Alternative fermentation feedstocks such as ethanol can be produced from CO2 via electrocatalytic processes that coproduce O2. In this study, industrial-scale fermentation of ethanol with pure O2 for single cell protein (SCP) production was studied using a modeling approach. This approach considered (i) microbial kinetics, (ii) gas-liquid transfer, and (iii) an exploration of potential operational constraints. The technical feasibility for producing up to 58 kt/y of SCP in a 600 m3 bubble column operating in continuous mode was assessed and attributed mainly to a high O2 transfer rate of 1.1 mol/(kg h) through the use of pure O2. However, most of the pure O2 fed to the fermenter remains unconsumed due to the large gas flows needed to maximize mass transfer. In addition, biomass production may be hampered by high dissolved CO2 concentrations and by large heat production. The model estimates a microbial biomass concentration of 114 g/kg, with a yield on ethanol of 0.61 gx/gethanol (> 95% Y x / s max ). Although the large predicted O2 transfer capacity seems technically feasible, it needs further experimental validation. The model structure allows the analysis of alternative substrates in the same way as identifying the best carbon feedstock.

Abstract Background Anaerobic Saccharomyces cerevisiae cultures require glycerol formation to re-oxidize NADH formed in biosynthetic processes. Introduction of the Calvin-cycle enzymes phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) has been shown to couple re-oxidation of biosynthetic NADH to ethanol production and improve ethanol yield on sugar in fast-growing batch cultures. Since growth rates in industrial ethanol production processes are not constant, performance of engineered strains was studied in slow-growing cultures. Results In slow-growing anaerobic chemostat cultures (D = 0.05 h−1), an engineered PRK/RuBisCO strain produced 80-fold more acetaldehyde and 30-fold more acetate than a reference strain. This observation suggested an imbalance between in vivo activities of PRK/RuBisCO and formation of NADH in biosynthesis. Lowering the copy number of the RuBisCO-encoding cbbm expression cassette from 15 to 2 reduced acetaldehyde and acetate production by 67% and 29%, respectively. Additional C-terminal fusion of a 19-amino-acid tag to PRK reduced its protein level by 13-fold while acetaldehyde and acetate production decreased by 94% and 61%, respectively, relative to the 15 × cbbm strain. These modifications did not affect glycerol production at 0.05 h−1 but caused a 4.6 fold higher glycerol production per amount of biomass in fast-growing (0.29 h−1) anaerobic batch cultures than observed for the 15 × cbbm strain. In another strategy, the promoter of ANB1, whose transcript level positively correlated with growth rate, was used to control PRK synthesis in a 2 × cbbm strain. At 0.05 h−1, this strategy reduced acetaldehyde and acetate production by 79% and 40%, respectively, relative to the 15 × cbbm strain, without affecting glycerol production. The maximum growth rate of the resulting strain equalled that of the reference strain, while its glycerol production was 72% lower. Conclusions Acetaldehyde and acetate formation by slow-growing cultures of engineered S. cerevisiae strains carrying a PRK/RuBisCO bypass of yeast glycolysis was attributed to an in vivo overcapacity of PRK and RuBisCO. Reducing the capacity of PRK and/or RuBisCO was shown to mitigate this undesirable byproduct formation. Use of a growth rate-dependent promoter for PRK expression highlighted the potential of modulating gene expression in engineered strains to respond to growth-rate dynamics in industrial batch processes.

Glycerol is the major organic byproduct of industrial ethanol production with the yeast Saccharomyces cerevisiae . Improved ethanol yields have been achieved with engineered S. cerevisiae strains in which heterologous pathways replace glycerol formation as the predominant mechanism for anaerobic re-oxidation of surplus NADH generated in biosynthetic reactions. Functional expression of heterologous phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (RuBisCO) genes enables yeast cells to couple a net oxidation of NADH to the conversion of glucose to ethanol. In another strategy, NADH-dependent reduction of exogenous acetate to ethanol is enabled by introduction of a heterologous acetylating acetaldehyde dehydrogenase (A-ALD). This study explores potential advantages of co-cultivating engineered PRK-RuBisCO-based and A-ALD-based strains in anaerobic bioreactor batch cultures. Co-cultivation of these strains, which in monocultures showed reduced glycerol yields and improved ethanol yields, strongly reduced the formation of acetaldehyde and acetate, two byproducts that were formed in anaerobic monocultures of a PRK-RuBisCO-based strain. In addition, co-cultures on medium with low acetate-to-glucose ratios that mimicked those in industrial feedstocks completely removed acetate from the medium. Kinetics of co-cultivation processes and glycerol production could be optimized by tuning the relative inoculum sizes of the two strains. Co-cultivation of a PRK-RuBisCO strain with a Δgpd1 Δgpd2 A-ALD strain, which was unable to grow in the absence of acetate and evolved for faster anaerobic growth in acetate-supplemented batch cultures, further reduced glycerol formation but led to extended fermentation times. These results demonstrate the potential of using defined consortia of engineered S. cerevisiae strains for high-yield, minimal-waste ethanol production.

