Optimization of carotenoid production by Rhodotorula mucilaginosa using sugarcane bagasse hydrolysate as carbon source and mutagen ethyl methanesulfonate for biosynthesis of antioxidant

In the world of food and medicine, nature provides a vibrant palette of pigments that do more than just color our world; they protect it. Among these are carotenoids, a family of antioxidant compounds found in carrots, tomatoes, and many other plants. These molecules act as a shield for living cells, neutralizing harmful substances that can damage tissues and contribute to chronic diseases. For decades, scientists have sought ways to produce these valuable compounds without relying solely on harvesting crops, which can be seasonal and land-intensive. Instead, they have turned to the microscopic world, using tiny organisms like yeast and bacteria as living factories. These microbes can be fed simple sugars and coaxed into producing carotenoids in large quantities, offering a sustainable alternative that does not compete with food supplies. The challenge, however, lies in finding a cheap and abundant food source for these microbes and then training them to work harder, producing more pigment than their natural counterparts ever could.

A team of researchers in Indonesia has taken a significant step forward in this effort by turning agricultural waste into a high-value product. They focused on sugarcane bagasse, the fibrous residue left over after sugarcane juice is squeezed out. This material is abundant, with hundreds of millions of tons produced globally every year, yet it is often underutilized. The researchers knew that bagasse is rich in cellulose, a complex sugar that can be broken down into simple sugars like glucose, which yeast loves to eat. Their goal was to transform this waste into a growth medium for Rhodotorula mucilaginosa, a red yeast known for its ability to produce carotenoids. To make the process even more efficient, they did not just rely on the natural yeast; they used a chemical tool to create a "super-yeast" mutant that produces far more pigment than the original strain.

The journey began with preparing the sugarcane waste. The researchers took the dried bagasse and treated it with a mild alkaline solution to loosen its tough structure, making it easier to break down. They then added a specific enzyme, cellulase, which acts like a pair of molecular scissors, cutting the long cellulose chains into the simple glucose and xylose sugars the yeast needs to survive. Through careful testing of different chemical concentrations, they identified the perfect recipe: a treatment with 0.75 molar sodium hydroxide followed by hydrolysis with 20 grams of enzyme per liter. This combination yielded a liquid rich in fermentable sugars, containing approximately 27.34 grams of glucose and 3.59 grams of xylose per liter. This sugary broth became the new home for the yeast, replacing expensive, refined sugars with a renewable, low-cost alternative.

With the food source ready, the researchers turned their attention to improving the yeast itself. They knew that the natural strain of Rhodotorula mucilaginosa was capable, but they wanted to push its limits. To do this, they exposed the yeast cells to ethyl methanesulfonate, a chemical mutagen that introduces small, random changes to the organism's genetic code. Think of this process as shuffling the deck of cards in the yeast's instruction manual; most shuffles result in no change or a broken hand, but occasionally, a shuffle creates a winning combination. They tested various concentrations of the chemical, finding that a 4 percent solution was the sweet spot. It was strong enough to kill off the vast majority of the cells—about 97.6 percent—ensuring that only the most robust survivors remained, but gentle enough to leave a few viable colonies that had undergone beneficial genetic changes.

From the survivors of this chemical challenge, the researchers selected seven distinct colonies to see which one had improved the most. They grew each one in a standard nutrient broth and measured how much carotenoid they produced. While several mutants showed promise, one strain, designated M7, stood out for its consistency and high output. When the team compared this mutant to the original, unmodified yeast, the difference was striking. The M7 mutant produced 33.12 micrograms of carotenoid for every gram of dry yeast biomass, a 1.46-fold increase over the native strain, which produced only 22.64 micrograms per gram. This improvement was not just a matter of having more pigment inside the cells; the mutant also converted the sugar in the bagasse broth into carotenoid much more efficiently. For every gram of glucose consumed, the mutant yielded 5.15 micrograms of carotenoid, whereas the native strain yielded less than half that amount.

The performance of the mutant extended to the overall volume of production as well. In the same amount of time, the M7 strain generated a total carotenoid concentration of 66.80 micrograms per liter of liquid, nearly double the 33.51 micrograms produced by the native strain. This translated to a production speed that was twice as fast, with the mutant creating 0.46 micrograms of carotenoid per liter every hour, compared to 0.23 for the original. Beyond the numbers, the researchers observed that the mutant was better adapted to the sugarcane environment. While the native yeast took six hours to adjust to the new broth before it started growing, the M7 mutant began multiplying immediately, suggesting that the genetic changes had made it more resilient and ready to work.

Finally, the team tested whether this increased production actually resulted in a more powerful antioxidant. They used a standard test that measures how well a substance can neutralize free radicals, a key function of antioxidants. The extract from the M7 mutant showed a radical-scavenging activity of 46.94 percent, higher than the 35.75 percent seen in the native strain. However, the statistical analysis revealed that this difference was not significant enough to be considered a definitive improvement over the original. This suggests that while the mutant produced more carotenoid overall, the specific mix of pigments it created might have shifted. The yeast produces several types of carotenoids, including beta-carotene, torulene, and torularhodin, which have different strengths as antioxidants. It is possible that the mutant produced more of a type that is less potent in this specific test, or that the total increase in pigment simply wasn't enough to show a clear statistical difference in this assay.

The study concludes that combining agricultural waste with genetic improvement is a viable path toward sustainable carotenoid production. By using sugarcane bagasse, the researchers demonstrated that expensive food-grade sugars are not necessary to fuel these microbial factories. By using chemical mutagenesis, they showed that natural yeast strains can be enhanced to work harder and more efficiently. While the antioxidant power of the final product requires further detailed study to understand exactly which pigments are present, the process itself offers a promising framework. It turns a waste product into a valuable resource and creates a more productive organism, pointing toward a future where natural pigments can be made cheaply and sustainably without straining the planet's food systems.