#FEMSmicroBlog: Cellobiose fermentation as a path towards sustainable biofuels


One of our major global challenges is the sustainable production of biofuels. An efficient option could be the fermentation of cellulosic hydrolysates from renewable biomass. However, microorganisms ferment the sugars in lignocellulosic hydrolysates poorly and inefficiently. The article “Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose” in FEMS Yeast Research reports the successful development of yeast strains capable of producing bioethanol from cellobiose at industrial conditions, as discussed by Roksolana Vasylyshyn, Olena Dmytruk, Andriy Sibirny, and Justyna Ruchala in this #FEMSmicroBlog. #FascinatingMicrobes


The challenges of producing bioethanol

Global ethanol production has risen to 120 billion liters per year, with 80% being used as liquid fuel. Almost all ethanol on the planet is currently obtained from sugar (sucrose) and starch as traditional raw materials. While some countries use sugar cane as a substrate, others use starch from corn, cereals, potatoes, or molasses.

However, the raw material base for obtaining sugar and starch is limited, driving up costs and potentially causing food and feed shortages. Therefore, in recent decades, plant biomass became a sustainable alternative as its material is readily available. Raw plant substrates for industrial ethanol production could include waste from agriculture, woodworking, pulp and paper industries, or municipal waste.

Yet, the process required for converting biomaterial into ethanol is currently economically inefficient. For example, microorganisms do not ferment all sugars in the raw material with the same efficiency, demanding to improve the metabolic fermentation pathways for industrialisation.

Another problem in modern biorefineries originates from the thermal sensitivity of the yeasts involved in fermentation. The first step of enzymatic hydrolysis occurs at 45-50°C, which is followed by the fermentation stage at 30-35°C. Hence, the reactors need to be cooled down in an energy-intensive process to reach the optimal growth and fermentation temperature of the yeast strains.

Additionally, yeast fermentation generates heat also requiring cooling of the fermentation reactor. If uncontrolled, the rising temperature would lead to reduced fermentation efficiency, altered product yields, and potential damage to the cells.


Microbial fermentation of cellobiose

As the primary source of sugars for biofuel production, cellulosic hydrolysates are derived from renewable biomass, which undergoes a series of biochemical processes. During enzymatic hydrolysis, the complex carbohydrates of the raw material are broken down into fermentable sugars, including cellodextrins, cellobiose, and xylose.

Cellodextrins are short chains of glucose molecules resulting from the partial hydrolysis of cellulose, while cellobiose consists of two linked glucose units. Xylose, on the other hand, is a pentose sugar derived from hemicellulose.

Several microorganisms, such as Saccharomyces cerevisiae, Myceliophthora thermophile, and Zymobacter palmae, have the natural or acquired abilities to effectively ferment the sugars and produce ethanol. As most of them require a lower fermentation temperature than at which hydrolysis occurs, the bioreactor need to be cooled. Other strains can be highly sensitive to some of the compounds formed during the process.

The study “Engineering of Ogataea polymorpha strains with ability for high-temperature alcoholic fermentation of cellobiose” in FEMS Yeast Research aimed to optimize the fermentation efficiency of a yeast strain already used to produce cellulose-derived biofuels.

Scheme of fuel ethanol production from lignocellulosic materials.
Scheme of fuel ethanol production from lignocellulosic materials. Courtesy of Justyna Ruchala.


Optimising the fermentation process of cellulosic hydrolysates

Ogataea polymorpha metabolises xylose efficiently and is one of the most thermotolerant yeasts known, with a maximum growth temperature above 50 °C. However, O. polymorpha is unable to ferment cellodextrins such as cellobiose.

For the study, genes encoding cellobiose transporters and the hydrolases ß-glucosidase or cellobiose phosphorylase were introduced into O. polymorpha. This gave the strains the ability to consume all fermentable sugars in lignocellulosic hydrolysates under high-temperature alcoholic conditions.

Even though cellobiose conversion was still incomplete and ethanol yields were comparatively low, these strains are promising tools for the sustainable biofuel production from lignocellulose. By demonstrating the ability of O. polymorpha strains to ferment sugars under industrial conditions, the study highlights the potential application of modified strains in real-world biorefinery settings while lowering the costs of cooling the reactor. Such strains represent another step towards sustainable biofuels and a healthier and greener planet.


About the authors of this blog

Roksolana Vasylyshyn holds a PhD in Microbiology and is currently working as an Associate Professor at the University of Rzeszów (Poland) and as a Research Fellow at the Institute of Cell Biology of the NAS of Ukraine. She is nominated for the MSCA4Ukraine program funded under the EU’s Marie Skłodowska-Curie Actions. She studies the regulation mechanisms of sugar metabolisms from cellulose hydrolysis to enhance bioethanol production in the methylotrophic thermotolerant yeast Ogataea (Hansenula) polymorpha, as well as the genetic control of the synthesis of other important metabolites.

Andriy Sibirny is the Director of the Institute of Cell Biology of the NAS of Ukraine and leader of many international scientific projects. He also volunteers as a Director of FEMS and is a Professor at the University of Rzeszów (Poland). He has described several new genes involved in autophagy and pexophagy in the model yeast organisms Komagataella phaffii, Yarrowia lipolytica, Ogataea polymorpha, and Saccharomyces cerevisiae. He studies high-temperature alcoholic fermentation of xylose and the synthesis of riboflavin and flavin nucleotides. He has isolated strains of O. polymorpha and Escherichia coli capable of producing various recombinant proteins of medical interest.

Justyna Ruchala is the Deputy Director of the Institute of Biotechnology at the University of Rzeszów (Poland). She participates in the international COST program (European cooperation in science and technology) and the WG4: Bioproducts generation from the sugars platform by non-conventional yeasts. She aims to develop thermotolerant Ogataea polymorpha strains capable of efficiently producing bioethanol from the planet’s second most abundant sugar, xylose, at high temperatures. She shed light on the molecular mechanisms of xylose fermentation transcription and the involvement of peroxisomal organelles. She also aims to engineer yeasts Komagataella phaffii and Candida famata to produce bacterial antibiotics aminoriboflavin and roseoflavin.

Olena Dmytruk is a specialist in biotechnology and biochemistry of non-conventional yeasts. She is an experienced Research Fellow at the Institute of Cell Biology of the NAS of Ukraine. Her main research topics involve the mechanisms of autophagic degradation of soluble cytosolic proteins in the methylotrophic yeast Komagataella phaffii (Pichia pastoris). Additionally, Olena works on the peroxisomal enzymes transketolase and transaldolase necessary for xylose fermentation in Ogataea polymorpha.

About this blog section

The section #FascinatingMicrobes for the #FEMSmicroBlog explains the science behind a paper and highlights the significance and broader context of a recent finding. One of the main goals is to share the fascinating spectrum of microbes across all fields of microbiology.

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