Biotechnology for Biofuels最新文献

Isoprenoids constitute a large and various number of bio-compounds, with many profitable applications in pharmaceutical, nutraceutical, and industrial fields. The complexity of isoprenoid molecules leads to a challenging, expensive, and environmentally unfriendly chemical synthesis of these metabolites. In addition, the awareness and desire of many consumers for products generated by natural microbial processes has increased recently. Metabolic engineering tools and synthetic biology strategies have been used as a means for the enhancement and optimization of the natural isoprenoid biosynthetic pathways of wild strains. Microalgae as production organisms have been manipulated for the bioproduction of diverse isoprenoids. Particularly when cultivated in unsuitable conditions (such as wastewater, unbalanced nutritional sources, and distinct environmental conditions), microalgae can adjust their metabolic pathways and generate compounds with significant technological potential. Several metabolic engineering approaches have been developed, modifying the metabolic pathways in microalgae to redirect the flow of carbon toward isoprenoid biosynthesis, including pathway engineering, strain improvement, and synthetic biology. In this review, some beneficial features of these high-value metabolites are summarized. Besides, recent advancements in metabolic engineering approaches for the biosynthesis of isoprenoids are discussed in detail. At last, the viewpoints and challenges for the biosynthesis of novel compositions with isoprene units in the microalgae are also included.

Background

Filamentous fungi form a range of macromorphologies during submerged cultivation including dispersed mycelia, loose clumps, and pellets. Macromorphological development is usually heterogenous, whereby mixtures form due to a complex interplay of growth, aggregation, and fragmentation. Submerged macromorphology strongly impacts product titres and rheological performance. Nevertheless, studies that systematically investigate the quantitative effect of cultivation parameters on macromorphology and heterogeneity are lacking.

Results

In this study, we have developed shake flask cultivation conditions which enable reproducible macromorphological control of the multipurpose cell factory Aspergillus niger. Tested culture parameters included various spore titres, concentration of talc microparticles, shaking frequency, and presence/absence of baffles (n = 48 conditions). We quantified macromorphology (e.g., pellet diameter) using high-throughput two-dimensional image analysis and report intra-flask heterogeneity and flask-to-flask variation. These data identified optimal culture conditions which cause minimal macromorphological variation within individual flasks and between technical replicates. We demonstrate that pellet diameter can be reproducibly adjusted between experiments using simple cultivation conditions, and use these parameters to prove larger pellets secrete more protein while consuming less glucose. Linear regression models allowed us to identify spore concentration, shaking frequency, and talc concentration as crucial parameters impacting pellet diameter. Finally, we used a newly developed microtomography (µ-CT) approach to quantify the three-dimensional internal architecture for thousands of pellets at the cellular level. Cultivation conditions drastically impacted internal architecture. For the first time we report distinct types of pellets- those formed from a single (I) or multi-spore (II) core, and additionally pellets formed by agglomeration of mature pellets (III). Remarkably, these data show that a pellet of 2 mm consists of up to about 30 m of total hyphal length and contain approximately 200,000 tips.

Conclusions

This study identifies simple methods for adjusting macromorphology and heterogeneity, which will enable facile testing of different macromorphologies for maximizing product titres. For the first time we have investigated how pellet internal architecture is impacted by numerous culture parameters. We propose a new pellet classification system based on internal spore core architecture, thus broadening our understanding of fungal macromorphological development and opening up new avenues for bioprocess or strain engineering.

Background

A key objective in microbial biorefinery technologies is to identify resilient microorganisms capable of simultaneously synthesizing diverse bioactive metabolites. Among these, Rhodotorula yeasts emerge as promising candidates for converting various waste streams and by-products into high-value chemicals. Their industrial potential stems from their ability to accumulate significant amounts of lipids and carotenoids while also secreting extracellular polymers such as exopolysaccharides, polyol esters of fatty acids, glycolipids, and enzymes—many of which remain to be fully characterized.

