Cell Wall Digestion Research Articles

Disruption of aldehyde dehydrogenase decreases cell wall-bound p-hydroxycinnamates and improves cell wall digestibility in rice.

In grass cell walls, ferulic acid (FA) serves as an important cross-linker between cell wall polymers, such as arabinoxylan (AX) and lignin, affecting the physicochemical properties of the cell walls as well as the utilization properties of grass lignocellulose for biorefinering. Here, we demonstrate that hydroxycinnamaldehyde dehydrogenase (HCALDH) plays a crucial role in the biosynthesis of the FA used for cell wall feruloylation in rice (Oryza sativa). Bioinformatic and gene expression analyses of aldehyde dehydrogenases (ALDHs) identified two rice ALDH subfamily 2C members, OsHCALDH2 (OsALDH2C2) and OsHCALDH3 (OsALDH2C3), potentially involved in cell wall feruloylation in major vegetative tissues of rice. CRISPR-Cas9 genome editing of OsHCALDH2 and OsHCALDH3 revealed that the contents of AX-bound ferulate were reduced by up to ~45% in the cell walls of the HCALDH-edited mutants, demonstrating their roles in cell wall feruloylation. The abundance of hemicellulosic sugars including arabinosyl units on AX was notably reduced in the cell walls of the HCALDH-edited mutants, whereas cellulose and lignin contents remained unaffected. In addition to reducing cell wall-bound ferulate, the loss of OsHCALDH2 and/or OsHCALDH3 also partially reduced cell wall-bound p-coumarate and sinapate in the vegetative tissues of rice, whereas it did not cause detectable changes in the amount of γ-oryzanol (feruloyl sterols) in rice seeds. Furthermore, the HCALDH-edited mutants exhibited improved cell wall saccharification efficiency, both with and without alkaline pretreatment, plausibly due to the reduction in cell wall cross-linking FA. Overall, HCALDH appears to present a potent bioengineering target for enhancing utilization properties of grass lignocellulose.

Synergistic wall digestion and cuproptosis against fungal infections using lywallzyme-induced self-assembly of metal-phenolic nanoflowers

Fungi are very common infectious pathogens, which may cause invasive and potentially life-threatening infections. However, the efficacy of antifungal medications remains limited. Herein, a Cu2+-phenolic nanoflower is designed to combat fungal infections by combining cuproptosis and cell wall digestion. Firstly, protocatechuic acid (PA)-Cu2+ (PC) nanopetals are prepared by coordination interaction. Lywallzyme (Lyw) is then added to induce the self-assembly of PC to form Lyw loaded PC (PCW) nanoflowers. PCW nanoflowers can effectively adhere to fungal surface and Lyw can digest fungal cell walls to facilitate Cu2+ to penetrate into fungal interior, thereby exerting a synergistic fungicidal effect. PCW nanoflowers exhibit excellent fungicidal activity even in protein-rich and high-salt conditions, where dissociative Cu2+ completely loses fungicidal activity. Transcriptome sequencing analysis reveals that PCW can lead to fungal cuproptosis. The in vivo fungicidal effect of PCW nanoflowers is confirmed on a murine skin fungal infection model and a murine fungal keratitis model.

Natural agents derived Pickering emulsion enabled by silica nanoparticles with enhanced antibacterial activity against drug-resistant bacteria

The emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a global health challenge due to the overuse of antibiotics. Natural substances including enzymes and essential oils have shown great potential as alternative treatment options. However, the combinational use of these natural agents remains challenging due to the denaturation of enzymes upon direct contact with oil. In this study, we report the design of a Pickering emulsion containing two natural antibacterial agents, lysozyme and tea tree oil, stabilized by fractal silica nanoparticles. In this design, the enzyme activity is kept and the volatility problem of tea tree oil is mitigated. Due to synergistic bacterial cell wall digestion and membrane disruption functions, potent bactericidal efficacy in vitro against drug-resistant bacteria is achieved. The therapeutic potential is further demonstrated in a wound healing model with drug-resistant bacteria infection, better than a synthetic antibiotic, Ampicillin. This study opens new avenues for the development of natural product-based antimicrobial treatments with promising application potential.

