Blocking Mitophagy Does Not Significantly Improve Fuel Ethanol Production in Bioethanol Yeast Saccharomyces cerevisiae.

Ethanolic fermentation is frequently performed under conditions of low nitrogen. In Saccharomyces cerevisiae, nitrogen limitation induces macroautophagy, including the selective removal of mitochondria, also called mitophagy. Previous research showed that blocking mitophagy by deletion of the mitophagy-specific gene ATG32 increased the fermentation performance during the brewing of Ginjo sake. In this study, we tested if a similar strategy could enhance alcoholic fermentation in the context of fuel ethanol production from sugarcane in Brazilian biorefineries. Conditions that mimic the industrial fermentation process indeed induce Atg32-dependent mitophagy in cells of S. cerevisiae PE-2, a strain frequently used in the industry. However, after blocking mitophagy, no significant differences in CO2 production, final ethanol titers, or cell viability were observed after five rounds of ethanol fermentation, cell recycling, and acid treatment, which is commonly performed in sugarcane biorefineries. To test if S. cerevisiae's strain background influenced this outcome, cultivations were carried out in a synthetic medium with strains PE-2, Ethanol Red (industrial), and BY (laboratory) with and without a functional ATG32 gene and under oxic and oxygen restricted conditions. Despite the clear differences in sugar consumption, cell viability, and ethanol titers, among the three strains, we did not observe any significant improvement in fermentation performance related to the blocking of mitophagy. We concluded, with caution, that the results obtained with Ginjo sake yeast were an exception and cannot be extrapolated to other yeast strains and that more research is needed to ascertain the role of autophagic processes during fermentation. IMPORTANCE Bioethanol is the largest (per volume) ever biobased bulk chemical produced globally. The fermentation process is well established, and industries regularly attain nearly 85% of maximum theoretical yields. However, because of the volume of fuel produced, even a small improvement will have huge economic benefits. To this end, besides already implemented process improvements, various free energy conservation strategies have been successfully exploited at least in laboratory strains to increase ethanol yields and decrease byproduct formation. Cellular housekeeping processes have been an almost unexplored territory in strain improvement. It was previously reported that blocking mitophagy by deletion of the mitophagy receptor gene ATG32 in Saccharomyces cerevisiae led to a 2.1% increase in final ethanol titers during Japanese sake fermentation. We found in two commercially used bioethanol strains (PE-2 and Ethanol Red) that ATG32 deficiency does not lead to a significant improvement in cell viability or ethanol levels during fermentation with molasses or in a synthetic complete medium. More research is required to ascertain the role of autophagic processes during fermentation conditions.

Ethanol yield calculations in biorefineries.

The ethanol yield on sugar during alcoholic fermentation allows for diverse interpretation in academia and industry. There are several different ways to calculate this parameter, which is the most important one in this industrial bioprocess and the one that should be maximized, as reported by Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91). On the one hand, the various methods currently employed in industry provide dissimilar results, and recent evidence shows that yield has been consistently overestimated in Brazilian sugarcane biorefineries. On the other hand, in academia, researchers often lack information on all the intricate aspects involved in calculating the ethanol yield in industry. Here, we comment on these two aspects, using fuel ethanol production from sugarcane in Brazilian biorefineries as an example, and taking the work of Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91.) as a starting point. Our work is an attempt to demystify some common beliefs and to foster closer interaction between academic and industrial professionals from the fermentation sector. Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91).

The protective role of intracellular glutathione in Saccharomyces cerevisiae during lignocellulosic ethanol production.

