Biotechnology for Biofuels最新文献

Background

Sugarcane plant engineered to accumulate lipids in its vegetative tissue is being developed as a new bioenergy crop. The new crop would be a source of juice, oil, and cellulosic sugars. However, limited tolerance of industrially recognized yeasts towards inhibitors generated during the processing of lignocellulosic biomass to produce fermentable sugars is a major challenge in developing scalable processes for second-generation drop-in fuel production. To this end, hydrolysates generated from engineered sugarcane—‘oilcane’ bagasse contain added phenolics and fatty acids that further restrict the growth of fermenting microorganisms and necessitate nutrient supplementation and/or detoxification of hydrolysate which makes the fermentation process expensive. Herein, we propose a resourceful and economical approach for growing lab and commercial strains of S. cerevisiae on unrefined cellulosic sugars aerobically and fermentatively.

Results

An equal ratio of hydrolysate and juice was found optimum for growth and fermentation by lab and commercial strains of Saccharomyces cerevisiae engineered for xylose fermentation. The industrial strain grew and fermented efficiently under low aeration conditions having an ethanol titer, yield, specific and volumetric productivities of 46.96 ± 0.19 g/l, 0.51 ± 0.00 g/g, 0.27 ± 0.02 g/g.h and 1.95 ± 0.01 g/l.h, respectively, while the lab strain grew better under higher aeration conditions having the ethanol titer, yield, specific and volumetric productivities of 24.93 ± 0.09, 0.27 ± 0.00 g/g, 0.17 ± 0.00 g/g.h and 1.04 ± 0.00 g/l.h, respectively. Acclimation of cultures in a blended medium significantly improved the performance of the yeast strains.

Conclusions

The addition of transgenic oilcane juice, which is inedible and rich in amino acids, to the hydrolysate averted the need for expensive nutrient supplementation and detoxification steps of hydrolysate. The approach provides an economical solution to reduce the cost of fermentation at an industrial scale for second-generation drop-in fuel production.

Graphical Abstract

Background

Fungal pretreatment for partial separation of lignocellulosic components may reduce lignocellulose recalcitrance during the production of biofuels and biochemicals. Quantitative and qualitative modification of plant lignin through genetic engineering or traditional breeding may also reduce the recalcitrance. This study was conducted to examine the effects of combining these two approaches using three white rot fungi and mulberry wood with an altered lignin structure.

Results

Mulberry wood prepared from homozygotes or heterozygotes with a loss-of-function in the cinnamyl alcohol dehydrogenase gene (CAD) was pretreated with three fungal species. Both heterozygous (CAD/cad) and homozygous (cad/cad, null mutant) mulberry plants were derived from the same parents via backcrossing between Sekizaisou (cad/cad, seed parent), a natural lignin mutant, and its F1 progeny (CAD/cad, pollen parent). Homozygote wood and the isolated lignin exhibited an abnormal color. Lignin in homozygotes without fungal pretreatment exhibited a lower syringyl/guaiacyl ratio, molar mass, and thioacidolysis product yield than those in heterozygotes. Pretreatment with Phanerochaete chrysosporium achieved the highest delignification efficiency with a significant reduction in the cellulose content in both mulberry genotypes. In contrast, Ceriporiopsis subvermispora selectively removed lignin, with a weaker reduction in the cellulose content. The degree of delignification by C. subvermispora was significantly higher in homozygotes than in heterozygotes. Trametes versicolor tended to have a lower delignification capacity and smaller effect of subsequent enzymatic sugar release toward the wood from both genotypes than the other two fungi, making it less suitable for fungal pretreatment. Thioacidolysis assays indicated that cinnamaldehyde β-O-4, a typical subunit in the homozygote lignin, did not contribute to the high degradability of the lignin. The saccharification efficiency tended to be higher in homozygote wood than in heterozygote wood under all fungal pretreatment conditions.

Conclusions

Although further optimization of various system conditions is required, our findings suggest that CAD deficiency promotes delignification and subsequent enzymatic saccharification and may improve the biorefining efficiency of wood when combined with fungal pretreatment.

