Search Results (22)

Bioethanol from sugarcane bagasse is a promising second-generation biofuel due to its abundance as a sugar industry by-product. Herein, enzymatic hydrolysate obtained from sugarcane bagasse pretreated with optimized hydrothermal alkaline sulfite (HAS) was evaluated for its fermentability using Saccharomyces cerevisiae PE-2 and Scheffersomyces stipitis CBS 5773. The HAS pretreatment achieved a high delignification rate (63%), resulting in a cellulose- and hemicellulose-enriched substrate (55% and 27%, respectively). While the cellulose content remained relatively constant, hemicellulose content was reduced by 25%, with significant removal of acetyl groups (80%) and arabinan groups (39%). The pretreated bagasse exhibited high digestibility, applying 10 FPU (filter paper unit) cellulase together with 10 CBU (cellobiose unit) β-glucosidase per gram of dry bagasse in the hydrolysis step, yielding 72% glucan and 66% xylan conversion within 72 h. The resulting hydrolysate was efficiently fermented by S. cerevisiae and S. stipitis, achieving ethanol yields of 0.51 and 0.43 g/g of sugars, respectively. The fermentation kinetics were comparable to those observed in a synthetic medium containing pure sugars, demonstrating the effectiveness of HAS pretreatment in generating readily fermentable, carbohydrate-rich substrates. HAS pretreatment enabled improved conversion of sugarcane bagasse into fermentation-ready sugars, constituting a potential resource for bioethanol synthesis applying both S. cerevisiae and S. stipitis in the future. Full article

Lactate is a valuable compound used in food, chemical, and pharmaceutical industries. High-value, optically pure L- or D-lactate can be synthesized microbially via specific dehydrogenases. The non-conventional yeast Scheffersomyces stipitis, which is known for fermenting both hexoses and pentoses, is a promising host for biochemical production from lignocellulosic biomass but does not naturally produce lactate. In this study, we engineered S. stipitis to produce lactate by expressing two codon-optimized bacterial L-lactate dehydrogenase genes under the control of strong native promoters. The engineered strain produced 7.42 g/L (0.46 g/g yield) and 11.67 g/L (0.58 g/g yield) lactate from glucose and xylose, respectively. The highest titer, 19.27 g/L (0.52 g/g yield), was achieved from 50 g/L xylose after 74 h. Increasing the fermentation temperature from 28 °C to 32 °C improved yield by 30%, while a neutralizing agent further enhanced yield by 25% and prevented lactate degradation following carbon depletion. Although the wildtype strain produced a significant amount of ethanol on both glucose and xylose, the engineered strain produced ethanol as a side product exclusively on glucose and not on xylose. This phenomenon could be advantageous for biotechnological applications and may reflect shifts in gene expression depending on the carbon source or even on the presence of lactate. Full article

This study evaluates the feasibility of using banana peels as a substrate for cultivating fodder yeast biomass. Banana peels (BPs), representing approximately 38% of the total fruit weight, are rich in cellulose and hemicellulose, thus presenting a significant opportunity for valorisation. The study investigates the effects of microwave and ultrasound treatments on the hydrolysis efficiency of banana peels and the subsequent cultivation of yeast. Two yeast strains, Scheffersomyces stipitis and Meyerozyma guilliermondii, were cultivated in hydrolysates prepared using various methods, including acid–thermal, enzymatic, microwave, and ultrasound treatments. The results demonstrate that enzymatic hydrolysis following microwave or ultrasound pretreatment significantly enhances sugar release, supporting higher biomass yields. The maximum biomass concentration achieved was 7.68 g DM/L, with crude protein content reaching up to 45.46% DM. These results indicate that banana peels can be effectively utilised for single-cell protein production, providing a sustainable alternative for animal feed. The study underscores the potential of integrating microwave and ultrasound technologies in bioprocessing to enhance the efficiency and environmental sustainability of yeast cultivation. Full article