To enhance the competitiveness of industrial lignocellulose ethanol production, robust enzymes and cell factories are vital. Lignocellulose derived streams contain a cocktail of inhibitors that drain the cell of its redox power and ATP, leading to a decrease in overall ethanol productivity. Many studies have attempted to address this issue, and we have shown that increasing the glutathione (GSH) content in yeasts confers tolerance towards lignocellulose inhibitors, subsequently increasing the ethanol titres. However, GSH levels in yeast are limited by feedback inhibition of GSH biosynthesis. Multidomain and dual functional enzymes exist in several bacterial genera and they catalyse the GSH biosynthesis in a single step without the feedback inhibition. To test if even higher intracellular glutathione levels could be achieved and if this might lead to increased tolerance, we overexpressed the genes from two bacterial genera and assessed the recombinants in simultaneous saccharification and fermentation (SSF) with steam pretreated spruce hydrolysate containing 10% solids. Although overexpressing the heterologous genes led to a sixfold increase in maximum glutathione content (18 µmol gdrycellmass-1) compared to the control strain, this only led to a threefold increase in final ethanol titres (8.5 g L- 1). As our work does not conclusively indicate the cause-effect of increased GSH levels towards ethanol titres, we cautiously conclude that there is a limit to cellular fitness that could be accomplished via increased levels of glutathione.

A microbubble-sparged yeast propagation-fermentation process for bioethanol production.

BACKGROUND: Industrial biotechnology will play an increasing role in creating a more sustainable global economy. For conventional aerobic bioprocesses supplying O2 can account for 15% of total production costs. Microbubbles (MBs) are micron-sized bubbles that are widely used in industry and medical imaging. Using a fluidic oscillator to generate energy-efficient MBs has the potential to decrease the costs associated with aeration. However, little is understood about the effect of MBs on microbial physiology. To address this gap, a laboratory-scale MB-based Saccharomyces cerevisiae Ethanol Red propagation-fermentation bioethanol process was developed and analysed. RESULTS: Aeration with MBs increased O2 transfer to the propagation cultures. Titres and yields of bioethanol in subsequent anaerobic fermentations were comparable for MB-propagated and conventional, regular bubble (RB)-propagated yeast. However, transcript profiling showed significant changes in gene expression in the MB-propagated yeast compared to those propagated using RB. These changes included up-regulation of genes required for ergosterol biosynthesis. Ergosterol contributes to ethanol tolerance, and so the performance of MB-propagated yeast in fed-batch fermentations sparged with 1% O2 as either RBs or MBs were tested. The MB-sparged yeast retained higher levels of ergosteryl esters during the fermentation phase, but this did not result in enhanced viability or ethanol production compared to ungassed or RB-sparged fermentations. CONCLUSIONS: The performance of yeast propagated using energy-efficient MB technology in bioethanol fermentations is comparable to that of those propagated conventionally. This should underpin the future development of MB-based commercial yeast propagation.

Neither 1G nor 2G fuel ethanol: setting the ground for a sugarcane-based biorefinery using an iSUCCELL yeast platform.

First-generation (1G) fuel ethanol production in sugarcane-based biorefineries is an established economic enterprise in Brazil. Second-generation (2G) fuel ethanol from lignocellulosic materials, though extensively investigated, is currently facing severe difficulties to become economically viable. Some of the challenges inherent to these processes could be resolved by efficiently separating and partially hydrolysing the cellulosic fraction of the lignocellulosic materials into the disaccharide cellobiose. Here, we propose an alternative biorefinery, where the sucrose-rich stream from the 1G process is mixed with a cellobiose-rich stream in the fermentation step. The advantages of mixing are 3-fold: (i) decreased concentrations of metabolic inhibitors that are typically produced during pretreatment and hydrolysis of lignocellulosic materials; (ii) decreased cooling times after enzymatic hydrolysis prior to fermentation; and (iii) decreased availability of free glucose for contaminating microorganisms and undesired glucose repression effects. The iSUCCELL platform will be built upon the robust Saccharomyces cerevisiae strains currently present in 1G biorefineries, which offer competitive advantage in non-aseptic environments, and into which intracellular hydrolyses of sucrose and cellobiose will be engineered. It is expected that high yields of ethanol can be achieved in a process with cell recycling, lower contamination levels and decreased antibiotic use, when compared to current 2G technologies.

Bacterial cellulose: Biosynthesis, production, and applications.