Applicability of non-invasive and live-cell holotomographic imaging on fungi

The ability to acquire three-dimensional (3D) information of cellular structures without the need for fluorescent tags or staining makes holotomographic imaging a powerful tool in cellular biology. It provides valuable insights by measuring the refractive index (RI), an optical parameter describing the phase delay of light that passes through the living cell.Here, we demonstrate holotomographic imaging on industrial relevant ascomycete fungi and study their development and morphogenesis. This includes conidial germination, subcellular dynamics, and cytoplasmic flow during hyphal growth in Aspergillus niger. In addition, growth and budding of Aureobasidium pullulans cells are captured using holotomographic microscopy. Coupled to fluorescence imaging, lipid droplets, vacuoles, the mitochondrial network, and nuclei are targeted and analyzed in the 3D RI reconstructed images. While lipid droplets and vacuoles can be assigned to a specific RI pattern, mitochondria and nuclei were not pronounced. We show, that the lower sensitivity of RI measurements derives from the fungal cell wall that acts as an additional barrier for the illumination light of the microscope. After cell wall digest of hyphae and protoplast formation of A. niger expressing GFP-tagged histone H2A, location of nuclei could be determined by non-invasive RI measurements. Furthermore, we used coupled fluorescence microscopy to observe migration of nuclei in unperturbed hyphal segments and duplication during growth on a single-cell level. Detailed micromorphological studies in Saccharomyces cerevisiae and Trichoderma reesei are challenging due to cell size restrictions.Overall, holotomography opens up new avenues for exploring dynamic cellular processes in real time and enables the visualization of fungi from a new perspective.

Modifying lignin composition and xylan O-acetylation induces changes in cell wall composition, extractability, and digestibility

BackgroundLignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing.ResultsArabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes.ConclusionsSimultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection.

Intracellularity, extracellularity, and squeezing in the symbiotic organ underpin nurturing and functioning of bacterial symbiont in leaf beetles

Cassidine leaf beetles are associated with genome-reduced symbiotic bacteria Stammera involved in pectin digestion. Stammera cells appear to be harbored in paired symbiotic organs located at the foregut-midgut junction either intracellularly or extracellularly, whereas the symbiont is extracellular in the ovary-accessory glands of adult females and during caplet transmission in eggs. However, using fluorescence and electron microscopy, an intracellular symbiotic configuration of Stammera was observed in Notosacantha species. Detailed inspection of other cassidine species revealed fragmented cell membrane and cytoplasm of the symbiotic organs, wherein Stammera cells are in an intermediate status between intracellularity and extracellularity. We also identified a mitochondria-rich region adjacent to the symbiont-filled region and well-developed muscle fibers surrounding the whole symbiotic organ. Based on these observations, we discuss why the Stammera genome has been reduced so drastically and how symbiont-derived pectinases are produced and supplied to the host's alimentary tract for plant cell wall digestion.

Fruit softening: evidence for rhamnogalacturonan lyase action in vivo in ripe fruit cell walls.

The softening of ripening fruit involves partial depolymerization of cell-wall pectin by three types of reaction: enzymic hydrolysis, enzymic elimination (lyase-catalysed) and non-enzymic oxidative scission. Two known lyase activities are pectate lyase and rhamnogalacturonan lyase (RGL), potentially causing mid-chain cleavage of homogalacturonan and rhamnogalacturonan-I (RG-I) domains of pectin respectively. However, the important biological question of whether RGL exhibits action in vivo had not been tested. We developed a method for specifically and sensitively detecting in-vivo RGL products, based on Driselase digestion of cell walls and detection of a characteristic unsaturated 'fingerprint' product (tetrasaccharide) of RGL action. In model experiments, potato RG-I that had been partially cleaved in vitro by commercial RGL was digested by Driselase, releasing an unsaturated tetrasaccharide ('ΔUA-Rha-GalA-Rha'), taken as diagnostic of RGL action. This highly acidic fingerprint compound was separated from monosaccharides (galacturonate, galactose, rhamnose, etc.) by electrophoresis at pH 2, then separated from ΔUA-GalA (the fingerprint of pectate lyase action) by thin-layer chromatography. The 'ΔUA-Rha-GalA-Rha' was confirmed as 4-deoxy-β-l-threo-hex-4-enopyranuronosyl-(1→2)-l-rhamnosyl-(1→4)-d-galacturonosyl-(1→2)-l-rhamnose by mass spectrometry and acid hydrolysis. Driselase digestion of cell walls from diverse ripe fruits [date, sea buckthorn, cranberry, yew (arils), mango, plum, blackberry, apple, pear and strawberry] yielded the same fingerprint compound, demonstrating that RGL had been acting in vivo in these fruits prior to harvest. The 'fingerprint' : (galacturonate + rhamnose) ratio in digests from ripe dates was approximately 1 : 72 (mol/mol), indicating that ~1.4 % of the backbone Rha→GalA bonds in endogenous RG-I had been cleaved by in-vivo RGL action. The results provide the first demonstration that RGL, previously known from studies of fruit gene expression, proteomic studies and in-vitro enzyme activity, exhibits enzyme action in the walls of soft fruits and may thus be proposed to contribute to fruit softening.