Results

Among the five Rhodotorula strains tested, three exhibited substantial exopolysaccharide production. Notably, Rhodotorula graminis CCY 20-2-47 strain was identified, for the first time, to produce two distinct extracellular biopolymers—exopolysaccharides or polyol esters of fatty acids—depending on the growth conditions. It was observed enhanced production of exopolysaccharides up to 7.2 g L⁻¹ and 14.7 g L⁻¹ lipid-rich biomass by Rhodotorula graminis CCY 20-2-47 using lignocellulose hydrolysate and urea by-product. This study, for the first time, reports triggering effect of Mn²⁺ on exopolysaccharide production in Rhodotorula. Glucose-based medium resulted in co-production of polyol esters of fatty acids (3.9 g L⁻¹) and lipid-rich biomass (15 g L⁻¹) for Rhodotorula graminis CCY 20-2-47. Batch bioreactor fermentation for Rhodotorula graminis CCY 20-2-47 resulted in production of 13.1 g L⁻¹ of exopolysaccharides and 50% w/w intracellular lipids when using lignocellulose hydrolysate and urea by-product. In contrast, 7.4 g L⁻¹ of polyol esters of fatty acids and 35% w/w intracellular lipids were produced by the same strain on medium with pure glucose.

Conclusions

In conclusion, Rhodotorula yeasts demonstrate significant potential for microbial biorefineries due to their ability to efficiently convert diverse waste substrates into valuable biomaterials, including lipids and extracellular biopolymers. This study provides new insights into a potential metabolic switch in extracellular polymer biosynthesis, driven by Mn²⁺ availability in the culture medium.

Graphical abstract

Background

2,3-butanediol (2,3-BDO) is an economically important platform chemical that can be produced by the fermentation of sugars using an engineered strain of Zymomonas mobilis. These fermentations require continuous monitoring and modification of fermentation conditions to maximize 2,3-BDO yields and minimize the production of the undesired coproducts glycerol and acetoin. Because of the time required for sampling and off-line chromatographic measurement of fermentation samples, the ability of fermentation scientists to modify fermentation conditions in a timely manner is limited. The goal of this study was to test if near-infrared spectroscopy (NIRS) along with multivariate statistics could reduce the time needed for this analysis and enable real-time monitoring and control of the fermentation.

Results

In this work we developed partial least squares (PLS) calibration models to predict the concentrations of glucose, xylose, 2,3-BDO, acetoin, and glycerol in fermentations via NIRS using two different spectrometers and two different spectroscopy modalities. We first evaluated the feasibility of rapid NIRS monitoring through experiments where we measured the signals from each analyte of interest and built NIRS-based PLS models using spectra from synthetic samples containing uncorrelated concentrations of these analytes. All analytes showed unique spectral signatures, and this initial modeling showed that all analytes could be detected simultaneously. We then began work with samples from laboratory fermentation experiments and tested the feasibility of regression model development across two spectral collection modalities (at-line and on-line) and two instruments: a laboratory-grade instrument and a low-cost instrument with a more limited spectral range. All modalities showed promise in the ability to monitor Z. mobilis fermentations of glucose and xylose to 2,3-BDO. The low-cost instrument displayed a lower signal-to-noise ratio than the laboratory-grade instrument, which led to comparatively lower performance overall, but still provided sufficient accuracy to monitor fermentation trends. While the ease of use of on-line monitoring systems was favored as compared to at-line systems due to the lack of sampling required and potential for automated process control, we observed some decrease in performance due to the additional complexity of the sample matrix.

Conclusion

We have demonstrated that NIRS combined with multivariate analysis can be used for at-line and on-line monitoring of the concentrations of glucose, xylose, 2,3-BDO, acetoin, and glycerol during Z. mobilis fermentations. The decrease in signal-to-noise ratio when using a low-cost spectrometer led to greater prediction error than the laboratory-grade spectrometer for at-line monitoring. The on-line monitoring modality showed great promise for real time process control via NIRS.

