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Biofuels from microalgal biomass is among some of the promising sustainable energy technologies that can significantly replace the dependence on fossil fuels worldwide due to potentiality to lower CO2 emissions. Nevertheless, the extraction of biomolecules for biofuel generation is inhibited by the rigidity of the cellular structure of microalgal biomass. Various pretreatment strategies have been evaluated for their efficacy in microalgal cell wall disruption to enhance microalgal bioenergy production. However, the efficiency of the pretreatment methods depend on the particular species being treated due to the inherent variability of the composition of the cell wall. This paper reviews pretreatment strategies (mainly novel physical, chemical and physicochemical) employed in bioenergy generation from microalgal biomass, address existing constraints and provides prospects for economic and industrial-scale production. The authors have also discussed the different pretreatment methods used for biodiesel, bioethanol, and biohydrogen production.

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Background and aim: The present study investigates Plectranthus scutellarioides (L.) R.Br. as potential antibacterial oral rinse against bacteria associated with peri-implantitis to prevent the initial infection as well as disease progression. Experimental procedure: Phytochemical screening was done on P. scutellarioides lyophilized extract to identify the presence of chemical constituent by using mass-based identification. The extract was screened for its antibacterial activity against 4 Gram-positive aerobes (early colonizer) and 5 Gram-negative facultative anaerobes as well as obligate anaerobes (late colonizer) using disc diffusion method. The extract was tested for minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), its cytotoxicity effects on human gingival fibroblast cell (HnGF) as well as bacteria morphological changes by scanning electron microscopy (SEM). Results and conclusion: Four flavonoid compounds were identified namely quercetin-3-glucoside, quercitrin, quercetin 3-(6″-acetylglucoside) and quercetin 3-O-acetyl-rhamnoside. The sensitivity test revealed that P. scutellarioides extract was effective against all the bacteria tested. MIC concentrations for the Gram-positive aerobes were in the range of 1.56-12.50 mg/mL, and the MBC concentrations were within 3.13-12.50 mg/mL. For Gram-negative obligate anaerobes, the MIC concentration were within 3.13-12.50 mg/mL and MBC within 6.25-200.00 mg/mL. The ethanolic extract did not have any cytotoxic effect on HnGF cells at the tested concentrations. SEM images showed bacterial cell wall disruption for all the bacteria tested. The results showed that P. scutellarioides extract exerts its antibacterial property by disrupting the cell wall of all the bacteria tested. Hence, P. scutellarioides may benefit from further investigations on its safety for oral use as an adjunctive treatment for peri-implantitis.

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Magnetic nanoparticles (MNPs) are becoming important DNA nanocarriers for genetic engineering of industrial fungi. However, the biological effect of MNPs on industrial fungi remains unknown. In this study, we prepared three kinds of magnetic nanoparticles with different sizes (i.e., 10 nm, 20 nm, and 200 nm) to investigate their impact on the growth and sporulation of the important industrial fungus Aspergillus niger. Transmission electron microscopy, X-ray diffraction analysis and Zeta potential analysis revealed that the three kinds of MNPs, including MNP10, MNP20 and MNP200, had uniform size distribution, regular Fe3O4 X-ray diffraction (XRD) patterns and similar Zeta potentials. Interestingly, although the three kinds of MNPs did not obviously inhibit growth of the fungus, the MNP20 at 500 mg/L strongly attenuated sporulation, leading to a remarkable decrease in spore numbers on culturing plates. Further investigation showed that MNP20 at the high concentration led to drastic chitin accumulation in the cell wall, indicating cell wall disruption of the MNP20-treated fungal cells. Moreover, the MNPs did not cause unusual iron dissolution and reactive oxygen species (ROS) accumulation, and the addition of ferrous ion, ferric ion or the reactive oxygen species scavenger N-acetyl-L-cysteine (NAC) had no impact on the sporulation of the fungus, suggesting that both iron dissolution and ROS accumulation did not contribute to attenuated sporulation by MNP20. This study revealed the size-dependent effect of MNPs on fungal sporulation, which was associated with MNP-induced cell wall disruption.

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Traditional methods for lipid extraction from microalgal biomass usually involve harsh reaction conditions or the use of contaminant reagents, which lead to enormous energy consumption and wastage. Hence, a novel strategy was presented, which combined water-plasma and three-phase partitioning (TPP) techniques. Benefiting from its unique advantages such as rapid and low cost, water-plasma strategy can disrupt microalgal cell wall and can thus greatly affect lipid extraction. As a result, assisted with the TPP method, excellent performance lipid recovery (74.34%) was obtained at 200 W in 10 min. The performance was superior to that achieved through cell disruption via water-plasma pretreatment. Importantly, the whole process of lipid extraction prevented the drying of microalgal biomass, contributing to reduced energy consumption in large-scale biodiesel production. Moreover, the high fatty acids content suggested that the extracted lipids are great potential candidate for biodiesel production.

