Biotechnology for Biofuels最新文献

Background

Clostridium thermocellum is a promising candidate for production of cellulosic biofuels, however, its final product titer is too low for commercial application, and this may be due to thermodynamic limitations in glycolysis. Previous studies in this organism have revealed a metabolic bottleneck at the phosphofructokinase (PFK) reaction in glycolysis. In the wild-type organism, this reaction uses pyrophosphate (PP_i) as an energy cofactor, which is thermodynamically less favorable compared to reactions that use ATP as a cofactor. Previously we showed that replacing the PP_i-linked PFK reaction with an ATP-linked reaction increased the thermodynamic driving force of glycolysis, but only had a local effect on intracellular metabolite concentrations, and did not affect final ethanol titer.

Results

In this study, we substituted PP_i-pfk with ATP-pfk, deleted the other PP_i-requiring glycolytic gene pyruvate:phosphate dikinase (ppdk), and expressed a soluble pyrophosphatase (PPase) and pyruvate kinase (pyk) genes to engineer PP_i-free glycolysis in C. thermocellum. We demonstrated a decrease in the reversibility of the PFK reaction, higher levels of lower glycolysis metabolites, and an increase in ethanol titer by an average of 38% (from 15.1 to 21.0 g/L) by using PP_i-free glycolysis.

Conclusions

By engineering PP_i-free glycolysis in C. thermocellum, we achieved an increase in ethanol production. These results demonstrate that optimizing the thermodynamic landscape through metabolic engineering can enhance product titers. While further increases in ethanol titers are necessary for commercial application, this work represents a significant step toward engineering glycolysis in C. thermocellum to increase ethanol titers.

Biofilm is a syntrophic community of microorganisms enveloped by extracellular polymeric substances and displays remarkable adaptability to dynamic environments. Implementing biofilm in anaerobic digestion has been widely investigated and applied as it promotes microbial retention time and enhances the efficiency. Previous studies on anaerobic biofilm primarily focused on application in wastewater treatment, while its role has been significantly extended to accelerate the degradation of lignocellulosic biomass, improve gas–liquid mass transfer for biogas upgrading, or enhance resistance to inhibitors or toxic pollutants. This work comprehensively reviewed the current applications of biofilm in anaerobic digestion and focused on impacting factors, optimization strategies, reactor set-up, and microbial communities. Moreover, a full-scale biofilm reactor case from Norway is also reported. This review provides a state of-the- art insight on the role of biofilm in anaerobic digestion.

Graphical Abstract

Background

Cyanobacteria have long been suggested as an industrial chassis for the conversion of carbon dioxide to products as part of a circular bioeconomy. The slow growth, carbon fixation rates, and limits of carbon partitioning between biomass and product in cyanobacteria must be overcome to fully realise this industrial potential. Typically, flux towards heterologous pathways is limited by the availability of core metabolites. Citramalate is produced in a single enzymatic step through the condensation of the central metabolites pyruvate and acetyl-CoA; improvements in citramalate productivity can, therefore, be used as a measure of overcoming this limitation. Furthermore, citramalate is a useful biomaterial precursor and provides a route to renewable methyl methacrylate and poly(methyl methacrylate), which is often traded as Perspex or Plexiglas.

Results

Here, we describe a phenomenon where the concerted optimisation of process parameters significantly increased citramalate production in Synechocystis sp. PCC 6803. Design of experiment principles were used to determine the optima for each parameter and the interplay between multiple parameters. This approach facilitated a ~ 23-fold increase in citramalate titre from initial unoptimised experiments. The process of scale-up from batch cultures to 0.5, 2, and 5 L photobioreactors is described. At the 2-L scale, citramalate titres from carbon dioxide reached 6.35 g/L with space–time yields of 1.59 g/L/day whilst 5-L PBRs yielded 3.96 ± 0.23 g/L with a productivity of 0.99 ± 0.06 g/L/day. We believe the decrease in productivity from 2-L to 5-L scale was likely due to the increased pathlength and shading for light delivery reducing incident light per cell. However, changes in productivity and growth characteristics are not uncommon when scaling up biotechnology processes and have numerous potential causes.