Background

Large-scale cultivation of microalgae provides a carbon–neutral source of biomass for extracting valuable compounds and producing renewable fuels. Owing to their high metabolic activity and rapid reproduction rates, Chlorella species are highly productive when grown in photobioreactors. However, wild-type strains have some biological limitations that make algal bioproducts more expensive than those from more traditional sources. Domestication is thus required for improving strains. Engineering Chlorella species has been made difficult by their chemically complex and highly resistant cell wall, making transformation difficult. Cell wall also restricts diffusion of organic solvents; thus, limiting the extraction of valuable intracellular compounds. Obtaining strains with weakened cell wall is crucial to enhance the extractability of intracellular molecules, reducing the costs of biomass disruption, and to improve genetic transformation efficiency.

Results

We developed a mutagenesis pipeline combined with single-cell fluorescence scanning on the microalga Chlorella vulgaris to identify mutants with altered cell wall properties. We used the fluorescent dyes erythrosin B and calcofluor white, as markers for cell wall permeability and for binding the structural polysaccharides of the cell wall, respectively. Flow cytometry with fluorescence-activated cell sorting was employed to enrich mutagenized populations with altered emission profiles. After a first round of mutagenesis, we found six mutants with significantly higher cell permeability to erythrosin B than the wild type (CWP lines) and altered cell wall structure and composition. A second round of mutagenesis on a selected CWP strain, followed by selection for lower calcofluor white signal, resulted in the isolation of CFW lines, which exhibited reduced mechanical resistance when the biomass was subjected to cell disruption procedures. This two-steps procedure allowed us to identify new mutant strains with both an increased cell wall permeability and a reduced mechanical resistance, making a novel step towards Chlorella domestication.

Conclusions

This study demonstrated the feasibility of using mutagenesis and phenotypic selection based on flow cytometry screening to alter the cell wall of C. vulgaris and identify promising strains with improved traits for industrial applications.

Bioenergy sorghum is a highly productive drought tolerant C4 grass that accumulates ~ 80% of its harvested biomass in ~ 4 m long stems comprised of > 40 internodes that develop sequentially during an extended vegetative growth phase. Following elongation of each internode, internode density increases ~ threefold to fourfold primarily due to the accumulation of cell walls composed of cellulose, glucuronoarabinoxylan and lignin. Lignin accumulates initially on cell walls of sclerenchyma cells surrounding vascular bundles and later on cell walls of the stem rind and stem core pith parenchyma. Many genes involved in cell wall biosynthesis were expressed continuously during the stem internode densification process whereas others showed dynamic patterns of expression (high to low, low to high). Several CESA genes involved in primary cell wall cellulose synthesis were expressed in the stem rind and core throughout the stem densification phase. In contrast, CESA genes involved in secondary cell wall biogenesis were expressed continuously in the stem rind but downregulated in the stem core shortly after completion of internode elongation. Overall, accumulation of cell wall biomass in elongated internodes during stem densification increases stem mechanical strength and biomass bulk density while modifying biomass composition in ways that could impact the amount and release of cellulosic sugars and lignin-derived bioproducts.