Breeding for improved digestibility and processing of lignocellulosic biomass in Zea mays.

Forage maize is a versatile crop extensively utilized for animal nutrition in agriculture and holds promise as a valuable resource for the production of fermentable sugars in the biorefinery sector. Within this context, the carbohydrate fraction of the lignocellulosic biomass undergoes deconstruction during ruminal digestion and the saccharification process. However, the cell wall's natural resistance towards enzymatic degradation poses a significant challenge during both processes. This so-called biomass recalcitrance is primarily attributed to the presence of lignin and ferulates in the cell walls. Consequently, maize varieties with a reduced lignin or ferulate content or an altered lignin composition can have important beneficial effects on cell wall digestibility. Considerable efforts in genetic improvement have been dedicated towards enhancing cell wall digestibility, benefiting agriculture, the biorefinery sector and the environment. In part I of this paper, we review conventional and advanced breeding methods used in the genetic improvement of maize germplasm. In part II, we zoom in on maize mutants with altered lignin for improved digestibility and biomass processing.

Cell wall digestion of the dinoflagellate Breviolum minutum

The mitigation of coral bleaching requires adaptation of its endosymbionts to rising ocean temperatures, acidity, and ultra-violet radiation. While genetic transformation may offer a means for enhancing endosymbiont resilience to these environmental stressors, the opportunity for doing so in dinoflagellates is likely hampered, in part, by their armored cortex, which may present a formidable barrier to intracellular delivery. Here, using Breviolum minutum (Clade B Symbiodiniaceae) as a model, we show that this barrier may be moderated by first disrupting the outer plasma membrane with the detergent octyl β-D-glucopyranoside, followed by enzymatic digestion of the underlying cellulose with Cellulase RS and Macerozyme R-10. Treatment using this new protocol results in 61% reduction in calcofluor-based cell wall staining (i.e., 25% staining for protocol vs. 64% for control), thus demonstrating the ability for considerable cell wall digestion. Furthermore, protoplasts isolated thereby exhibit rapid cell wall recovery, as well as comparable PSII activity and cell growth as the control, thus suggesting that the protocol minimally affects acute- and long-term cellular function. Finally, the isolated protoplasts also show a potentially slight increase in permeability to a model exogenous cargo after electroporation with a non-optimized protocol. Collectively, this constitutes the first known successful generation of viable B. minutum protoplasts, and thus, serves as a foundation for future studies seeking to lower the barrier for transformation of these and possibly other Symbiodiniaceae.

Effect of Different Additives on the Quality of Rehydrated Corn Grain Silage: A Systematic Review

This review aimed to analyze the effects of additives in producing silage from rehydrated corn grains for ruminants. The control treatment studies used in this analysis involved corn grain rehydrated with water only. To be included in the review, the studies needed to follow standardized criteria, including the absence of additives in the control treatment and the silage evaluation of the in animals such as cattle, goats, and sheep. A total of fifteen publications between 2014 and 2023 were included in the final dataset. The PROC ANOVA of SAS was used to compare the results, which included a random effect of comparison within the study, performing a paired comparison. It was observed that additives did not influence the chemical composition, pH, organic acid, ethanol content, microbial population, fermentative losses, aerobic stability, and dry matter in vitro digestibility of rehydrated corn grain silage (p > 0.05). Using additives in corn silage is a promising practice that can significantly benefit silage fermentation. Moisture silage additives mitigate high mycotoxin levels, enhance aerobic stability, improve cell wall digestibility, and increase the efficiency of utilization of silage nitrogen by ruminants. Using fermentation-stimulating additives (Lactobacillus buchneri) can improve the quality of rehydrated corn grain silage. There are still a few studies and more research to elucidate the best additives and the ideal amount to be added to ground corn grain silage.