Background

The gradual extrusion of water-soluble intracellular components (such as proteins) from microalgae after pulsed electric field (PEF) treatment is a well-documented phenomenon. This could be utilized in biorefinery applications with lipid extraction taking place after such an ‘incubation’ period, i.e., a post-PEF-treatment step during which the biomass is left undisturbed before any further processing. The goal of this work was to further explore how this incubation could improve lipid extraction.

Results

Experiments were conducted on wet, freshly harvested Auxenochlorella protothecoides, treated with 0.25 or 1.5 MJ/kg_DW and incubated for 24 h. Lipid extraction took place with a monophasic ethanol:hexane:water, 1:0.41:0.04 vol/vol/vol mixture with a 75.6 mL solvent per 1 g of dry biomass ratio. The kinetics of the extraction were studied with samples taken between 10 and 1080 min from fresh and incubated biomass. The yields at 10 min were significantly increased with incubation compared to without (31.2% dry weight compared to 1.81%, respectively). The experimental data were fitted with the Patricelli model where extraction occurs in two steps, a rapid washing of immediate available lipids and a slower diffusion one. During Nile-Red staining of microalgae and microscopy imaging, a shift of emission from both GFP and RFP channels to mostly RFP was observed indicating an increase in the polarity of the environment of Nile-Red. These led to an adaption of a biphasic ethanol:hexane:water 1:6:0.4 vol/vol/vol solvent with 37 mL solvent per 1 g of dry biomass ratio which while ineffective on fresh biomass, achieved a 27% dry weight yield from incubated microalgae. The extraction efficiency in the biphasic route was lower compared to the monophasic (i.e., 69% and 95%, respectively). It was compensated however, by the significant solvent reduction (37 mL to 75.6 mL respectively), in particular the ethanol minimization. For the extraction of 1 L lipids, it was estimated that the energy consumption ratio for the biphasic process was 1.6 compared to 9.9 for monophasic, making clearly the most preferential one.

Conclusions

This biphasic approach significantly reduces solvent consumption and the respective energy requirement for solvent recovery. Incubation thus could majorly improve the commercialization prospects of the process.

Graphical abstract

Background

Aspergillus niger is an important lignocellulose-degrading enzyme-producing strain. Multiple regulatory factors regulate the synthesis of lignocellulose-degrading enzymes in A. niger. We previously found that A. niger possessed an intracellular β-glucosidase BGL1B, and the intracellular localization of BGL1B and its active transglycosylation action prompted us to explore whether BGL1B was involved in the regulation of the synthesis of lignocellulose-degrading enzymes in A. niger.

Results

In this study, by investigating the production of lignocellulose-degrading enzymes of bgl1B knockout strain (Δbgl1B) and overexpression strain (OE::bgl1B), it was found that BGL1B exhibited a repressive role on the expression of lignocellulose-degrading enzyme genes through carbon catabolite repression (CCR) way. On the other hand, BGL1B’s transglycosylation products sophorose and laminaribiose were proved to be able to induce the expression of lignocellulose-degrading enzyme genes, which explained why OE::bgl1B showed the same enhanced enzyme activity and gene expression as Δbgl1B strain compared to the starting strain (WT).

Conclusions

The present study demonstrates that BGL1B plays dual regulatory roles in the regulation of the synthesis of lignocellulose-degrading enzymes in A. niger: the repressive role caused by BGL1B’s hydrolysis product glucose and the induction role caused by BGL1B’s transglycosylation products sophorose and laminaribiose. This study broadens the understanding of the regulatory network of the synthesis of lignocellulose-degrading enzymes in A. niger. Also, it provides a strategy to create an engineered strain with high production of lignocellulose-degrading enzymes.