Fossil fuel combustion is a major contributor to the greenhouse effect, which drives global environmental challenges such as climate change. The rapid depletion of fossil fuel reserves necessitates the urgent management of greenhouse gas emissions and the development of sustainable alternatives. Green algae are a promising resource for biofuel production because of their high lipid content (up to 70% dry weight), which can be converted into biofuel. This study investigated the lipid production potential of Tetraselmis sp. under different nutrient media conditions to determine the glucose concentration that maximizes lipid accumulation to advance biofuel research. To determine the effect of glucose concentration on lipid accumulation, Tetraselmis sp. was cultured in three different nutrient media: standard microalgal culture medium (F/2), seawater, and nitrogen-deficient medium (NDM) supplemented with different glucose concentrations. The glucose concentration that maximized lipid accumulation was incorporated into NDM (NDM+G) and effect of the medium was compared with the effects of other media over 9 days. Additionally, reactive oxygen species (ROS) levels and apoptosis rates were measured to assess the cellular effects of glucose supplementation and nitrogen deprivation. NDM+G, with 2 mg/mL glucose, was the most effective medium for lipid accumulation in Tetraselmis sp., with lipid levels peaking significantly (p < 0.05) at 79.8% on day 6 post-glucose supplementation. This suggests that maximum lipid yield can be achieved by harvesting Tetraselmis sp. cultured in glucose-supplemented NDM on day 6. However, ROS levels were elevated significantly (p < 0.05) by day 4, and apoptosis rate reached 31% by day 9, indicating potential cellular stress under the conditions. The use of seawater and cost-effective nutrient formulations improves the industrial feasibility of the approach, while the high lipid yield within a short cultivation period supports its potential application in sustainable large-scale biofuel production. Further research is required to optimize culture conditions using low-cost nitrogen and carbon sources. Such optimization should aim to reduce costs and cellular damage while maximizing lipid production, ultimately enabling more sustainable biofuel solutions.

Fungi play a pivotal role in ecosystem functionality, driving processes such as decomposition, nutrient cycling, and symbiotic interactions. Their wide enzymatic strategies enable the breakdown of complex organic materials and the valorization of organic waste streams, providing sustainable pathways for bioproduct development. Fungi also exhibit significant potential in industrial applications, particularly in biofuel and nutraceutical production, owing to their high lipid content and adaptability to diverse feedstocks. Genera such as Aspergillus, Mortierella, and Linnemannia have demonstrated exceptional lipid production capabilities and unique fatty acid profiles, including high yields of nutraceuticals like arachidonic acid (ARA) and oleic acid. This study explored uncharacterized fungal strains isolated from California grassland soils, analyzing their phylogeny, morphology, growth rates, lipid content, and fatty acid profiles. Results revealed notable genetic and physiological diversity among the isolates, with Mortierella strains emerging as the most promising for industrial applications due to their superior lipid content and productivity of ARA and oleic acid. Confocal microscopy confirmed consistent lipid droplet morphology, while phylogenetic analysis uncovered novel species-level diversity. Key strains were identified for biofuel and nutraceutical production, highlighting their industrial potential. These findings underscore the versatility of fungi as biotechnological tools and provide a foundation for further exploration and utilization of these promising strains in industrial processes.

Graphical Abstract

To propose a strategy for the commercial cultivation of a Korean strain of Nannochloropsis oceanica, the growth, fatty acid content and bacterial community of N. oceanica cultures exposed to different light sources were investigated. Significant growth of N. oceanica cultured under blue (450 nm), red (620 nm) and white (cool-white fluorescent; control) light was observed, whereas growth with relatively low densities was observed in N. oceanica cultured under purple (415 nm) and yellow (592 nm) light. Cells cultured under white and blue light began growing again at day 26, after experiencing stationary phases for 7 days, indicating that day 26 may be a switching point for the growth trajectory in batch culture of N. oceanica. White light also produced the highest biomass of N. oceanica, followed by blue, red, and yellow light. These results indicate that blue and red light, excluding the white light characterized by a wide spectral band, can ensure a high growth rate and biomass of a Korean strain of N. oceanica. With respect to fatty acid content, eicosapentaenoic acid (EPA) was the most dominant under the yellow and red light with N. oceanica exhibiting relatively low biomass dry weight and growth rates. In bacterial communities in N. oceanica cultures exposed to different light sources, the genus Roseovarius appeared to promote the growth of N. oceanica. Based on the results of this study, the most advantageous EPA production system for a Korean strain of N. oceanica initially uses white or blue light to produce the desired cell concentration and rapid growth, then switches to red or yellow light to enhance EPA content. This two-phase cultivation approach offers a viable pathway for large-scale EPA production from native strains, with potential application in nutraceutical or aquaculture industries.

Graphical Abstract