Bacterial cellulose (BC) is a natural polymer produced by the acetic acid producing bacterium and has gathered much interest over the last decade for its biomedical and biotechnological applications. Unlike the plant derived cellulose nanofibres, which require pretreatment to deconstruct the recalcitrant lignocellulosic network, BC are 100% pure, and are extruded by cells as nanofibrils. Moreover, these nanofibrils can be converted to macrofibers that possess excellent material properties, surpassing even the strength of steel, and can be used as substitutes for fossil fuel derived synthetic fibers. The focus of the review is to present the fundamental long-term research on the influence of environmental factors on the organism's BC production capabilities, the production methods that are available for scaling up/scaled-up processes, and its use as a bulk commodity or for biomedical applications.

Forever panting and forever growing: physiology of Saccharomyces cerevisiae at extremely low oxygen availability in the absence of ergosterol and unsaturated fatty acids.

We sought to investigate how far the growth of Saccharomyces cerevisiae under full anaerobiosis is dependent on the widely used anaerobic growth factors (AGF) ergosterol and oleic acid. A continuous cultivation setup was employed and, even forcing ultrapure N2 gas through an O2 trap upstream of the bioreactor, neither cells from S. cerevisiae CEN.PK113-7D (a lab strain) nor from PE-2 (an industrial strain) washed out after an aerobic-to-anaerobic switch in the absence of AGF. S. cerevisiae PE-2 seemed to cope better than the laboratory strain with this extremely low O2 availability, since it presented higher biomass yield, lower specific rates of glucose consumption and CO2 formation, and higher survival at low pH. Lipid (fatty acid and sterol) composition dramatically altered when cells were grown anaerobically without AGF: saturated fatty acid, squalene and lanosterol contents increased, when compared to either cells grown aerobically or anaerobically with AGF. We concluded that these lipid alterations negatively affect cell viability during exposure to low pH or high ethanol titers.

Lignin-first biomass fractionation using a hybrid organosolv - Steam explosion pretreatment technology improves the saccharification and fermentability of spruce biomass.

For a transition to a sustainable society, fuels, chemicals, and materials should be produced from renewable resources. Lignocellulosic biomass constitutes an abundant and renewable feedstock; however, its successful application in a biorefinery requires efficient fractionation into its components; cellulose, hemicellulose and lignin. Here, we demonstrate that a newly established hybrid organosolv - steam explosion pretreatment can effectively fractionate spruce biomass to yield pretreated solids with high cellulose (72% w/w) and low lignin (delignification up to 79.4% w/w) content. The cellulose-rich pretreated solids present high saccharification yields (up to 61% w/w) making them ideal for subsequent bioconversion processes. Moreover, under high-gravity conditions (22% w/w) we obtained an ethanol titer of 61.7 g/L, the highest so far reported for spruce biomass. Finally, the obtained high-purity lignin is suitable for various advanced applications. In conclusion, hybrid organosolv pretreatment could offer a closed-loop biorefinery while simultaneously adding value to all biomass components.

A comparative study of the enzymatic hydrolysis of batch organosolv-pretreated birch and spruce biomass.

A shift towards a sustainable and green society is vital to reduce the negative effects of climate change associated with increased CO2 emissions. Lignocellulosic biomass is both renewable and abundant, but is recalcitrant to deconstruction. Among the methods of pretreatment available, organosolv (OS) delignifies cellulose efficiently, significantly improving its digestibility by enzymes. We have assessed the hydrolysability of the cellulose-rich solid fractions from OS-pretreated spruce and birch at 2% w/v loading (dry matter). Almost complete saccharification of birch was possible with 80 mg enzyme preparation/gsolids (12 FPU/gsolids), while the saccharification yield for spruce was only 70%, even when applying 60 FPU/gsolids. As the cellulose content is enriched by OS, the yield of glucose was higher than in their steam-exploded counterparts. The hydrolysate was a transparent liquid due to the absence of phenolics and was also free from inhibitors. OS pretreatment holds potential for use in a large-scale, closed-loop biorefinery producing fuels from the cellulose fraction and platform chemicals from the hemicellulose and lignin fractions respectively.