This study explores the use of conductive material in scaling up anaerobic digestion for enhanced biogas production. Focusing on Direct Interspecies Electron Transfer (DIET), the research employs a syntrophic DIET-able consortium formed by Shewanella oneidensis and Methanosarcina barkerii in 3.8-L experiments utilizing reticulated vitreous carbon (RVC) as conductive material. In short-term tests with acetate the syntrophic co-culture with RVC resulted in 86% higher maximum velocity of methane production, while in long term with real feed 13% increased rate was observed: the addition of 1.77 (S/m)*m² RVC resulted in a faster methane production of 2.39 mL/gVS*h compared to 2.08 mL/gVS*h of the reference. The experimental conditions of syntrophic inoculum and RVC as conductive material gave a benefit in terms of process rate compared to the reference, considering the inoculum fate, Methanosarcina barkerii was among the dominant taxa at the end of the experiment, while Shewanella oneidensis was outcompeted. Among the methanogenesis production pathways, an increase of hydrogenotrophic methanogenesis has been observed in presence of conductive material. Further research is needed to understand the role of RVC in sulfur compounds production. Utilization of RVC to augment methane production yielded interesting results for real-scale application. As an added carrier, RVC remains unaltered and can be readily recuperated and reused multiple times.

Coastal wetlands are rich in terrestrial organic carbon. Recent studies suggest that microbial consortia play a role in lignin degradation in coastal wetlands, where lignin turnover rates are likely underestimated. However, the metabolic potentials of these consortia remain elusive. This greatly hinders our understanding of the global carbon cycle and the “bottom-up” design of synthetic consortia to enhance lignin conversion. Here, we developed two groups of lignin degrading consortia, L6 and L18, through the 6- and 18-month in situ lignin enrichments in the coastal East China Sea, respectively. Lignin degradation by L18 was 3.6-fold higher than L6. Using read-based analysis, 16S rRNA amplicon and metagenomic sequencing suggested that these consortia possessed varied taxonomic compositions, yet similar functional traits. Further comparative metagenomic analysis, based on metagenomic assembly, revealed that L18 harbored abundant metagenome-assembled genomes (MAGs) that encoded diverse and unique lignin degradation gene clusters (LDGCs). Importantly, anaerobic MAGs were significantly enriched in L18, highlighting the role of anaerobic lignin degradation. Furthermore, the generalist taxa, which possess metabolic flexibility, increased during the extended enrichment period, indicating the advantage of generalists in adapting to heterogenous resources. This study advances our understanding of the metabolic strategies of coastal prokaryotic consortia and lays a foundation for the design of synthetic communities for sustainable lignocellulose biorefining.

For biofuels and nutraceuticals, the green microalga Haematococcus pluvialis (Chlorophyceae) is a prospective source of biomass and lipids. This study examined how biomass production and lipid accumulation were affected by temperature (10 °C, 20 °C, and 30 °C) and potassium nitrate (KNO₃) concentrations (0.41 g/L, 0.31 g/L, 0.21 g/L, 0.10 g/L, and 0). The findings showed that the largest biomass (0.665 ± 0.200 g/L) was produced at a potassium nitrate concentration of 0.21 g/L at 20 °C, whereas the highest lipid content (46.31 ± 0.026% dry weight) was produced at a temperature without nitrate. Notably, a balanced result was obtained with a modest nitrate content (0.10 g/L) at 20 °C, yielding significant biomass (0.560 ± 0.136 g/L) and lipids (40.30 ± 0.012% dry weight). These results highlight how crucial it is to optimize cultivation settings in order to increase H. pluvialis's dual productivity, offering important new information for its industrial-scale use. By adjusting growing conditions, this research helps meet the need for renewable resources worldwide by promoting the production of high-value bioproducts and sustainable, commercially viable algae-based biofuels.

Background

Phaeodactylum tricornutum is a versatile marine microalga renowned for its high-value metabolite production, including omega-3 fatty acids and fucoxanthin, with emerging potential for integrated biorefinery approaches that encompass biofuel and bioproduct generation. Therefore, in this study we aimed to optimize the cultivation conditions for boosting biomass, lipid, and fucoxanthin production in P. tricornutum, focusing on the impacts of different nutrient ratios (nitrogen, phosphorus, silicate), glycerol supplementation, and light regimes.

Results

Optimized medium (− 50%N%, + 50% P, Zero-Si, 2 g glycerol) under low-intensity blue light (100 μmol m⁻² s⁻¹) improved biomass to 1.6 g L⁻¹, with lipid productivity reaching 539.25 mg g⁻¹, while fucoxanthin increased to 20.44 mg g⁻¹. Total saturated fatty acid (ΣSFA) content in the optimized culture increased approximately 2.4-fold compared to the control F/2 medium. This change in fatty acid composition led to improved biodiesel properties, including a higher cetane number (59.18 vs. 56.04) and lower iodine value (53.96 vs 88.99 g I₂/100 g oil). The optimized conditions also altered the biodiesel characteristics, such as kinematic viscosity, cloud point, and higher heating value.

