Biotechnology for Biofuels最新文献

Background

Parageobacillus thermoglucosidasius is a facultatively anaerobic thermophile that is able to produce hydrogen (H₂) gas from the oxidation of carbon monoxide through the water–gas shift reaction when grown under anaerobic conditions. The water–gas shift (WGS) reaction is driven by a carbon monoxide dehydrogenase–hydrogenase enzyme complex. Previous experiments exploring hydrogenogenesis with P. thermoglucosidasius have relied on batch fermentations comprising defined media compositions and gas atmospheres. This study evaluated the effects of a semi-continuous feeding strategy on hydrogenogenesis.

Results

A batch and two semi-continuous fermentations, with feeding of the latter fresh media (with glucose) in either 24 h or 48 h intervals were undertaken and H₂ production, carbon monoxide dehydrogenase (CODH) activity, and metabolite consumption/production were monitored throughout. Maximum H₂ production rates (HPR) of 0.14 and 0.3 mmol min⁻¹, were observed for the batch and the semi-continuous fermentations, respectively. Daily feeding attained stable H₂ production for 7 days, while feeding every 48 h resulted in high variations in H₂ production. CODH enzyme activity correlated with H₂ production, with a maximum of 1651 U mL⁻¹ on day 14 with the 48 h feeding strategy, while CODH activity remained relatively constant throughout the fermentation process with the 24 h feeding strategy.

Conclusions

The results emphasize the significance of a semi-continuous glucose-containing feed for attaining stable hydrogen production with P. thermoglucosidasius. The semi-continuous fermentations achieved a 46% higher HPR than the batch fermentation. The higher HPRs achieved with both semi-continuous fermentations imply that this approach could enhance the biohydrogen platform. However, optimizing the feeding interval is pivotal to ensuring stable hydrogen production.

Background

Spent coffee grounds (SCG) are the most abundant waste byproducts generated from coffee beverage production worldwide. Typically, these grounds are seen as waste and end up in landfills. However, SCG contain valuable compounds that can be valorized and used in different applications. Notably, they are rich in carbohydrates, primarily galactomannan, arabinogalactan type II, and cellulose. Within the framework of a circular bioeconomy, the targeted degradation of these polysaccharides via a tailored cocktail of carbohydrate-active enzymes offers a promising strategy for producing high-value saccharides from coffee waste.

Results

In this study, various mild pretreatments were evaluated to increase the enzyme accessibility of SCG-derived biomass, reduce lignin content, and minimize hemicellulose loss. Thermostable enzymes were selected to construct an enzymatic cocktail specifically targeting cellulose and hemicelluloses in pretreated SCGs. The approach used achieved a conversion of 52% of the polysaccharide content to oligo- and monosaccharides, producing 17.4 mg of reducing sugars and 5.1 mg of monosaccharides from 50 mg of SCG. Additionally, microwave pretreatment followed by the application of a thermostable endo β-mannanase resulted in the production of 62.3 mg of mannooligosaccharides from 500 mg of SCG. In vitro experiments demonstrated that the produced mannooligosaccharides exhibited prebiotic activity, promoting the growth and biofilm formation of five probiotic bacterial strains.

Conclusions

This study highlights an effective strategy for the valorization of SCG polysaccharides through mild pretreatment and customized enzymatic cocktails in a circular bioeconomic context. The production of both monosaccharides and oligosaccharides with prebiotic activity illustrates the versatility and commercial potential of SCG as a substrate for high-value saccharides. Furthermore, the use of mild pretreatment methods and thermostable enzymes minimizes chemical inputs and energy demands, aligning with sustainable processing practices. The ability to selectively target and degrade specific polysaccharides within SCG not only enhances the yield of desirable products, but also preserves key structural components, reducing waste and promoting resource efficiency.

Background

Crocetin is a multifunctional apocarotenoid natural product derived from saffron, holding significant promises for protection against various diseases and other nutritional applications. Historically, crocetin has been extracted from saffron stigmas, but this method is hindered by the limited availability of high-quality raw materials and complex extraction processes. To overcome these challenges, metabolic engineering and synthetic biology can be applied to the sustainable production of crocetin.

Results

We constructed a Yarrowia lipolytica strain using hybrid promoters and copy number adjustment, which was able to produce 2.66 g/L of β-carotene, the precursor of crocetin. Next, the crocetin biosynthetic pathway was introduced, and we observed both the production and secretion of crocetin. Subsequently, the metabolite profiles under varied temperatures were studied and we found that low temperature was favorable for crocetin biosynthesis in Y. lipolytica. Therefore, a two-step temperature-shift fermentation strategy was adopted to optimize yeast growth and biosynthetic enzyme activity, bringing a 2.3-fold increase in crocetin titer. Lastly, fermentation media was fine-tuned for an optimal crocetin output of 30.17 mg/L, bringing a 51% higher titer compared with the previous highest report in shake flasks. Concomitantly, we also generated Y. lipolytica strains capable of achieving substantial zeaxanthin production, yielding 1575.09 mg/L, doubling the previous highest reported titer.