Tracking digestible and non-digestible cell wall components during protein concentrate production from grass-clover and alfalfa

AbstractGreen biomass from leguminous and gramineous forage crops, such as alfalfa and grass-clover, has been proposed as a potential new source of feed protein concentrates for non-ruminant livestock. However, the efficient separation of the protein fraction from the non-digestible cell components, primarily comprising cell walls (fiber) and starch, presents a significant technological challenge. Moreover, it is crucial to optimize the process to preserve the optimal nutritional value of the final product. This study comprehensively analyzed the non-digestible fiber content and composition across all biorefinery fractions using two different feedstocks: green biomass from alfalfa and grass-clover. The pilot scale refining process involved a combination of screw pressing, lactic acid fermentation, and protein separation via centrifugation. We observed variations in carbohydrate composition and abundance between alfalfa and grass-clover. The lactic acid fermentation led to a reduction in cellulose and total glucose content. Our findings indicate that the final protein concentrate still contains residual cell wall components, including lignin, indicating potential inefficiencies in the filtration, fermentation, and isolation steps. The presented analytical approach provides a valuable framework for optimizing processing conditions and tailoring enzyme cocktails for enhanced valorization of the by-products.

Alfalfa Stem Cell Wall Digestibility: Current Knowledge and Future Research Directions

Alfalfa (Medicago sativa L.) is considered as the most important forage legume with high biomass yield and nutritional quality for ruminants. The alfalfa leaf cell walls are highly digestible, but stem cell walls of alfalfa are not readily digestible. The cell wall component of alfalfa has a large source of dietary energy, but ruminant animals can digest less than half of this component due to the presence of high lignin content. The main goal of this review is to make a summary of existing knowledge of alfalfa cell wall thickening and lignification patterns and suggest future directions for improving alfalfa stem cell wall digestibility. We describe alfalfa cell wall biochemistry, alfalfa stem morphology, stem tissue degradation, and existing methods to improve alfalfa digestibility, and discuss the potential future strategies for improving alfalfa cell wall digestibility. Information on these will help alfalfa breeders and producers identify superior alfalfa cultivars with improved stem cell wall digestibility. Concentrating future efforts on the selection and identification of traits and associated genes that affect cell wall digestibility could improve alfalfa cell wall digestibility.

An elastic proteinaceous envelope encapsulates the early Arabidopsis embryo.

Plant external surfaces are often covered by barriers that control the exchange of molecules, protect from pathogens, and offer mechanical integrity. A key question is when and how such surface barriers are generated. Post-embryonic surfaces have well- studied barriers, including the cuticle, and late Arabidopsis embryo was shown to be protected by an endosperm-derived sheath deposited onto a primordial cuticle. Here we show that both cuticle and sheath are preceded by another structure during the earliest stages of embryogenesis. This structure, which we named the embryonic envelope, is tightly wrapped around the embryonic surface but can be physically detached by cell wall digestion. We show that this structure is composed primarily of Extensin and Arabinogalactan O-glycoproteins and lipids, which appear to form a dense and elastic crosslinked embryonic envelope. The envelope forms in cuticle-deficient mutants and in a mutant that lacks endosperm. This embryo-derived envelope is therefore distinct from previously described cuticle and sheath structures. We propose that it acts as an expandable diffusion barrier, as well as a means to mechanically confine the embryo to maintain its tensegrity during early embryogenesis.

Automation protocol for high-efficiency and high-quality genomic DNA extraction from Saccharomyces cerevisiae.