In anaerobic Saccharomyces cerevisiae cultures, NADH-cofactor balancing by glycerol formation constrains ethanol yields. Introduction of an acetate-to-ethanol reduction pathway based on heterologous acetylating acetaldehyde dehydrogenase (A-ALD) can replace glycerol formation as ‘redox-sink’ and improve ethanol yields in acetate-containing media. Acetate concentrations in feedstock for first-generation bioethanol production are, however, insufficient to completely replace glycerol formation. An alternative glycerol-reduction strategy bypasses the oxidative reaction in glycolysis by introducing phosphoribulokinase (PRK) and ribulose-1,5-bisphosphate carboxylase (RuBisCO). For optimal performance in industrial settings, yeast strains should ideally first fully convert acetate and, subsequently, continue low-glycerol fermentation via the PRK-RuBisCO pathway. However, anaerobic batch cultures of a strain carrying both pathways showed inferior acetate reduction relative to a strain expressing only the A-ALD pathway. Complete A-ALD-mediated acetate reduction by a dual-pathway strain, grown anaerobically on 50 g L -1 glucose and 5 mmol L -1 acetate, was achieved upon reducing PRK abundance by a C-terminal extension of its amino-acid sequence. Yields of glycerol and ethanol on glucose were 55% lower and 6% higher, respectively, than those of a non-engineered reference strain. The negative impact of the PRK-RuBisCO pathway on acetate reduction was attributed to sensitivity of the reversible A-ALD reaction to intracellular acetaldehyde concentrations.

Product yield on carbohydrate feedstocks is a key performance indicator for industrial ethanol production with the yeast Saccharomyces cerevisiae. This paper reviews pathway engineering strategies for improving ethanol yield on glucose and/or sucrose in anaerobic cultures of this yeast by altering the ratio of ethanol production, yeast growth and glycerol formation. Particular attention is paid to strategies aimed at altering energy coupling of alcoholic fermentation and to strategies for altering redox-cofactor coupling in carbon and nitrogen metabolism that aim to reduce or eliminate the role of glycerol formation in anaerobic redox metabolism. In addition to providing an overview of scientific advances we discuss context dependency, theoretical impact and potential for industrial application of different proposed and developed strategies.

Background and objectives This study demonstrates ethanol production using a new yeast strain called eBOOST (TM) GT, that can self-produce glucoamylase and reduce glycerol production. The advanced yeast's potential with added glucoamylase expression was investigated by observing and comparing its final ethanol and glycerol concentration against granular starch hydrolyzing enzyme (GSHE) catalyzed Ethanol Red fermentation. Findings The ethanol concentration in advanced yeast fermentation with self-produced glucoamylase was measured at 10.03% v/v, which is 40% less than GSHE catalyzed Ethanol Red fermentation. However, upon externally providing additional glucoamylase (at 50% of the recommended dosage), eBOOST (TM) GT was able to produce similar ethanol concentration, compared to Ethanol Red fermentation catalyzed with GSHE enzyme. Furthermore, in this scenario, the glycerol concentration at the end of fermentation was 31% less than observed concentrations in operating with only self-produced glucoamylase and 72% less than conventional GSHE fermentation. Conclusions The newly engineered yeast strain proves its capability to meet GSHE catalyzed ethanol yield at reduced enzyme consumption, and therefore, reduction in enzyme costs. The rate of substrate consumption and process economics are areas of future research for eBOOST (TM) GT ethanol fermentation. Significance and novelty This study provides important information on enzyme reduction and ethanol production, while using a self-enzyme producing yeast.

