Study broadens biotechnological production of xylitol

Brazilian researchers increase the production capacity to a scale one thousand times larger than in typical experiments.

By Noêmia Lopes

Agência FAPESP – The idea of using sugarcane bagasse to produce xylitol emerged from the studies in the 1980s that focused on the better utilization of plant biomass left over from ethanol production.

Xylitol is a sweetener with very peculiar characteristics, such as reducing cavities and replacing glucose in the diets of diabetics, and is effective in the treatment and prevention of osteoporosis and respiratory diseases – but with high production costs. These high production costs reflect the chemical process by which xylitol is produced, involving the use of catalyzers in a process that requires extensive purification stages.

In the pursuit of alternatives, researchers from the group of Applied Microbiology and Bioprocesses at the Lorena School of Engineering at Universidade de São Paulo (EEL/USP) have been refining the biotechnological production method.

The method consists of conversion of xylose sugar – which abundant in nature and found in the walls of such plants as sugarcane, wheat, oats, rice, eucalyptus and pine – to xylitol using microorganisms that have the capacity to metabolize xylose under certain growing conditions.

Until recently, the process was conducted on a small scale, most often in a 125-millimeter Erlenmeyer flask. It was only through an FAPESP-funded study from 2010-2012 that the team managed to expand the production capacity to 125-liter reactors, a scale some one thousand times larger than in the original experiments.

“We established important parameters for the manufacture of xylitol through biotechnology with a goal of increasing the scale using sugarcane bagasse but not exclusively. This also allowed the possibility of treating renewable vegetal biomass, such as cane, rice and wheat straw, apple bagasse and oat and coffee husks,” said Maria das Graças de Almeida Felipe, professor at EEL/USP and coordinator of the study.

The study required the establishment of the necessary conditions for the utilization of different lignocellulosic materials, which are nothing more than plant biomass, for example, sugarcane, composed of three main fractions: cellulose, hemicellulose and lignin.

The first two, cellulose and hemicellulose, are carbohydrates (sugars like glucose and xylose), which are found together in chains and can be used as a means of growing microorganisms to obtain different products.

In the case of xylitol, one must first break down the sugar chains, releasing them from the plant cell walls. The EEL/USP team achieved this through hydrolysis with diluted sulfuric acid, which allows for the solubilization of sugars to produce hydrolyzed hemicellulose. 

“Because acid hydrolysis results in unwanted byproducts, we subjected the hydrolyzed items to different detoxification processes,” stated Felipe.

The hydrolyzed hemicellulose of sugarcane bagasse without toxins was then fermented using Candida guilliermondii, a yeast, grown in several media with different carbon sources (xylose, glucose and a mixture of xylose and glucose).

According to the study’s coordinator, “This allowed the scientists to evaluate the joint effects of the carbon source employed in the preparation of yeast (innocuous) and the detoxifying agent.” These fermentations replicated the conditions utilized in previous studies, such as Erlenmeyer flasks in triplicate at an agitation speed of 200 rotations per minute for 120 hours. 

As a result, xylose was the carbon source that best favored the production of xylitol. “We opted for coal as the detoxification agent for the subsequent fermentations because of the lower cost when compared to resin,” said Felipe. 

Increased scale

The better conditions observed in the first stage of the project were employed to evaluate the same bioprocess on a large scale in 2.4-liter, 16-liter and 125-liter reactors. In the first two cases, the fermentations occurred over 144 hours; the third case was for 86 hours.

“We concluded that is possible to increase the scale under the conditions evaluated, as the results obtained for xylitol production were similar using fermenters of different capacities,” affirmed the researcher.

The fermentation in 125 liters was the most challenging stage for the team. This was because, until then, no one knew how the yeast would behave on a reactor scale. In the experiment, 70 liters of hydrolyzed hemicellulose was used, with detoxification using activated coal over seven days. 

Secondly, “There could have been problems in the aeration of the system, as the availability of oxygen is the main fermentative parameter to be controlled so that metabolism is directed toward the conversion of xylose to xylitol and not to cellular growth,” commented Felipe. To avoid setbacks, the team used volumetric oxygen transfer coefficients (kLa) for monitoring the gas in the liquid medium with the increase in scale from 2.4 to 125 liters.

In terms of reducing the cost of the biotechnological production of xylitol in relation to the chemical process, the researcher stressed that additional studies are needed to examine the entire production chain with regard to detoxification of the hydrolyzed residue.

Continuity and the impact

In the last few decades, the studies conducted by EEL/USP have indicated the technical and economic feasibility of increasing the production scale of xylitol. Currently, these studies are resulting in opportunities for other research frontiers. According to Felipe, “The knowledge that we obtained has contributed to the development of a project on second-generation ethanol production using sugarcane bagasse in its cellulosic and hemicellulosic fractions.” 

To date, pre-scientific initiation, scientific initiation, master’s, doctoral and post-doctoral students at EEL/USP had been involved in research on xylitol. The project to expand the production scale had 15 participants, including researchers and technicians from the Brazilian Bioethanol Science and Technology Laboratory (CTBE).

“Part of the results have been released in the form of complete works or summaries in indexed periodicals, book chapters and the annals of national and international meetings. We published an international book, registered patents and integrated two important projects: ‘Public Policy Guidelines for Sugarcane Agroindustry in São Paulo State’ and ‘CYTED Thematic Network 310RT0397 – the Ibero-American Society for the Development of Biorefineries (Siadeb)’,” explained Felipe.

Xylitol applications

Because of its anti-cavity properties, xylitol does not pose risks to dental health, unlike conventional sugars, such a sucrose. This is because the sweetener is not fermented by bacteria in the buccal flora – a process that contributes to the formation of the acid that attacks tooth enamel. 

“This property was discovered in the 1970s in research with children who chewed gum containing xylitol. The results demonstrated a reduction in cavities and the remineralization of newly formed cavity lesions, with a prolonged effect months after the tests,” said Felipe.

A second relevant characteristic of xylitol is that it is not related to insulin. Absorbed slowly, the sweetener does not cause rapid alterations in blood sugar levels. Furthermore, the glucose formed based on xylitol metabolism is not directly released into the blood stream. These factors make xylitol a sweetener that is well tolerated by diabetics. 

Another application is the treatment of osteoporosis. Studies conducted in rats showed that the sweetener contributes to increases in bone mass and the biomechanical properties of bone. The gains are attributed to calcium absorption through the intestine (easing the passage into blood vessels) stimulated by xylitol.

The sweetener is also utilized in the treatment of respiratory diseases. Research has proven that the presence of xylitol in nasal sprays reduces the number of mucosa, helping to reduce the risk of pulmonary infections. “One could say, therefore, that xylitol strengthens the natural defense system of lungs, delaying or preventing the establishment of bacterial infections, such as pneumonia,” stated Felipe.