Although many protocols have been previously developed for genomic DNA (gDNA) extraction from S. cerevisiae, to take advantage of recent advances in laboratory automation and DNA-barcode sequencing, there is a need for automated methods that can provide high-quality gDNA at high efficiency. Here, we describe and demonstrate a fully automated protocol that includes five basic steps: cell wall and RNA digestion, cell lysis, DNA binding to magnetic beads, washing with ethanol, and elution. Our protocol avoids the use of hazardous reagents (e.g., phenol, chloroform), glass beads for mechanical cell disruption, or incubation of samples at 100°C (i.e., boiling). We show that our protocol can extract gDNA with high efficiency both from cells grown in liquid culture and from colonies grown on agar plates. We also show results from gel electrophoresis that demonstrate that the resulting gDNA is of high quality.

MicroMagnify: A Multiplexed Expansion Microscopy Method for Pathogens and Infected Tissues.

Super-resolution optical imaging tools are crucial in microbiology to understand the complex structures and behavior of microorganisms such as bacteria, fungi, and viruses. However, the capabilities of these tools, particularly when it comes to imaging pathogens and infected tissues, remain limited. MicroMagnify (µMagnify) is developed, a nanoscale multiplexed imaging method for pathogens and infected tissues that are derived from an expansion microscopy technique with a universal biomolecular anchor. The combination of heat denaturation and enzyme cocktails essential is found for robust cell wall digestion and expansion of microbial cells and infected tissues without distortion. µMagnify efficiently retains biomolecules suitable for high-plex fluorescence imaging with nanoscale precision. It demonstrates up to eightfold expansion with µMagnify on a broad range of pathogen-containing specimens, including bacterial and fungal biofilms, infected culture cells, fungus-infected mouse tone, and formalin-fixed paraffin-embedded human cornea infected by various pathogens. Additionally, an associated virtual reality tool is developed to facilitate the visualization and navigation of complex 3D images generated by this method in an immersive environment allowing collaborative exploration among researchers worldwide. µMagnify is a valuable imaging platform for studying how microbes interact with their host systems and enables the development of new diagnosis strategies against infectious diseases.

An enzyme-based system for extraction of small extracellular vesicles from plants

Plant-derived nanovesicles (NVs) and extracellular vesicles (EVs) are the next generation of nanocarrier platforms for biotherapeutics and drug delivery. EVs exist not only in the extracellular space, but also within the cell wall. Due to the limitations of existing isolation methods, the EVs extraction efficiency is low, and a large amount of plant material is wasted, which is of concern for rare and expensive medicinal plants. We proposed and validated a novel method for isolation of plant EVs by enzyme degradation of the plant cell wall to release the EVs. The released EVs can easily be collected. The new method was used for extraction of EVs from the roots of Morinda officinalis (MOEVs). For comparison, nanoparticles from the roots (MONVs) were extracted using the grinding method. The new method yielded a greater amount of MOEVs, and the vesicles had a smaller diameter compared to MONVs. Both MOEVs and MONVs were readily absorbed by endothelial cells without cytotoxic effect and promoted the expression of miR-155. The promotion of miR-155 by MOEVs was dose-dependent. More importantly, we found that MOEVs and MONVs were enriched toward bone tissue. These results support our hypothesis that EVs in plants could be efficiently extracted by enzymatic cell wall digestion and confirm the potential of MOEVs as therapeutic agents and drug carriers.

Development of a protoplast isolation system for functional gene expression and characterization using petals of Camellia Oleifera

Protoplasts preparation and purification have been frequently used in plant genetics and breeding studies, whereas application of protoplasts in woody plants is still in its infancy. Although transient gene expression using purified protoplasts is well-documented and widely used in model plants and agriculture crops, no instance of either stable transformation or transient gene expression in the woody plant Camellia Oleifera has as of yet been reported. Here, we developed a protoplast preparation and purification method using C. oleifera petals by optimizing osmotic condition with D-mannitol and polysaccharide-degrading enzyme concentrations for petal cell wall digestion, to reach a high efficiency of protoplast productivity and viability. The achieved protoplasts yield was approximately 1.42 × 107 cells per gram of petal material and the viability of protoplasts was up to 89%. In addition, we explored influencing factors of protoplast transformation, including concentrations of PEG4000 and plasmid DNA. The transformation efficiency of 81% could be reached under the optimized condition. This protoplast isolation and transient expression system were deployed to further identify the functional regulation of C. oleifera related genes and the subcellular distribution of their encoded products. In summary, the protoplast isolation and transient expression system we established using oil-tea tree petals is an efficient, versatile and time-saving system, being suitable for gene function characterization and molecular mechanism analysis.