Poland is one of the leading apple-producing countries, both in Europe and around the world. One of the main byproducts of apple processing is pomace, which can account for 20–35% of the harvested apples. Pomace is a potential source of many valuable bioactive components and can also serve as a food ingredient, either directly or indirectly (after bioconversion with fodder yeast). This study aimed to evaluate the possibility of converting polysaccharides contained in apple pomace to yeast biomass. Meyerozyma guilliermondii and Scheffersomyces stipitis yeasts were grown in a medium prepared by pretreatment of the raw material with water or 2% sulphuric acid at 120 °C. Subsequently, enzymatic hydrolysis was performed using a Cellic CTec2 preparation at 30 °C or 50 °C. The resulting hydrolysates were enriched with ammonium salts, and shaken yeast cultures were incubated at 30 °C for 72 h. Based on the results, it can be concluded that acid pretreatment of apple pomace is more effective than water pretreatment under the same time and temperature conditions. The Meyerozyma guilliermondii strain grows in apple pomace hydrolysates more efficiently (16.29 g/L) than Scheffersomyces stipitis cells do (14.63 g/L). Full article

In this study, the green macroalgae Ulva rigida, which contains 34.9% carbohydrates, underwent treatment with commercial hydrolytic enzymes. This treatment yielded a hydrolysate that contained 23 ± 0.6 g·L⁻¹ of glucose, which was subsequently fermented with Saccharomyces cerevisiae. The fermentation process resulted in an ethanol concentration of 9.55 ± 0.20 g·L⁻¹. The optimal conditions for ethanol production by S. cerevisiae were identified as follows: non-sterilized conditions, an absence of enrichment, and using an inoculum size of 118 mg·L⁻¹. Under these conditions, the fermentation of the green macroalgal hydrolysate achieved a remarkable conversion efficiency of 80.78%. The ethanol o/t ratio, namely the ratios of the experimental to theoretical ethanol produced, for Scheffersomyces stipitis, Candida guilliermondii, Kluyveromyces marxianus, and S. cerevisiae after 48 h of fermentation were 52.25, 63.20, 70.49, and 82.87%, respectively. Furthermore, S. cerevisiae exhibited the best outcomes in terms of ethanol production (9.35 g·L⁻¹) and conversion efficiency (80.78%) after 24 h (optimal time) of fermentation. Full article

Despite the significant progress in the research, the problem of finding an efficient method for producing bioethanol from renewable lignocellulosic waste materials remains unresolved. Our investigation aimed to assess the efficacy of ethanol production from sugar beet pulp (SBP) utilising various approaches, including pretreatment variations, enzymatic processes, and microbial hydrolysis. Our research involved using the post-cultivation concentrate of T. viride LOCK 0588 grown in the SBP-based medium as a source of enzymes. The SBP hydrolysis process was carried out for 48 h at 50 °C. The quantity of sugar released, up to 61 g dm⁻³, through the utilisation of this extract proved to be on par with the outcomes achieved by the application of the commercial Cellic Ctec2 preparation. The final yields of the ethanol production with the use of the coculture of S. cerevisiae (Ethanol Red) and Scheffersomyces stipitis LOCK 0047 strain were in the range 5.1 ± 0.11 kg 100 kg⁻¹ ÷ 5.38 ± 0.11 kg 100 kg⁻¹. These results provide a solid basis for improving larger-scale industrial procedures that involve converting SBP into bioethanol using a cost-efficient approach of microbial hydrolysis with T. viride and a blend of pentose and hexose fermenting yeast. Full article

Rapeseed meal (RM) is produced in large quantities as a byproduct of oil extraction from rapeseeds. However, the efficient utilization of RM as animal feed is limited by its low metabolizable energy, poor palatability, and high levels of fiber and anti-nutritional components. Here, we investigate the potential of enriching RM with single-cell protein through fermentation with conventional and unconventional yeasts. The process of simultaneous saccharification and fermentation improved the parameters of the waste biomass, especially the protein content, while reducing the amount of crude fiber and enhancing the biotransformation of isoflavone compounds present in the waste. Fermentation yielded the highest protein gain for the Saccharomyces cerevisiae Ethanol Red strain (ΔN = 2.38%) at a biomass load of 12.5 g and for Scheffersomyces stipitis (ΔN = 2.34%) at an enzyme dose of 0.125 mL/10 g DM. The crude fiber content (CF) was reduced by 2.55–7.18%. The simultaneous saccharification and fermentation (SSF) process resulted in the conversion of isoflavones to forms with fewer adverse effects and a lower estrogenic activity. The results show the potential of using RM as a substrate for making a nutritionally improved feed components. Full article

