Sustainable polyesters via direct functionalization of lignocellulosic sugars

The development of sustainable plastics from abundant renewable feedstocks has been limited by the complexity and efficiency of their production as well as their lack of competitive material properties. Here, we demonstrate the direct transformation of the hemicellulosic fraction of non-edible biomass into a diester plastic precursor at 83% yield (95% from commercial xylose) during integrated plant fractionation with glyoxylic acid. Melt polycondensation of the resulting xylose-based diester with a range of aliphatic diols led to high-molecular weight amorphous polyesters with combined high glass transition temperatures, tough mechanical properties, and strong gas barriers, which could be processed by injection-molding, thermoforming, and 3D-printing. These polyesters could then be chemically recycled from mixed plastic waste streams or digested under biologically relevant conditions. The transformation’s simplicity led to projected costs that were competitive with fossil alternatives and significantly reduced associated greenhouse gas emissions, especially if glyoxylic acid was sourced from CO2.

Principles and practice of designing microbial biocatalysts for fuel and chemical production

ABSTRACT The finite nature of fossil fuels and the environmental impact of its use have raised interest in alternate renewable energy sources. Specifically, non-food carbohydrates, such as lignocellulosic biomass, can be used to produce next generation biofuels, including cellulosic ethanol and other non-ethanol 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.

Butanol production from lignocellulosic sugars by Clostridium beijerinckii in microbioreactors

Background Butanol (n-butanol) has been gaining attention as a renewable energy carrier and an alternative biofuel with superior properties to the most widely used ethanol. We performed 48 anaerobic fermentations simultaneously with glucose and xylose as representative lignocellulosic sugars by Clostridium beijerinckii NCIMB 8052 in BioLector® microbioreactors to understand the effect of different sugar mixtures on fermentation and to demonstrate the applicability of the micro-cultivation system for high-throughput anaerobic cultivation studies. We then compared the results to those of similar cultures in serum flasks to provide insight into different setups and measurement methods. Results ANOVA results showed that the glucose-to-xylose ratio affects both growth and production due to Carbon Catabolite Repression. The study demonstrated successful use of BioLector® system for the first time for screening several media and sugar compositions under anaerobic conditions by using online monitoring of cell mass and pH in real-time and at unprecedented time-resolution. Fermentation products possibly interfered with dissolved oxygen (DO) measurements, which require a careful interpretation of DO monitoring results. Conclusions The statistical approach to evaluate the microbioreactor setup, and information obtained in this study will support further research in bioreactor and bioprocess design, which are very important aspects of industrial fermentations of lignocellulosic biomass.

Deletion of pgi gene in E. coli increases tolerance to furfural and 5-hydroxymethyl furfural in media containing glucose-xylose mixture

Background: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together.Results: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96-120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144-168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration.Conclusions: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

Deletion of Pgi Gene in E. coli Increases Tolerance to Furfural and 5-Hydroxymethyl Furfural in Media Containing Glucose-Xylose Mixture

Background: Furfural and 5-hydroxymethyl furfural (5-HMF) are key furan inhibitors that are generated due to breakdown of lignocellulosic sugars at high temperature and acidic treatment conditions. Both furfural and 5-HMF act in a synergistic manner to inhibit microbial metabolism and resistance to both is a desirable characteristic for efficient conversion of lignocellulosic carbon to ethanol. Genetic manipulations targeted toward increasing cellular NADPH pools have successfully imparted tolerance against furfural and 5-HMF. In present study, deletion of pgi gene as a strategy to augment carbon flow through pentose phosphate pathway (PPP) was studied in ethanologenic Escherichia coli strain SSK101 to impart tolerance towards either furfural or 5-HMFor both inhibitors together.Results: A key gene of EMP pathway, pgi, was deleted in an ethanologenic E. coli strain SSK42 to yield strain SSK101. In presence of 1 g/L furfural in minimal AM1 media, the rate of biomass formation for strain SSK101 was up to 1.9-fold higher as compared to parent SSK42 strain, and it was able to clear furfural in half the time. Tolerance to inhibitor was associated with glucose as carbon source and not xylose, and the tolerance advantage of SSK101 was neutralized in LB media. Bioreactor studies were performed under binary stress of furfural and 5-HMF (1 g/L each) and different glucose concentrations in a glucose-xylose mixture with final sugar concentration of 5.5%, mimicking major components of dilute acid treated biomass hydrolysate. In the mixture having 6 g/L and 12 g/L glucose, SSK101 strain produced ~ 18 g/L and 20 g/L ethanol, respectively. Interestingly, the maximum ethanol productivity was better at lower glucose load with 0.46 g/(L.h) between 96–120 h, as compared to higher glucose load where it was 0.33 g/(L.h) between 144–168 h. Importantly, parent strain SSK42 did not exhibit significant metabolic activity under similar conditions of inhibitor load and sugar concentration.Conclusions: E. coli strain SSK101 with pgi deletion had enhanced tolerance against both furfural and 5-HMF, which was associated with presence of glucose in media. Strain SSK101 also had improved fermentation characteristics under both hyperosmotic as well as binary stress of furfural and 5-HMF in media containing glucose-xylose mixture.

Butanol Production from Lignocellulosic Sugars by Clostridium beijerinckii in Microbioreactors

Background: Butanol ( n- butanol) has been gaining attention as a renewable energy carrier and an alternative biofuel with superior properties to the most widely used ethanol. We performed 48 anaerobic fermentations simultaneously with glucose and xylose as representative lignocellulosic sugars by Clostridium beijerinckii NCIMB 8052 in BioLector® microbioreactors to understand the effect of different sugar mixtures on fermentation and to demonstrate the applicability of the micro-cultivation system for high-throughput anaerobic cultivation studies. We then compared the results to those of similar cultures in serum flasks to provide insight into scalability.Results: ANOVA results showed that the glucose to xylose ratio affects both growth and production due to Carbon Catabolite Repression . The study showed that the BioLector® system is well suited for screening several media and sugar compositions under anaerobic conditions.Conclusions: The approach of, and information obtained in this study will support further research in bioreactor and bioprocess design and scale-up that are very important aspects of industrial fermentations of lignocellulosic biomass.