Conclusions

Through metabolic engineering and fermentation optimization, we demonstrated the first de novo biosynthesis of crocetin in the industrial yeast Yarrowia lipolytica. In addition, we achieved a higher crocetin titer in flasks than all our known reports. This work not only represents a high production of crocetin, but also entails a significant simultaneous zeaxanthin production, setting the stage for sustainable and cost-effective production of these valuable compounds.

Background

Currently, efficient technologies producing useful chemicals from alternative carbon resources, such as methanol, to replace petroleum are in demand. The methanol-utilizing yeast, Komagataella phaffii, is a promising microorganism to produce chemicals from methanol using environment-friendly microbial processes. In this study, to achieve efficient D-lactic acid production from methanol, we investigated a combination of D-lactate dehydrogenase (D-LDH) genes and promoters in K. phaffii. The yeast strain was constructed by integrating a gene cassette containing the identified gene and promoter into the rDNA locus of K. phaffii, followed by post-transformational gene amplification. Subsequently, D-lactic acid production from methanol was evaluated.

Results

Among the five D-LDH genes and eight promoters tested, the combination of LlDLDH derived from Leuconostoc lactis and CAT1 and FLD1 promoters was suitable for expression in K. phaffii. GS115_CFL/Z3/04, the best-engineered strain constructed via integration of LlDLDH linked to CAT1 and FLD1 promoters into the rDNA locus and post-transformational gene amplification, produced 5.18 g/L D-lactic acid from methanol. To the best of our knowledge, the amount of D-lactic acid from methanol produced by this engineered yeast is the highest reported value to date when utilizing methanol as the sole carbon source.

Conclusions

This study demonstrated the effectiveness of combining different enzyme genes and promoters using multiple promoters with different induction and repression conditions, integrating the genes into the rDNA locus, and further amplifying the genes after transformation in K. phaffii. Using our established method, other K. phaffii strains can be engineered to produce various useful chemicals in the future.

Inspired by the natural symbiotic relationships between diverse microbial members, researchers recently focused on modifying microbial chassis to create artificial coculture systems using synthetic biology tools. An increasing number of scientists are now exploring these systems as innovative biosynthetic platforms for biomass conversion. While significant advancements have been achieved, challenges remain in maintaining the stability and productivity of these systems. Sustaining an optimal population ratio over a long time period and balancing anabolism and catabolism during cultivation have proven difficult. Key issues, such as competitive or antagonistic relationships between microbial members, as well as metabolic imbalances and maladaptation, are critical factors affecting the stability and productivity of artificial coculture systems. In this article, we critically review current strategies and methods for improving the stability and productivity of these systems, with a focus on recent progress in biomass conversion. We also provide insights into future research directions, laying the groundwork for further development of artificial coculture biosynthetic platforms.

Graphical Abstract

Originally native to South America, tobacco and is now distributed worldwide as a major cash crop. Nicotine is the main harmful component of tobacco leaves, cigarette smoke and tobacco waste, which severely affects not only the flavor of the tobacco leaf, but also causes great damage to human health. As the anti-smoking movement continued to grow since the 1950s, and consumers become more aware of their health and environmental protection, the world tobacco industry has been committed to research, develop and produce low nicotine cigarette products with relatively low risk to human health. Among various approaches, the use of microorganisms to reduce nicotine content and improve tobacco quality has become one of the most promising methods. Due to increasing interest in nicotine-degrading microorganisms (NDMs), this article reviews recent reports on NDMs, nicotine-degrading enzymes, regulation of nicotine-degrading bacterial consortia and optimization of fermentation conditions, aiming to provide updated references for the in-depth research and application of microorganisms for the degradation of nicotine.

Graphical Abstract

Background

Biorefineries usually focus on the production of low-value commodities, such as bioethanol, platform chemicals or single cell protein. Shifting production to bioactive compounds, such as antimicrobial peptides, could provide an opportunity to increase the economic viability of biorefineries.

Results

Recombinant production of the antimicrobial peptide pediocin PA-1 in Corynebacterium glutamicum was transferred from yeast extract-based media to minimal media based on lignocellulosic spent sulfite liquor. Induced batch, fed batch, and extended batch process modes were compared for highest pediocin PA-1 production.

Conclusion

For pediocin PA-1 production on lignocellulosic residues, extended batch cultivation was identified as the optimal process mode, producing up to (simeq) 104 mg/L active pediocin PA-1. Moreover, the production of pediocin PA-1 on this sustainable second generation resource exceeded its state-of-the-art production on yeast extract-based media (simeq) 1.5-fold.

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