Vinasse: current concepts, challenges and opportunities for the sustainability

The objective was to highlight the historical documents that foresee the use and abuse of ethanol production (vinasse) in Brazil according to literature published in the period 1934-2020. The methodology used was documentary, the review and analysis of which brought with it the synthesis and perspectives of vinasse as well as its uses and abuses. Results: a) there are legal instruments in Brzil since 1934 whose goal is to care for the environment; b) about in 1978 was prohibited the direct dumping of stillage into bodies of water and c) in 2011 environmental guidelines were provided on this topic to achieve sustainability in the Brazilian space. Conclusions: 1) as can be seen in the cited literature, vinasse is one of the most impressive respurces for ethanol production; 2) Brazil is the second largest ethanol producer worldwide; 3) the use of stillage for cooling sugarcane fiekds, its organic content, chemical and biochemical oxygen demand rates are high and 4) the synergy between academics from universities, the government sector, civil society, companies in the productive chain of the sugar energy sector, among others, is important.

An assessment of serial co-cultivation approach for generating novel Zymomonas mobilis strains

Objective The alphaproteobacterium Zymomonas mobilis is an efficient ethanol producer, and Z. mobilis-based biorefinery shows great potential for biofuel production. Serial co-cultivation is an emerging approach that promotes inter-species interactions which can improve or rewire the metabolic features in industrially useful microorganisms by inducing frequent mutations. We applied this method to assess if it improves or rewires the desirable physiological features of Z. mobilis, especially ethanol production. Results We performed serial co-culture of Z. mobilis with the baker’s yeast, Saccharomyces cerevisiae. We observed filamentation of Z. mobilis cells in the co-culture, indicating that the Z. mobilis cells were exposed to stress due to the presence of a competitor. After 50 times of serial transfers, we characterized the generated Z. mobilis strains, showing that long term co-culture did not drive significant changes in either the growth or profile of excreted metabolites in the generated strains. In line with this, whole genome sequencing of the generated Z. mobilis strains revealed only minor genetic variations from the parental strain. 50 generations of Z. mobilis monoculture did not induce morphological changes or any significant genetic variations. The result indicates that the method needs to be carefully optimized for Z. mobilis strain improvement.

An assessment of serial co-cultivation approach for generating novel Zymomonas mobilis strains

Objective The alphaproteobacterium Zymomonas mobilis is an efficient ethanol producer. By utilizing its distinctive physiological features, Z. mobilis-based biorefinery shows great potential for large scale industrial biofuel production. Serial co-cultivation is an emerging approach that promotes inter-species interactions which can improve or rewire the metabolic features in industrially useful microorganisms by inducing frequent mutations. We applied this method to assess if adaptation to long term co-culture improves or rewires the desirable physiological features of Z. mobilis.Results We performed serial co-culture of Z. mobilis with the baker’s yeast, Saccharomyces cerevisiae. We observed filamentation of Z. mobilis cells in the co-culture, indicating that the Z. mobilis cells were exposed to a selective pressure due to the presence of a competitor. After 50 times of serial transfers, we characterized the generated Z. mobilis strains. The analysis showed that long term co-culture did not drive significant changes in either the growth or profile of excreted metabolites in the generated strains. In line with this, whole genome sequencing of the generated Z. mobilis strains revealed only minor genetic variations from the parental strain. The result indicates that co-culture method needs to be carefully optimized for Z. mobilis strain improvement.

Metabolic Profiling of Glucose-Fed Metabolically Active Resting Zymomonas mobilis Strains

Zymomonas mobilis is the most efficient bacterial ethanol producer and its physiology is potentially applicable to industrial-scale bioethanol production. However, compared to other industrially important microorganisms, the Z. mobilis metabolome and adaptation to various nutritional and genetic perturbations have been poorly characterized. For rational metabolic engineering, it is essential to understand how central metabolism and intracellular redox balance are maintained in Z. mobilis under various conditions. In this study, we applied quantitative mass spectrometry-based metabolomics to explore how glucose-fed non-growing Z. mobilis Zm6 cells metabolically adapt to change of oxygen availability. Mutants partially impaired in ethanol synthesis (Zm6 adhB) or oxidative stress response (Zm6 cat) were also examined. Distinct patterns of adaptation of central metabolite pools due to the change in cultivation condition and between the mutants and Zm6 reference strain were observed. Decreased NADH/NAD ratio under aerobic incubation corresponded to higher concentrations of the phosphorylated glycolytic intermediates, in accordance with predictions of the kinetic model of Entner–Doudoroff pathway. The effects on the metabolite pools of aerobic to anaerobic transition were similar in the mutants, yet less pronounced. The present data on metabolic plasticity of non-growing Z. mobilis cells will facilitate the further metabolic engineering of the respective strains and their application as biocatalysts.