An example of the implementation of the principles of the circular economy is the use of sugar beet pulp as animal feed. Here, we investigate the possible use of yeast strains to enrich waste biomass in single-cell protein (SCP). The strains were evaluated for yeast growth (pour plate method), protein increment (Kjeldahl method), assimilation of free amino nitrogen (FAN), and reduction of crude fiber content. All the tested strains were able to grow on hydrolyzed sugar beet pulp-based medium. The greatest increases in protein content were observed for Candida utilis LOCK0021 and Saccharomyces cerevisiae Ethanol Red (ΔN = 2.33%) on fresh sugar beet pulp, and for Scheffersomyces stipitis NCYC1541 (ΔN = 3.04%) on dried sugar beet pulp. All the strains assimilated FAN from the culture medium. The largest reductions in the crude fiber content of the biomass were recorded for Saccharomyces cerevisiae Ethanol Red (Δ = 10.89%) on fresh sugar beet pulp and Candida utilis LOCK0021 (Δ = 15.05%) on dried sugar beet pulp. The results show that sugar beet pulp provides an excellent matrix for SCP and feed production. Full article

Cytosolic pyruvate is an essential metabolite in lactic acid production during microbial fermentation. However, under aerobiosis, pyruvate is transported to the mitochondrial matrix by the mitochondrial pyruvate carrier (MPC) and oxidized in cell respiration. Previous reports using Saccharomyces cerevisiae or Aspergillus oryzae have shown that the production of pyruvate-derived chemicals is improved by deleting the MPC1 gene. A previous lactate-producing K. phaffii strain engineered by our group was used as a host for the deletion of the MPC1 gene. In addition, the expression of a bacterial hemoglobin gene under the alcohol dehydrogenase 2 promoter from Scheffersomyces stipitis, known to work as a hypoxia sensor, was used to evaluate whether aeration would supply enough oxygen to meet the metabolic needs during lactic acid production. However, unlike S. cerevisiae and A. oryzae, the deletion of Mpc1 had no significant impact on lactic acid production but negatively affected cell growth in K. phaffii strains. Furthermore, the relative quantification of the VHb gene revealed that the expression of hemoglobin was detected even in aerobic cultivation, which indicates that the demand for oxygen in the bioreactor could result in functional hypoxia. Overall, the results add to our previously published ones and show that blocking cell respiration using hypoxia is more suitable than deleting Mpc for producing lactic acid in K. phaffii. Full article

Economic conversion of biomass to biofuels and chemicals requires efficient and complete utilization of xylose. Saccharomyces cerevisiae strains engineered for xylose utilization are still considerably limited in their overall ability to metabolize xylose. In this study, we identified causative mutations resulting in improved xylose fermentation of an adapted S. cerevisiae strain expressing codon-optimized xylose isomerase and xylulokinase genes from the rumen bacterium Prevotella ruminicola. Genome sequencing identified single-nucleotide polymorphisms in seven open reading frames. Tetrad analysis showed that mutations in both PBS2 and PHO13 genes were required for increased xylose utilization. Single deletion of either PBS2 or PHO13 did not improve xylose utilization in strains expressing the xylose isomerase pathway. Saccharomyces can also be engineered for xylose metabolism using the xylose reductase/xylitol dehydrogenase genes from Scheffersomyces stipitis. In strains expressing the xylose reductase pathway, single deletion of PHO13 did show a significant increase xylose utilization, and further improvement in growth and fermentation was seen when PBS2 was also deleted. These findings will extend the understanding of metabolic limitations for xylose utilization in S. cerevisiae as well as understanding of how they differ among strains engineered with two different xylose utilization pathways. Full article