A novel hybrid organosolv: steam explosion method for the efficient fractionation and pretreatment of birch biomass.

BACKGROUND: The main role of pretreatment is to reduce the natural biomass recalcitrance and thus enhance saccharification yield. A further prerequisite for efficient utilization of all biomass components is their efficient fractionation into well-defined process streams. Currently available pretreatment methods only partially fulfill these criteria. Steam explosion, for example, excels as a pretreatment method but has limited potential for fractionation, whereas organosolv is excellent for delignification but offers poor biomass deconstruction. RESULTS: In this article, a hybrid method combining the cooking and fractionation of conventional organosolv pretreatment with the implementation of an explosive discharge of the cooking mixture at the end of pretreatment was developed. The effects of various pretreatment parameters (ethanol content, duration, and addition of sulfuric acid) were evaluated. Pretreatment of birch at 200 °C with 60% v/v ethanol and 1% w/wbiomass H2SO4 was proven to be the most efficient pretreatment condition yielding pretreated solids with 77.9% w/w cellulose, 8.9% w/w hemicellulose, and 7.0 w/w lignin content. Under these conditions, high delignification of 86.2% was demonstrated. The recovered lignin was of high purity, with cellulose and hemicellulose contents not exceeding 0.31 and 3.25% w/w, respectively, and ash to be < 0.17% w/w in all cases, making it suitable for various applications. The pretreated solids presented high saccharification yields, reaching 68% at low enzyme load (6 FPU/g) and complete saccharification at high enzyme load (22.5 FPU/g). Finally, simultaneous saccharification and fermentation (SSF) at 20% w/w solids yielded an ethanol titer of 80 g/L after 192 h, corresponding to 90% of the theoretical maximum. CONCLUSIONS: The novel hybrid method developed in this study allowed for the efficient fractionation of birch biomass and production of pretreated solids with high cellulose and low lignin contents. Moreover, the explosive discharge at the end of pretreatment had a positive effect on enzymatic saccharification, resulting in high hydrolyzability of the pretreated solids and elevated ethanol titers in the following high-gravity SSF. To the best of our knowledge, the ethanol concentration obtained with this method is the highest so far for birch biomass.

Correction to: Anaerobiosis revisited: growth of Saccharomyces cerevisiae under extremely low oxygen availability.

The published online version contains mistake in Figure1. In the x-axis, instead of "1000", the number should be "100".

Anaerobiosis revisited: growth of Saccharomyces cerevisiae under extremely low oxygen availability.

The budding yeast Saccharomyces cerevisiae plays an important role in biotechnological applications, ranging from fuel ethanol to recombinant protein production. It is also a model organism for studies on cell physiology and genetic regulation. Its ability to grow under anaerobic conditions is of interest in many industrial applications. Unlike industrial bioreactors with their low surface area relative to volume, ensuring a complete anaerobic atmosphere during microbial cultivations in the laboratory is rather difficult. Tiny amounts of O2 that enter the system can vastly influence product yields and microbial physiology. A common procedure in the laboratory is to sparge the culture vessel with ultrapure N2 gas; together with the use of butyl rubber stoppers and norprene tubing, O2 diffusion into the system can be strongly minimized. With insights from some studies conducted in our laboratory, we explore the question 'how anaerobic is anaerobiosis?'. We briefly discuss the role of O2 in non-respiratory pathways in S. cerevisiae and provide a systematic survey of the attempts made thus far to cultivate yeast under anaerobic conditions. We conclude that very few data exist on the physiology of S. cerevisiae under anaerobiosis in the absence of the anaerobic growth factors ergosterol and unsaturated fatty acids. Anaerobicity should be treated as a relative condition since complete anaerobiosis is hardly achievable in the laboratory. Ideally, researchers should provide all the details of their anaerobic set-up, to ensure reproducibility of results among different laboratories.

A simple scaled down system to mimic the industrial production of first generation fuel ethanol in Brazil.