Construction of advanced producers of first- and second-generation ethanol in Saccharomyces cerevisiae and selected species of non-conventional yeasts (Scheffersomyces stipitis, Ogataea polymorpha)

This review summarizes progress in the construction of efficient yeast ethanol producers from glucose/sucrose and lignocellulose. Saccharomyces cerevisiae is the major industrial producer of first-generation ethanol. The different approaches to increase ethanol yield and productivity from glucose in S. cerevisiae are described. Construction of the producers of second-generation ethanol is described for S. cerevisiae, one of the best natural xylose fermenters, Scheffersomyces stipitis and the most thermotolerant yeast known Ogataea polymorpha. Each of these organisms has some advantages and drawbacks. S. cerevisiae is the primary industrial ethanol producer and is the most ethanol tolerant natural yeast known and, however, cannot metabolize xylose. S. stipitis can effectively ferment both glucose and xylose and, however, has low ethanol tolerance and requires oxygen for growth. O. polymorpha grows and ferments at high temperatures and, however, produces very low amounts of ethanol from xylose. Review describes how the mentioned drawbacks could be overcome.

Novel Approach in the Construction of Bioethanol-Producing Saccharomyces cerevisiae Hybrids

Bioethanol production from lignocellulosic hydrolysates requires a producer strain that tolerates both the presence of growth and fermentation inhibitors and high ethanol concentrations. Therefore, we constructed heterozygous intraspecies hybrid diploids of Saccharomyces cerevisiae by crossing two natural S. cerevisiae isolates, YIIc17_E5 and UWOPS87-2421, a good ethanol producer found in wine and a strain from the flower of the cactus Opuntia megacantha resistant to inhibitors found in lignocellulosic hydrolysates, respectively. Hybrids grew faster than parental strains in the absence and in the presence of acetic and levulinic acids and 2-furaldehyde, inhibitors frequently found in lignocellulosic hydrolysates, and the overexpression of YAP1 gene increased their survival. Furthermore, although originating from the same parental strains, hybrids displayed different fermentative potential in a CO2 production test, suggesting genetic variability that could be used for further selection of desirable traits. Therefore, our results suggest that the construction of intraspecies hybrids coupled with the use of genetic engineering techniques is a promising approach for improvement or development of new biotechnologically relevant strains of S. cerevisiae. Moreover, it was found that the success of gene targeting (gene targeting fidelity) in natural S. cerevisiae isolates (YIIc17_E5α and UWOPS87-2421α) was strikingly lower than in laboratory strains and the most frequent off-targeting event was targeted chromosome duplication.

Genome Sequence of Zymomonas mobilis subsp. mobilis NRRL B-1960

ABSTRACT Zymomonas mobilis subsp. mobilis is an efficient ethanol producer with application for industrial production of biofuel. To supplement existing Z. mobilis genomic resources and to facilitate genomic research, we used Oxford Nanopore and Illumina sequencing to assemble the complete genome of the beer spoilage isolate Z. mobilis subsp. mobilis strain NRRL B-1960.

Ethanol production from olive stone hydrolysates by xylose fermenting microorganisms

Olive stones are the main solid byproducts obtained from olive oil production and from table olives production. As a lignocellulosic material, the use of olive stones for ethanol and other chemicals production has been proposed, particularly under the biorefinery concept. As part of such a process, this work deals with the fractionation of the lignocellulosic material by dilute acid autoclave pretreatment at 2% sulfuric acid, 130°C, 60 min and 1:1 liquid to solid ratio. Moreover, the work addresses the fermentation of the liquors obtained after pretreatment. The released sugars are composed mainly by xylose and other hemicellulosic sugars. The fermentation performance of three xylose-fermenting microorganisms, e.g. two Escherichia coli species and Scheffersomyces stipitis, are compared. The study analyzes in a first step the microorganism behavior on synthetic liquors, with a similar composition to that of the real liquors. Finally, and taken into account the results from the previous steps, the real liquor obtained from olive stones pretreatment is fermented. Results show that E. coli MM160 is the best ethanol producer out of the three microorganisms studied. Globally, the pretreatment produced a liquor containing 140 g hemicellulosic sugars/l and requiring firstly dilution by 50% and a detoxification step by overliming. The fermentation of this liquor by E. coli MM160 results in a 25 g ethanol/l solution equivalent to 50 g ethanol/kg olive stone, in spite of 20 g acetic acid/l also present. These results confirm both olive stones and E. coli MM160 as promising feedstock and microorganism for ethanol production.