Conclusions

This work demonstrates that the use of a process parameter control regime can ameliorate precursor limitation and enhance citramalate production. Since pyruvate and/or acetyl-CoA give rise to numerous products of biotechnological interest, the workflow presented here could be employed to optimise flux towards other heterologous pathways. Understanding the factors controlling and thus increasing carbon partitioning to product will help progress cyanobacteria as part of a carbon–neutral circular bioeconomy. This is the first study using design of experiment to optimise overall carbon fixation rate and carbon partitioning to product, with the goal of improving the performance of a cyanobacterium as a host for biological carbon capture.

Background

Elimination of greenhouse gas emissions in industrial biotechnology requires replacement of carbohydrates by alternative carbon substrates, produced from CO₂ and waste streams. Ethanol is already industrially produced from agricultural residues and waste gas and is miscible with water, self-sterilizing and energy-dense. The yeast C. jadinii can grow on ethanol and has a history in the production of single-cell protein (SCP) for feed and food applications. To address a knowledge gap in quantitative physiology of C. jadinii during growth on ethanol, this study investigates growth kinetics, growth energetics, nutritional requirements, and biomass composition of C. jadinii strains in batch, chemostat and fed-batch cultures.

Results

In aerobic, ethanol-limited chemostat cultures, C. jadinii CBS 621 exhibited a maximum biomass yield on ethanol ((Y_{X/S}^{max})) of 0.83 g_biomass (g_ethanol)⁻¹ and an estimated maintenance requirement for ATP (m_ATP) of 2.7 mmol_ATP (g_biomass)⁻¹ h⁻¹. Even at specific growth rates below 0.05 h⁻¹, a stable protein content of approximately 0.54 g_protein (g_biomass)⁻¹ was observed. At low specific growth rates, up to 17% of the proteome consisted of alcohol dehydrogenase proteins, followed by aldehyde dehydrogenases and acetyl-CoA synthetase. Of 13 C. jadinii strains evaluated, 11 displayed fast growth on ethanol (μ_max > 0.4 h⁻¹) in mineral medium without vitamins, and CBS 621 was found to be a thiamine auxotroph. The prototrophic strain C. jadinii CBS 5947 was grown on an inorganic salts medium in fed-batch cultures (10-L scale) fed with pure ethanol. Biomass concentrations in these cultures increased up to 100 g_biomass (kg_broth)⁻¹, with a biomass yield of 0.65 g_biomass (g_ethanol)⁻¹. Model-based simulation, based on quantitative parameters determined in chemostat cultures, adequately predicted biomass production. A different protein content of chemostat- and fed-batch-grown biomass (54 and 42%, respectively) may reflect the more dynamic conditions in fed-batch cultures.

Conclusions

Analysis of ethanol-grown batch, chemostat and fed-batch cultures provided a quantitative physiology baseline for fundamental and applied research on C. jadinii. Its high maximum growth rate, high energetic efficiency of ethanol dissimilation, simple nutritional requirements and high protein content, make C. jadinii a highly interesting platform for production of SCP and other products from ethanol.

Medium chain fatty acids (MCFAs) are valuable platform compounds for the production of biotechnologically relevant chemicals such as biofuels and biochemicals. Two distinct pathways have been implemented in the yeast Saccharomyces cerevisiae for the biosynthetic production of MCFAs: (i) the mutant fatty acid biosynthesis (FAB) pathway in which the fatty acid synthase (FAS) complex is mutated and (ii) a heterologous multispecies-derived reverse β-oxidation (rBOX) pathway. Hexanoic acid has become of great interest as its acyl-CoA ester, hexanoyl-CoA, is required for the biosynthesis of olivetolic acid (OA), a cannabinoid precursor. Due to insufficient endogenous synthesis of hexanoyl-CoA, recombinant microbial systems to date require exogenous supplementation of cultures with hexanoate along with the overexpression of an acyl-CoA ligase to allow cannabinoid biosynthesis. Here, we engineer a recombinant S. cerevisiae strain which was metabolically optimized for the production of hexanoic acid via the FAB and rBOX pathways and we combine both pathways in a single strain to achieve titers of up to 120 mg L⁻¹. Moreover, we demonstrate the biosynthesis of up to 15 mg L⁻¹ OA from glucose using hexanoyl-CoA derived from the rBOX pathway.