Background and objectives Reducing glycerol production, a major by-product produced during fermentation by yeastSaccharomyces cerevisiae,can improve the ethanol productivity in corn dry grind process. This study investigates the potential of glycerol reduction using an advanced engineered yeast strain and determines the effect of enzyme dosage, pH, and temperature on the granular starch hydrolysis (GSH) process efficiency using both conventional yeast and advanced yeast. Findings Ethanol yields in GSH process were found maximum at enzyme dosage 4.8 kg/MT grains and remained unchanged with further increase in dosage. The glycerol production was reduced by more than 50% with use of advanced enzymes compared to the conventional yeast and resulted in 2.2-3.8% higher final ethanol concentrations. Performances of both yeast were reduced and lead to stuck fermentation at 36 degrees C temperature. Conclusions Use of advanced yeast strains can improve the ethanol yields by reducing glycerol production in GSH process; however, the ethanol yields are highly dependent on the process conditions, especially enzyme loadings and temperature. Significance and novelty GSH process is expected to produce lower glycerol compared to conventional dry grind process. This research demonstrated that advanced engineered yeasts can reduce the glycerol production in GSH process also, improving the process yields and profitability.

The recent start-up of several full-scale ‘second generation’ ethanol plants marks a major milestone in the development of Saccharomyces cerevisiae strains for fermentation of lignocellulosic hydrolysates of agricultural residues and energy crops. After a discussion of the challenges that these novel industrial contexts impose on yeast strains, this minireview describes key metabolic engineering strategies that have been developed to address these challenges. Additionally, it outlines how proof-of-concept studies, often developed in academic settings, can be used for the development of robust strain platforms that meet the requirements for industrial application. Fermentation performance of current engineered industrial S. cerevisiae strains is no longer a bottleneck in efforts to achieve the projected outputs of the first large-scale second-generation ethanol plants. Academic and industrial yeast research will continue to strengthen the economic value position of second-generation ethanol production by further improving fermentation kinetics, product yield and cellular robustness under process conditions.

The metabolic engineering of Saccharomyces cerevisiae for the production of succinic acid has progressed dramatically, and a series of high-producing hosts are available. At low cultivation pH and high titers, the product transport can become bidirectional, i.e. the acid is reentering the cell and is again exported or even catabolized. Here, a quantitative approach for the identification of product recycling fluxes is developed.

Fermentative production of succinic acid (SA) from renewable carbohydrate feed-stocks can have the economic and sustainability potential to replace petroleum-based production in the future, not only for existing markets, but also new larger volume markets. To accomplish this, extensive efforts have been undertaken in the field of strain construction and metabolic engineering to optimize SA production in the last decade. However, relatively little effort has been put into fermentation process development. The choice for a specific host organism determines to a large extent the process configuration, which in turn influences the environmental impact of the overall process. In the last five years, considerable progress has been achieved towards commercialization of fermentative production of SA. Several companies have demonstrated their confidence about the economic feasibility of fermentative SA production by transferring their processes from pilot to production scale.

The fission yeast Schizosaccharomyces pombe CBS 356 exhibits extracellular maltase activity. This activity may be of commercial interest as it exhibited a low pH optimum (3.5) and a high affinity for maltose (Km of 7.0+/-1.8 mM). N-terminal sequencing of the protein indicates that it is the product of the AGL1 gene. Regulation of this gene occurs via a derepression/repression mechanism. In sugar- or nitrogen-limited chemostat cultures, the specific rate of enzyme production (q(p)) was independent of the nature of the carbon source (i.e. glucose or maltose), but synthesis was partially repressed by high sugar concentrations. Furthermore, q(p) increased linearly with specific growth rate (mu) between 0.04 and 0.10 h(-1). The enzyme is easily mass-produced in aerobic glucose-limited fed-batch cultures, in which the specific growth rate is controlled to prevent alcoholic fermentation. In fed-batch cultures in which biomass concentrations of 83 g L(-1) were attained, the enzyme concentration reached 58,000 Units per liter culture supernatant. Extracellular maltase may be used as a dough additive in order to prevent mechanisms such as maltose-induced glucose efflux and maltose-hypersensitivity that occur in maltose-consuming Saccharomyces cerevisiae.

The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h−1. It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5–10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5–10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.