Non-conventional yeasts are increasingly being investigated and used as producers in biotechnological processes which often offer advantages in comparison to traditional and well-established systems. Most biotechnologically interesting non-conventional yeasts belong to the Saccharomycotina subphylum, including those already in use (Pichia pastoris, Yarrowia lypolitica, etc.), as well as those that are promising but as yet insufficiently characterized. Moreover, for many of these yeasts the basic tools of genetic engineering needed for strain construction, including a procedure for efficient genetic transformation, heterologous protein expression and precise genetic modification, are lacking. The first aim of this study was to construct a set of integrative and replicative plasmids which can be used in various yeasts across the Saccharomycotina subphylum. Additionally, we demonstrate here that the electroporation procedure we developed earlier for transformation of B. bruxellensis can be applied in various yeasts which, together with the constructed plasmids, makes a solid starting point when approaching a transformation of yeasts form the Saccharomycotina subphylum. To provide a proof of principle, we successfully transformed three species from the Schwanniomyces genus (S. polymorphus var. polymorphus, S. polymorphus var. africanus and S. pseudopolymorphus) with high efficiencies (up to 8 × 10³ in case of illegitimate integration of non-homologous linear DNA and up to 4.7 × 10⁵ in case of replicative plasmid). For the latter two species this is the first reported genetic transformation. Moreover, we found that a plasmid carrying replication origin from Scheffersomyces stipitis can be used as a replicative plasmid for these three Schwanniomyces species. Full article

Advanced biofuels incorporation into the transportation sector, particularly cellulosic bioethanol, is crucial for attaining carbon neutrality by 2050, contributing to climate changes mitigation and wastes minimization. The world needs biofuel to be commercially available to tackle the socioeconomic challenges coming from the continued use of fossil fuels. Cynara cardunculus (cardoon) is a cheap lignocellulosic raw biomass that easily grows in Mediterraneous soils and is a potential renewable resource for a biorefinery. This work aimed to study the bioethanol production from cardoon hemicellulosic hydrolysates, which originated from dilute sulfuric acid hydrolysis pretreatment. A detoxification step to remove released microbial fermentative inhibitors was evaluated by using both activated carbon adsorption and a nanofiltration membrane system. The Scheffersomyces stipitis CBS5773 yeast and the modified Escherichia coli MS04 fermentation performances at different experimental conditions were compared. The promising results with E. coli, using detoxified cardoon by membrane nanofiltration, led to a bioethanol volumetric productivity of 0.30 g·L⁻¹·h⁻¹, with a conversion efficiency of 94.5%. Regarding the S. stipitis, in similar fermentation conditions, volumetric productivity of 0.091 g·L⁻¹·h⁻¹ with a conversion efficiency of 64.9% was obtained. Concluding, the production of bioethanol through detoxification of hemicellulosic cardoon hydrolysate presents a suitable alternative for the production of second-generation bioethanol, especially using the modified E. coli. Full article

In previous work, we developed a Saccharomyces cerevisiae strain (DLG-K1) lacking the main monosaccharide transporters (hxt-null) and displaying high xylose reductase, xylitol dehydrogenase and xylulokinase activities. This strain proved to be a useful chassis strain to study new glucose/xylose transporters, as SsXUT1 from Scheffersomyces stipitis. Proteins with high amino acid sequence similarity (78–80%) to SsXUT1 were identified from Spathaspora passalidarum and Spathaspora arborariae genomes. The characterization of these putative transporter genes (SpXUT1 and SaXUT1, respectively) was performed in the same chassis strain. Surprisingly, the cloned genes could not restore the ability to grow in several monosaccharides tested (including glucose and xylose), but after being grown in maltose, the uptake of ¹⁴C-glucose and ¹⁴C-xylose was detected. While SsXUT1 lacks lysine residues with high ubiquitinylation potential in its N-terminal domain and displays only one in its C-terminal domain, both SpXUT1 and SaXUT1 transporters have several such residues in their C-terminal domains. A truncated version of SpXUT1 gene, deprived of the respective 3′-end, was cloned in DLG-K1 and allowed growth and fermentation in glucose or xylose. In another approach, two arrestins known to be involved in the ubiquitinylation and endocytosis of sugar transporters (ROD1 and ROG3) were knocked out, but only the rog3 mutant allowed a significant improvement of growth and fermentation in glucose when either of the XUT permeases were expressed. Therefore, for the efficient heterologous expression of monosaccharide (e.g., glucose/xylose) transporters in S. cerevisiae, we propose either the removal of lysines involved in ubiquitinylation and endocytosis or the use of chassis strains hampered in the specific mechanism of membrane protein turnover. Full article