Background

Cellulose, an abundant biopolymer, has great potential to be utilized as a renewable fuel feedstock through its enzymatic degradation into soluble sugars followed by sugar fermentation into liquid biofuels. However, crystalline cellulose is highly resistant to hydrolysis, thus industrial-scale production of cellulosic biofuels has been cost-prohibitive to date. Mechanistic studies of enzymes that break down cellulose, called cellulases, are necessary to improve and adapt such biocatalysts for implementation in biofuel production processes. Thermobifida fusca Cel6B (TfCel6B) is a promising candidate for industrial use due to its thermostability and insensitivity to pH changes. However, mechanistic studies probing TfCel6B hydrolytic activity have been limited to ensemble-scale measurements.

Results

We utilized optical tweezers to perform single-molecule, nanometer-scale measurements of enzyme displacement during cellulose hydrolysis by TfCel6B. Records featured forward motility on the order of 0.17 nm s⁻¹ interrupted by backward motions and long pauses. Processive run lengths were on the order of 5 nm in both forward and backward directions. Motility records also showed rapid bidirectional displacements greater than 5 nm. Single-enzyme velocity and bulk ensemble activity were assayed on multiple crystalline cellulose allomorphs revealing that the degree of crystallinity and hydrogen bonding have disparate effects on the single-molecule level compared to the bulk scale. Additionally, we isolated and monitored the catalytic domain of TfCel6B and observed a reduction in velocity compared to the full-length enzyme that includes the carbohydrate-binding module. Applied force has little impact on enzyme velocity yet it readily facilitates dissociation from cellulose. Preliminary measurements at elevated temperatures indicated enzyme velocity strongly increases with temperature.

Conclusions

The unexpected motility patterns of TfCel6B are likely due to previously unknown mechanisms of processive cellulase motility implicating irregularities in cellulose substrate ultrastructure. While TfCel6B is processive, it has low motility at room temperature. Factors that most dramatically impact enzyme velocity are temperature and the presence of its native carbohydrate-binding module and linker. In contrast, substrate ultrastructure and applied force did not greatly impact velocity. These findings motivate further study of TfCel6B for its engineering and potential implementation in industrial processes.

Background

Acetone–butanol–ethanol (ABE) fermentation by solventogenic clostridia can be harnessed to produce CO₂ emission neutral bio-based 1-butanol, a valuable compound with a broad range of applications, e.g., in industrial production, as a solvent and as a fuel additive or replacement. However, the relatively low butanol titers and high feedstock costs prevent bio-butanol production on an industrial scale. Agricultural side-stream materials, like milling byproducts, are starch-rich, low-cost and produced all year round. They could be suitable substrates for bio-butanol production by ABE fermentation.

Results

The milling byproducts wheat red dog (WRD), rye second flour (RSF), wheat bran (WB), rye bran (RB) and ergot sclerotia-containing rye waste stream (ER) were found to contain between ~ 30 and ~ 85% glucan, most of which was starch based. WRD, RSF and ER had the highest glucan content, while the brans contained significant xylan concentrations. Four strains selected from the collection of solventogenic clostridia available in our group produced > 6 g/L butanol on the majority of these substrates, with Clostridium beijerinckii NCIMB 8052 showing the best ABE production performance when regarding all tested substrates. Toxic ergot sclerotia-containing waste material was found to be a suited substrate for ABE fermentation. Strain NCIMB 8052 exhibited butanol titers of up to 9 g/L on substrate mixtures of WRD plus ER and the highest butanol yield per used sugars. Finally, a semi-continuous ABE fermentation of C. beijerinckii NCIMB 8052 on WRD plus ER could be maintained for 96 h. The volumetric ABE productivity during the continuous phase of fermentation was ~ 0.41 g L⁻¹ h⁻¹ and a total of 37.7 g ABE was produced out of 168.2 g substrate.

Conclusions

Based on their carbohydrate composition, WRD, RSF and ER were the milling byproducts best suited as substrates for bio-butanol production by clostridial ABE fermentation. Importantly, also ergot sclerotia-containing waste materials can be used as substrates, which can help to reduce process costs. The semi-continuous fermentation showed that clostridial ABE fermentation on milling byproducts may represent a suitable avenue for commercial butanol production after further process and/or strain optimization.