Although first-generation fuel ethanol is produced in Brazil from sugarcane-based raw materials with high efficiency, there is still little knowledge about the microbiology, the biochemistry and the molecular mechanisms prevalent in the non-aseptic fermentation environment. Learning-by-doing has hitherto been the strategy to improve the process so far, with further improvements requiring breakthrough technologies. Performing experiments at an industrial scale are often expensive, complicated to set up and difficult to reproduce. Thus, developing an appropriate scaled down system for this process has become a necessity. In this paper, we present the design and demonstration of a simple and effective laboratory-scale system mimicking the industrial process used for first generation (1G) fuel ethanol production in the Brazilian sugarcane mills. We benchmarked this system via the superior phenotype of the Saccharomyces cerevisiae PE-2 strain, compared to other strains from the same species: S288c, baker's yeast, and CEN.PK113-7D. We trust that such a system can be easily implemented in different laboratories worldwide, and will allow a better understanding of the S. cerevisiae strains that can persist and dominate in this industrial, non-aseptic and peculiar environment.

Sucrose and Saccharomyces cerevisiae: a relationship most sweet.

Sucrose is an abundant, readily available and inexpensive substrate for industrial biotechnology processes and its use is demonstrated with much success in the production of fuel ethanol in Brazil. Saccharomyces cerevisiae, which naturally evolved to efficiently consume sugars such as sucrose, is one of the most important cell factories due to its robustness, stress tolerance, genetic accessibility, simple nutrient requirements and long history as an industrial workhorse. This minireview is focused on sucrose metabolism in S. cerevisiae, a rather unexplored subject in the scientific literature. An analysis of sucrose availability in nature and yeast sugar metabolism was performed, in order to understand the molecular background that makes S. cerevisiae consume this sugar efficiently. A historical overview on the use of sucrose and S. cerevisiae by humans is also presented considering sugarcane and sugarbeet as the main sources of this carbohydrate. Physiological aspects of sucrose consumption are compared with those concerning other economically relevant sugars. Also, metabolic engineering efforts to alter sucrose catabolism are presented in a chronological manner. In spite of its extensive use in yeast-based industries, a lot of basic and applied research on sucrose metabolism is imperative, mainly in fields such as genetics, physiology and metabolic engineering.

Mitochondrial import and accumulation of alpha-synuclein impair complex I in human dopaminergic neuronal cultures and Parkinson disease brain.

Alpha-synuclein, a protein implicated in the pathogenesis of Parkinson disease (PD), is thought to affect mitochondrial functions, although the mechanisms of its action remain unclear. In this study we show that the N-terminal 32 amino acids of human alpha-synuclein contain cryptic mitochondrial targeting signal, which is important for mitochondrial targeting of alpha-synuclein. Mitochondrial imported alpha-synuclein is predominantly associated with the inner membrane. Accumulation of wild-type alpha-synuclein in the mitochondria of human dopaminergic neurons caused reduced mitochondrial complex I activity and increased production of reactive oxygen species. However, these defects occurred at an early time point in dopaminergic neurons expressing familial alpha-synuclein with A53T mutation as compared with wild-type alpha-synuclein. Importantly, alpha-synuclein that lacks mitochondrial targeting signal failed to target to the mitochondria and showed no detectable effect on complex I function. The PD relevance of these results was investigated using mitochondria of substantia nigra, striatum, and cerebellum of postmortem late-onset PD and normal human brains. Results showed the constitutive presence of approximately 14-kDa alpha-synuclein in the mitochondria of all three brain regions of normal subjects. Mitochondria of PD-vulnerable substantia nigra and striatum but not cerebellum from PD subjects showed significant accumulation of alpha-synuclein and decreased complex I activity. Analysis of mitochondria from PD brain and alpha-synuclein expressing dopaminergic neuronal cultures using blue native gel electrophoresis and immunocapture technique showed the association of alpha-synuclein with complex I. These results provide evidence that mitochondrial accumulated alpha-synuclein may interact with complex I and interfere with its functions.