Conclusion

Our optimization approach reveals the significant potential of P. tricornutum as a versatile microbial platform for biomass, lipid, and fucoxanthin production. The tailored cultivation strategy successfully enhanced biomass and lipid accumulation, with notable improvements in biodiesel properties through strategic nutrient and light regime manipulation. These findings demonstrate the critical role of precise cultivation conditions in optimizing microalgal metabolic performance for biotechnological applications.

Background

The climate crisis and depleting fossil fuel reserves have led to a drive for ‘green’ alternatives to the way we manufacture chemicals, and the formation of a bioeconomy that reduces our reliance on petrochemical-based feedstocks. Advances in Synthetic biology have provided the opportunity to engineer micro-organisms to produce compounds from renewable feedstocks, which could play a role in replacing traditional, petrochemical based, manufacturing routes. However, there are few examples of bio-manufactured products achieving commercialisation. This may be partially due to a disparity between academic and industrial focus, and a greater emphasis needs to be placed on economic feasibility at an earlier stage. Terpenoids are a class of compounds with diverse use across fuel, materials and pharmaceutical industries and can be manufactured biologically from the key intermediate mevalonate.

Results

Here, we report on a method of utilising parallel bioreactors to rapidly map the growth-coupling relationship between the specific product formation rate, specific substrate utilisation rate and specific growth rate. Using mevalonate as an example product, a maximum product yield coefficient of 0.18 g_p/g_s was achieved at a growth rate ((mu)) of 0.34 h⁻¹. However, this process also led to the formation of the toxic byproduct acetate, which can slow growth and cause problems during downstream processing. By using gene editing to knock out the ackA-pta operon and poxB from E. coli BW25113, we were able to achieve the same optimum production rate, without the formation of acetate.

Conclusions

We demonstrated the power of using parallel bioreactors to assess productivity and the growth-coupling relationship between growth rate and product yield coefficient of mevalonate production. Using genetic engineering, our resultant strain demonstrated rapid mevalonate formation without the unwanted byproduct acetate. Mevalonate production is quantified and reported in industrially relevant units, including key parameters like conversion efficiency that are often omitted in early-stage publications reporting only titre in g/L.

Antimicrobial peptides (AMPs) are renowned for their potent bacteriostatic activity and safety, rendering them invaluable in animal husbandry, food safety, and medicine. Despite their potential, the physiological toxicity of AMPs to host cells significantly hampers their biosynthetic production. This study presents a novel approach for the biosynthesis of the antimicrobial peptide Kiadin by engineering a DAMP4–DPS–Kiadin fusion protein to mitigate host cell toxicity and achieve high-level expression. Leveraging the unique properties of the DAMP4 protein, we developed a non-chromatographic purification method to isolate the DAMP4–DPS–Kiadin fusion protein with high purity. The instability of the D–P peptide bond under acidic conditions, combined with the thermal and saline stability of DAMP4, enabled efficient separation of Kiadin through acid cleavage and isoelectric precipitation, yielding Kiadin with 96% purity and a production yield of 29.3 mg/L. Our optimization of acid cleavage temperature, duration, and isoelectric precipitation conditions proved critical for maximizing the purification efficiency and expression levels of Kiadin. The biosynthesized Kiadin exhibited robust bacteriostatic activity against Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, Bacillus cereus and Staphylococcus aureus. Notably, Kiadin demonstrated significant post-antibiotic effects by disrupting bacterial membrane integrity, inducing cytoplasmic leakage, and inhibiting biofilm formation in E. coli K88 and S. aureus Mu50, without cytotoxicity towards mouse macrophages. In vivo studies further confirmed Kiadin's exceptional therapeutic efficacy against abdominal infections caused by E. coli K88. The acid cleavage and non-chromatographic purification techniques developed in this study offer a cost-effective and efficient strategy for the high-purity production of AMPs.