The demand for greener energy sources necessitates the development of more efficient processes. Lignocellulosic biomass holds significant potential for biofuels production, but improvements in its enzymatic degradation are required to mitigate the susceptibility of enzymes by reaction products and pretreatment impurities. In this work, two cellobiohydrolases (CBHs) from the basidiomycete Phanerochaete chrysosporium (PcCel7C and PcCel7D) were heterologously expressed, characterized, and analyzed in the presence of their products (glucose and cellobiose) and harmful compounds commonly found in industrial processes (phenolics), as well as their adsorption to lignin and cellulose. The enzymes exhibited an optimum temperature of 55 °C and displayed a pH profile similar to the model CBHI from Trichoderma reesei (TrCel7A). Activity decreased consistently for all CBHs in the presence of cellobiose, while glucose significantly impacted the basidiomycete CBHs. Phenolic compounds with a higher content of OH groups were found to be more detrimental to the enzymes, with the location of the OH group on the phenolic ring playing a crucial role in enzyme deactivation. Molecular docking simulations predicted that the product-binding site of CBHs has the highest affinity for interaction with phenolics; however, they are unlikely to interact at this site in the presence of substrate. PcCel7C and PcCel7D exhibited poorer adsorption on cellulose compared to the TrCel7A enzyme. These findings provide insights into how the structure of CBHs influences their susceptibility to inhibitors and deactivating compounds present in saccharification reaction medium.

Background

The yeast Saccharomyces cerevisiae, commonly used in industry, exhibits complex metabolism due to the Crabtree effect, fermenting alcohol even under aerobic conditions when glucose exceeds 0.10-0.15 g/L. The heat released by the biological activity is a signal very easy to collect, given the minimal instrumentation requirements. However, this heat depends on the activated metabolic pathways and provides only an indirect indicator, that cannot be used in a simple way. This study demonstrated the potential of a mechanistic model to control the process by measuring the heat released by the biological activity.

Results

The complexity arising from coexisting metabolic pathways was addressed by a comprehensive model of Saccharomyces cerevisiae together with the heat of reaction included in a rigorous enthalpy balance of the bioreactor. Batch cultures were performed in an insulated bioreactor to trigger a temperature signal. The heat of individual metabolic pathways was determined by inverse analysis of these tests using Particle Swarm Optimization (PSO): -101.28 ±0.02kJ/mol for anaerobic fermentation, -231.27±0.06kJ/mol for aerobic fermentation, and -662.94 ± 0.54kJ/mol for ethanol respiration. Finally, the model was successfully applied and validated for online training under different operating conditions.

Conclusions

The model demonstrates remarkable accuracy, with a mean relative error under 0.38% in temperature predictions for both anaerobic and aerobic conditions. The viscous dissipation is a key parameter specific to the bioreactor and the growth conditions. However, we demonstrated that this parameter could be fitted accurately from the early stages of the experiment for further prediction of the remaining part. This model introduces temperature, or the thermal power required to maintain temperature, as a measurable parameter for online feedback model training to provide increasingly precise feed-forward control.

Graphical Abstract

Background

White rot fungi produce various reactive oxygen species and ligninolytic enzymes for lignocellulose deconstruction. However, their interactions during the deconstruction of lignocellulosic structural barriers for efficient enzymatic saccharification remain unclear.

Results

Herein, the extracellular enzyme activities and secretomic analysis revealed the sequential expression of hydroxyl radical (⋅OH) and manganese peroxidases (MnPs) for lignocellulose deconstruction by the white rot fungus Irpex lacteus. Subsequently, in vitro functional studies found that ⋅OH possessed the ability to disrupt the smooth surface structure of corn stover, resulting in increased enzymatic saccharification and cellulose accessibility. Purified recombinant MnPs from I. lacteus were able to cleave the β-O-4 bond in phenolic and non-phenolic lignin model dimers without the help of any mediators. Furthermore, the sequential pretreatment of corn stover with ⋅OH and MnP exhibited significant synergistic effects, increasing enzymatic saccharification and cellulose accessibility by 2.9-fold and 1.8-fold, respectively.

Conclusions

These results proved for the first time the synergistic effects of ⋅OH and MnP pretreatment in improving the enzymatic saccharification and cellulose accessibility of corn stover. These findings also demonstrated the potential application of ⋅OH and MnP pretreatment for the efficient enzymatic saccharification of corn stover.

Graphical Abstract