Pharmacological Modulation of Host Immunity with Hen Egg White Lysozyme (HEWL)-A Review.

In the 100 years since its discovery, lysozyme has become an important molecule, both as model for studies in different fields and as a candidate for the therapy of various pathological conditions. Of the dozens of known lysozymes, in this review we focus on one in particular, lysozyme extracted from hen egg white (HEWL), and its interaction with the immune system when it is administered orally. Experimental data show that there is an axis that directs immune system activation from GALT (gut-associated lymphoid tissue) and the intestinal lymphocyte clusters. Although a contribution of peptidoglycans from digestion of the bacterial cell wall in the intestinal lumen cannot be excluded, immune stimulation is not dependent on the enzymatic activity of HEWL. The immune responses suggest that HEWL is able to recover from immunodepression caused by tumor growth or immunosuppressants, and that it also improves the success of chemotherapy. The positive results obtained in a small Phase 2 study in patients, the ease of oral administration of this protein, and the absence of adverse effects suggest that HEWL may play an important role in all diseases where the immune system is weakened or where its enhancement plays a critical role in the resolution of the pathology.

Development of an in vitro regeneration system from immature inflorescences and CRISPR/Cas9-mediated gene editing in sudangrass

BackgroundSudangrass (Sorghum sudanense) is a major biomass producer for livestock feed and biofuel in many countries. It has a wide range of adaptations for growing on marginal lands under biotic and abiotic stresses. The immature inflorescence is an explant with high embryogenic competence and is frequently used to regenerate different sorghum cultivars. Caffeic acid O-methyl transferase (COMT) is a key enzyme in the lignin biosynthesis pathway, which limits ruminant digestion of forage cell walls and is a crucial barrier in the conversion of plant biomass to bioethanol. Genome editing by CRISPR/Cas9-mediated mutagenesis without a transgenic footprint will accelerate the improvement and facilitate regulatory approval and commercialization of biotech crops. Methods and resultsWe report the overcome of the recalcitrance in sudangrass transformation and regeneration in order to use genome editing technique. Hence, an efficient regeneration system has been established to induce somatic embryogenesis from the immature inflorescence of two sudangrass cultivars on four MS-based media supplemented with different components. Our results indicate an interaction between genotype and medium composition. The combination of Giza-1 cultivar and M4 medium produces the maximum frequency of embryogenic calli of 80% and subsequent regeneration efficiency of 22.6%. Precise mutagenesis of the COMT gene is executed using the CRISPR/Cas9 system with the potential to reduce lignin content and enhance forage and biomass quality in sudangrass. ConclusionA reliable regeneration and transformation system has been established for sudangrass using immature inflorescence, and the CRISPR/Cas9 system has demonstrated a promising technology for genome editing. The outcomes of this research will pave the road for further improvement of various sorghum genotypes to meet the global demand for food, feed, and biofuels, achieving sustainable development goals (SDGs).

A biphasic photobioreactor system for consecutive extraction of lipids and carotenoids from pre-hydrolysed microalgae and evaluation of its biodiesel potential

A green extraction method is developed using partially hydrolysed microalgal cells grown in biocompatible solvent for simultaneous cultivation and extraction of bioproducts from a highly efficient permeabilized microalgal cell with enhanced biomass and lipid content for potential use in biodiesel production. Incomplete digestion of cell wall was achieved by regulating the incubation time of the enzymatic pretreatment of the microalgal cells. 15.77% increase in lipid content was seen when untreated cells were cultured with biocompatible solvent, while cultivation of these enzymatically pretreated cells with biocompatible solvent, the lipid content increased by 53.33% and 22% higher carotenoid content was observed as compared to conventional extraction. The total fatty acids obtained after 1st and 2nd extractions in untreated samples were 67.82%, while those in enzymatically partially digested samples were 91.94%. The untreated and partially enzymatically predigested strain showed suitable properties for quality biodiesel production as per international recommendations. The cost benefit analysis of the overall process showed the use of biocompatible solvent coupled to enzymatically predigested biomass was a favorable option as compared to conventional extraction.