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•A simplified (L+SScF) process with phosphoric acid was modelled using Aspen.•A $10mil savings in capital cost was achieved by using phosphoric acid.•Capital investment ranged from ...$169 to $197mil for various scenarios.•Ethanol production cost varied between 50 and 63 cents per liter.•Overall ethanol yield had the biggest impact on production cost.
A techno-economic analysis was conducted for a simplified lignocellulosic ethanol production process developed and proven by the University of Florida at laboratory, pilot, and demonstration scales. Data obtained from all three scales of development were used with Aspen Plus to create models for an experimentally-proven base-case and 5 hypothetical scenarios. The model input parameters that differed among the hypothetical scenarios were fermentation time, enzyme loading, enzymatic conversion, solids loading, and overall process yield. The minimum ethanol selling price (MESP) varied between 50.38 and 62.72US cents/L. The feedstock and the capital cost were the main contributors to the production cost, comprising between 23–28% and 40–49% of the MESP, respectively. A sensitivity analysis showed that overall ethanol yield had the greatest effect on the MESP. These findings suggest that future efforts to increase the economic feasibility of a cellulosic ethanol process should focus on optimization for highest ethanol yield.
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•Glucose and xylose were co-fermented by a co-culture of engineered E. coli and yeast.•Co-fermentation of sugars from sugar cane bagasse slurry at 20% solids was achieved.•Xylose ...fermentation rate of E. coli in media with glucose was 1.58 ± 0.21 g.L−1.h−1.
Microorganisms ferment xylose at high rate only when glucose concentration in the medium falls below a critical level. Since the specific productivity of product is highest during exponential to early stationary phase of growth, a glucose utilization negative ethanologenic E. coli (strain LW419a) was constructed for high rate of xylose fermentation in combination with Turbo yeast. This co-culture fermented all the released sugars in an acid/enzyme-treated sugar cane bagasse slurry (10% solids) to an ethanol titer of 24.9 ± 0.8 g.L−1 (70% of the theoretical yield) in <30 h. Ethanol titer increased to 48.6 ± 1.04 g.L−1 (yield, 0.45 g.g−1 sugars) at a solids content of 20% and the highest rate of xylose consumption was 1.58 ± 0.21 g.L−1.h−1. This study demonstrates the potential of a co-culture of strain LW419a and yeast to rapidly ferment all the sugars in pretreated biomass slurries to ethanol at their respective highest rates.
Pretreatments such as dilute acid at elevated temperature are effective for the hydrolysis of pentose polymers in hemicellulose and also increase the access of enzymes to cellulose fibers. However, ...the fermentation of resulting syrups is hindered by minor reaction products such as furfural from pentose dehydration. To mitigate this problem, four genetic traits have been identified that increase furfural tolerance in ethanol-producing Escherichia coli LY180 (strain W derivative): increased expression of fucO , ucpA , or pntAB and deletion of yqhD . Plasmids and integrated strains were used to characterize epistatic interactions among traits and to identify the most effective combinations. Furfural resistance traits were subsequently integrated into the chromosome of LY180 to construct strain XW129 (LY180 Δ yqhD ackA:: P yₐdC′fucO-ucpA) for ethanol. This same combination of traits was also constructed in succinate biocatalysts (Escherichia coli strain C derivatives) and found to increase furfural tolerance. Strains engineered for resistance to furfural were also more resistant to the mixture of inhibitors in hemicellulose hydrolysates, confirming the importance of furfural as an inhibitory component. With resistant biocatalysts, product yields (ethanol and succinate) from hemicellulose syrups were equal to control fermentations in laboratory media without inhibitors. The combination of genetic traits identified for the production of ethanol (strain W derivative) and succinate (strain C derivative) may prove useful for other renewable chemicals from lignocellulosic sugars.
Advances in ethanol production Geddes, Claudia C; Nieves, Ismael U; Ingram, Lonnie O
Current opinion in biotechnology,
06/2011, Volume:
22, Issue:
3
Journal Article
Peer reviewed
Open access
Highlights ► Improved biocatalysts enable process simplification for cellulosic ethanol. ► Dual use of water and chemicals for process and for crops can reduce costs. ► Cellulase production by ...biocatalysts can reduce external cellulase requirements.
The fermentative metabolism of Escherichia coli was reengineered to efficiently convert glycerol to succinate under anaerobic conditions without the use of foreign genes. Formate and ethanol were the ...dominant fermentation products from glycerol in wild-type Escherichia coli ATCC 8739, followed by succinate and acetate. Inactivation of pyruvate formate-lyase (pflB) in the wild-type strain eliminated the production of formate and ethanol and reduced the production of acetate. However, this deletion slowed growth and decreased cell yields due to either insufficient energy production or insufficient levels of electron acceptors. Reversing the direction of the gluconeogenic phosphoenolpyruvate carboxykinase reaction offered an approach to solve both problems, conserving energy as an additional ATP and increasing the pool of electron acceptors (fumarate and malate). Recruiting this enzyme through a promoter mutation (pck*) to increase expression also increased the rate of growth, cell yield, and succinate production. Presumably, the high NADH/NAD⁺ ratio served to establish the direction of carbon flow. Additional mutations were also beneficial. Glycerol dehydrogenase and the phosphotransferase-dependent dihydroxyacetone kinase are regarded as the primary route for glycerol metabolism under anaerobic conditions. However, this is not true for succinate production by engineered strains. Deletion of the ptsI gene or any other gene essential for the phosphotranferase system was found to increase succinate yield. Deletion of pflB in this background provided a further increase in the succinate yield. Together, these three core mutations (pck*, ptsI, and pflB) effectively redirected carbon flow from glycerol to succinate at 80% of the maximum theoretical yield during anaerobic fermentation in mineral salts medium.