In order to exploit a fast-growing Paulownia hardwood as an energy crop, a xylose-enriched hydrolysate was obtained in this work to increase the ethanol concentration using the hemicellulosic fraction, besides the already widely studied cellulosic fraction. For that, Paulownia elongata x fortunei was submitted to autohydrolysis treatment (210 °C or S₀ of 4.08) for the xylan solubilization, mainly as xylooligosaccharides. Afterwards, sequential stages of acid hydrolysis, concentration, and detoxification were evaluated to obtain fermentable sugars. Thus, detoxified and non-detoxified hydrolysates (diluted or not) were fermented for ethanol production using a natural xylose-consuming yeast, Scheffersomyces stipitis CECT 1922, and an industrial Saccharomyces cerevisiae MEC1133 strain, metabolic engineered strain with the xylose reductase/xylitol dehydrogenase pathway. Results from fermentation assays showed that the engineered S. cerevisiae strain produced up to 14.2 g/L of ethanol (corresponding to 0.33 g/g of ethanol yield) using the non-detoxified hydrolysate. Nevertheless, the yeast S. stipitis reached similar values of ethanol, but only in the detoxified hydrolysate. Hence, the fermentation data prove the suitability and robustness of the engineered strain to ferment non-detoxified liquor, and the appropriateness of detoxification of liquor for the use of less robust yeast. In addition, the success of hemicellulose-to-ethanol production obtained in this work shows the Paulownia biomass as a suitable renewable source for ethanol production following a suitable fractionation process within a biorefinery approach. Full article

The aim of this paper is to study the effect of reinking and pretreatment of waste banknote paper on its usability in the bioethanol production process. To this end, the tensile strength of worn banknote paper was first studied at different pH values. The sample with the lowest tensile strength was considered for the next sections. In the deinking process, NaOH at different concentrations (1%, 2%, 3%, and 4%) and in combination with ultrasonic treatment was applied. After deinking the pulp, two acidic and alkaline chemical pretreatments with concentrations of 1%, 2%, 3%, and 4% were used independently and in combination with ultrasonic. Enzymatic hydrolysis, following fermentation with Scheffersomyces stipitis, and crystallinity measurements were used to confirm the efficiency of the pretreatments. RSM Design Expert software was used to determine the optimal values by considering the three variables—enzyme loading, ultrasonic loading, and contact time for waste paper deinked (WPD) and waste paper blank (WPB) pulps. The results indicated that repulping was the most efficient at pH = 2. In deinking, the highest brightness was obtained using 3% NaOH in combination with ultrasonic. Between the acid and alkaline pretreatment, the acid treatment was more appropriate according to the resulting sugar concentration and weight loss. XRD tests confirmed that the lowest crystallinity index was obtained in the sample pretreated with 4% sulfuric acid in combination with ultrasonic. The highest sugar concentration in the enzymatic hydrolysis step was 92 g/L for WPD and 81 g/L for WPB. For the fermentation at 96 h, the highest ethanol concentration and process efficiency achieved were 38 g/L and 80.9% for WPD and 31 g/L and 75.04% for WPB, respectively. Our research shows that the deinking process can widen the utilization potential of waste banknote paper in biorefinery processes. Full article