Currently, there is a growing interest in science and industry to generate new products and processes that can be used as efficient and green sources of energy. This is also important to find avenues to substitute fossil fuel consumption as their reserves are rapidly declining and the environmental impact issues associated with their intensive use have been much criticized worldwide [1]. Over the past few years, bioethanol has attracted significant attention due to its wide range of applicability, the lower CO2 released in comparison with that produced from the combustion of traditional hydrocarbons such as oil and gas [2], and the use of renewable sources for its production. As a result, bioethanol production has been widely considered environmentally sustainable and consequently has spurred a number of renewable energy initiatives worldwide. A major issue in the production of bioethanol is the relatively low production yields. Among many others, this has been attributed to declining cell viability levels throughout the process. An avenue to overcome this issue is the encapsulation of yeast cells into polymer matrices based on agar, carrageenan, alginate, and polyacrylamide [3]. This allows to maintain higher levels of viable cells while removing the produced bioethanol.

Accordingly, the main objective of the present study was to develop a crosslinked polymeric matrix from commercially-available collagen, to encapsulate the bioethanol-producing yeast Saccharomyces cerevisiae. The thermal and mechanical properties, as well as the microscopic features of the prepared matrices, were evaluated prior to encapsulating cells. Upon encapsulation, cell viability and biocompatibility were determined.

The gels were prepared by mixing 3.0, 5.0 and 7.5% (w/v) collagen with 1.0%, 3.0% and 5.0% (w/w) of the chemical crosslinker glutaraldehyde. The crosslinking reaction was conducted under mechanical agitation between 100 and 200 rpm, and at a temperature of 55 °C for 4 hours. A two-factor experimental design was performed here with 3 levels (32), where the factors were the concentration of glutaraldehyde and the concentration of collagen in the gel, including samples without addition of crosslinker. Near (NIR) and far infrared (IR) spectroscopy were used to identify the functional groups present in the hydrogels. In this case, we tracked NH2, NH, CH2, CH3, H2O, and CN, in bands in the range of 800 to 2,500 nm (NIR), and 2,500 to 20,000 nm for the infrared (IR).

SEM micrographs allowed us to estimate the average pore size, which is a critical parameter to determine the viability of yeast cells encapsulated. The analyses confirmed that an increase in the concentration of glutaraldehyde leads to a decrease in the average pore size of the gel. Gels to continue with cell immobilization were those with average pore size values in the range between 3 and 8 μm (Figure 1).

Figure 1. Surface morphology described by the electron scanning microscope (SEM) for collagen hydrogels at 7.5% (w/v) and crosslinked with 3.0% glutaraldehyde (A) and 5.0% (B) (w/w) . 

Bloom tests on the gels indicated that an increase in the crosslinking agent concentration led to more stable and elastic materials, however; but such an increase in stiffness promoted an increase in the tendency to fracture in the presence of plastic deformation.

Thermogravimetric analyses were conducted in the range from room temperature to 800ºC to estimate the thermal stability of the gels. The collected thermograms showed an initial weight loss at about 100 °C, which can be correlated to water in the sample. This was followed by the decomposition of the collagen network. Our results suggest an increase in thermal resistance for gels with higher crosslinking levels.

Hydrogels were subjected to swelling in an aqueous medium at different pH values (2.0, 7.4 and 9.0). Higher swelling was achieved for gels with lower crosslinking degrees. Additionally, superior structural stability in such medium (about six days) was observed for gels with a 7.5% (w/v) collagen content.

Yeast cells were then grown in a Yeast extract Peptone Dextrose (YDP) culture medium. Encapsulation of S. cerevisiae proceeded in collagen hydrogels with 7.5% (w/v) collagen contents at 3% and 5% (w/w) glutaraldehyde. To evaluate viability, cells within the matrix were stained and observed under optical and confocal microscopes and then counted to estimate the live/dead ratio.

The approach presented here provides a suitable route for the preparation of chemically crosslinked collagen hydrogels with applications in yeast encapsulation. The obtained encapsulates can be potentially useful to enable the next-generation bioethanol packed-bed bioreactors. This is critical to assure the sustainable production of biofuels from natural sources.

Keywords: Hydrogels, matrix, cross-linking, yeast, immobilization.

References

[1] S. Mun Tan, P. W. Sia Heng y L. Wah Chan, «Development of Re-Usable Yeast-Gellan Gum Micro-Bioreactors for Potential Application in Continuous Fermentation to Produce Bio-Ethanol,» Pharmaceutics, vol. 3, pp. 731-744, 2011.

[2] M. Inal y M. Yigitoglu, «Production of bioethanol by immobilized Saccharomyces Cerevisiae onto modified sodium alginate gel,» J Chem Technol Biotechnol, vol. 86, pp. 1548-1554, 2011.

[3] I. B. Holcberg y P. Margalith , «Alcoholic Fermentation by Immobilized Yeast at High Sugar Concentrations,» European J Appl Microbiol Biotechnol , vol. 13, pp. 133-140, 1981.