During metabolic evolution to improve succinate production in Escherichia coli strains, significant changes in cellular metabolism were acquired that increased energy efficiency in two respects. The ...energy-conserving phosphoenolpyruvate (PEP) carboxykinase (pck), which normally functions in the reverse direction (gluconeogenesis; glucose repressed) during the oxidative metabolism of organic acids, evolved to become the major carboxylation pathway for succinate production. Both PCK enzyme activity and gene expression levels increased significantly in two stages because of several mutations during the metabolic evolution process. High-level expression of this enzyme-dominated CO₂ fixation and increased ATP yield (1 ATP per oxaloacetate). In addition, the native PEP-dependent phosphotransferase system for glucose uptake was inactivated by a mutation in ptsI. This glucose transport function was replaced by increased expression of the GalP permease (galP) and glucokinase (glk). Results of deleting individual transport genes confirmed that GalP served as the dominant glucose transporter in evolved strains. Using this alternative transport system would increase the pool of PEP available for redox balance. This change would also increase energy efficiency by eliminating the need to produce additional PEP from pyruvate, a reaction that requires two ATP equivalents. Together, these changes converted the wild-type E. coli fermentation pathway for succinate into a functional equivalent of the native pathway that nature evolved in succinate-producing rumen bacteria.
The finite nature of fossil fuels and the environmental impact of its use have raised interest in alternate renewable energy sources. Specifically, nonfood carbohydrates, such as lignocellulosic ...biomass, can be used to produce next generation biofuels, including cellulosic ethanol and other nonethanol fuels like butanol. However, currently there is no native microorganism that can ferment all lignocellulosic sugars to fuel molecules. Thus, research is focused on engineering improved microbial biocatalysts for production of liquid fuels at high productivity, titer, and yield. A clear understanding and application of the basic principles of microbial physiology and biochemistry are crucial to achieve this goal. In this review, we present and discuss the construction of microbial biocatalysts that integrate these principles with ethanol-producing Escherichia coli as an example of metabolic engineering. These principles also apply to fermentation of lignocellulosic sugars to other chemicals that are currently produced from petroleum.
Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of ...lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(–)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L-1 of optically pure D(–)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 μmoles min-1 (mg protein)-1. By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(–) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.
ABSTRACT
Poly lactic acid (PLA) based plastics is renewable, bio‐based, and biodegradable. Although present day PLA is composed of mainly L‐LA, an L‐ and D‐ LA copolymer is expected to improve the ...quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D‐LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D‐lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D‐LDHs were purified and biochemically characterized. Among these, D‐LDH from L. delbrueckii subspecies bulgaricus supported the highest D‐LA titer (about 1M) and productivity (2 g h−1 g cells−1) at 37°C (B. subtilis strain DA12). The D‐LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D‐LA g−1glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g−1 glucose and the D‐lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost.
D‐lactic acid is used to improve the quality and expand the range of use of bio‐based PLA plastics. Due to the paucity of microbial biocatalysts that ferment sugars to D‐lactic acid at temperatures close to 50°C in mineral salts medium to lower process cost, Bacillus subtilis, used extensively by industry for production of enzymes and products, was engineered to produce D‐lactic acid at 48°C. The figure shows fermentation profile in LB of an engineered B. subtilis at 37 and 48°C.
•An experimental design was applied for the pretreatment of Eucalyptus benthamii.•Two models were developed to find the optimum conditions for pretreatment.•A L+SScF process was successfully carried ...out using the pretreated Eucalyptus.•Overall yields of 304 and 271L ethanol/tonne were obtained from the biomass.
This work deals with the production of ethanol from phosphoric acid-impregnated, steam-exploded Eucalyptus benthamii. The whole conversion process, addressing pretreatment, enzymatic hydrolysis of the whole slurry, and fermentation of both C5 and C6-sugars including a presaccharification step, is covered in this study.
Two separate models were developed to maximize sugar content and minimize inhibitor concentrations, resulting in xylose yields of ∼50% and ∼60% after pretreatment. In addition, a Liquefaction plus Simultaneous Saccharification and co-Fermentation (L+SScF) was performed to compare the fermentability of the resulting pretreated biomass. After the 6-h liquefaction step using the Cellic CTec2 enzyme from Novozyme and 10% DW pretreated biomass, the total sugar concentration in the slurry was 47g/L and 51g/L for the two conditions respectively. Enzymatic hydrolysis continued during fermentation using an ethanologenic derivative of Escherichia coli KO11. The sugars were completely consumed in 96h with product yields of 0.217 and 0.243g ethanol/g DW biomass for each condition, respectively. These yields are equivalent to 275 and 304L/tonne DW, confirming the effectiveness of the L+SScF process using phosphoric-acid-pretreated Eucalyptus.
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