/fenrg.2022.840418

Maitra S, Viswanathan MB, Park K, Kannan B, Alfanar SC, McCoy SM, Cahoon EB, Altpeter F, Leakey ADB, Singh V (2022b) Bioprocessing, Recovery, and Mass Balance of Vegetative Lipids from Metabolically Engineered “Oilcane” Demonstrates Its Potential as an Alternative Feedstock for Drop-In Fuel Production. ACS Sustain Chem Eng 10:16833–16844 . https://doi.org/10.1021/acssuschemeng.2c05327

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