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Biotechnological application of microalgae cultures at large scale has significant potential in the various fields of biofuels, food and feed, cosmetic, pharmaceutic, environmental remediation and water treatment. Despite this great potential application, industrialisation of microalgae culture and valorisation is still faced with serious remaining challenges in culture scale-up, harvesting and extraction of target molecules. This review presents a general summary of current techniques for harvesting and extraction of biomolecules from microalgae, their relative merits and potential for industrial application. The cell wall composition and its impact on microalgae cell disruption is discussed. Additionally, more recent progress and promising experimental methods and studies are summarised that would allow the reader to further investigate the state of the art. A final survey of energetic assessments of the different techniques is also made. Bead milling and high-pressure homogenisation seem to give clear advantages in terms of target high value compounds extraction from microalgae, with enzyme hydrolysis as a promising emerging technique. Future industrialisation of microalgae for high scale biotechnological processing will require the establishment of universal comparison-standards that would enable easy assessment of one technique against another.

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Microalgal cell wall integrity and composition have a significant impact on the fermentation process and biofuel recovery. In this study, various biofuels (bioethanol, higher alcohols (C3-C5), and biodiesel) were produced by the fermentation of carbohydrates and proteins, and transesterification of lipids from three different microalgal strains (Pseudochlorella sp., Chlamydomonas mexicana, and Chlamydomonas pitschmannii), each possessing different proportions of bioconstituents (carbohydrates, proteins, and lipids). Changes in the cell wall structure and thickness were observed before and after fermentation using transmission electron microscopy. Pseudochlorella sp. showed the highest yields of bioethanol (0.45 g-ethanol/g-carbohydrates), higher alcohols (0.44 g-higher alcohols/g-proteins), and biodiesel (0.55 g-biodiesel/g-lipids), which consequently revealed a maximum energy recovery (42%) from whole constituents. This study suggests that different physiological properties, including cell wall thickness and the proportion of bioconstituents in microalgae, could have a significant impact on the pretreatment and fermentation efficiencies for biofuels production.

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The algal cell wall is a potent barrier for delivery of transgenes for genetic engineering. Conventional methods developed for higher plant systems are often unable to penetrate or remove algal cell walls owing to their unique physical and chemical properties. Therefore, we developed a simple transformation method for Chlamydomonas reinhardtii using commercially available enzymes. Out of 7 enzymes screened for cell wall disruption, a commercial form of subtilisin (Alcalase) was the most effective at a low concentration (0.3 Anson units/mL). The efficiency was comparable to that of gamete lytic enzyme, a protease commonly used for the genetic transformation of C. reinhardtii. The transformation efficiency of our noninvasive method was similar to that of previous methods using autolysin as a cell wall-degrading enzyme in conjunction with glass bead transformation. Subtilisin showed approximately 35% sequence identity with sporangin, a hatching enzyme of C. reinhardtii, and shared conserved active domains, which may explain the effective cell wall degradation. Our trans-formation method using commercial subtilisin is more reliable and time saving than the conventional method using autolysin released from gametes for cell wall lysis.

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With increasing global population and depleting resources, there is an apparent demand for radical unprecedented innovation to satisfy the basal needs of lives. Hence, non-conventional renewable energy resources like biodiesel have been worked out in past few decades. Biofuel (e.g. Biodiesel) serves to be the most sustainable answer to solve "food vs. fuel crisis". In biorefinery process, lipid extraction from oleaginous microbial lipids is an integral part as it facilitates the release of fatty acids. Direct lipid extraction from wet cell-biomass is favorable in comparison to dry-cell biomass because it eliminates the application of expensive dehydration. However, this process is not commercialized yet, instead, it requires intensive research and development in order to establish robust approaches for lipid extraction that can be practically applied on an industrial scale. This review aims for the critical presentation on cell disruption, lipid recovery and purification to support extraction from wet cell-biomass for an efficient transesterification.

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Microalgae are the fledging feedstocks yielding raw materials for the production of third generation biofuel. Assorted and conventional cell wall disruption techniques were helpful in extracting lipids and carbohydrates, nevertheless the disadvantages have led the biotechnologists to explore new process to lyse cell wall in a faster and an economical manner. Silver nanoparticles have the ability to break the cell wall of microalgae and release biomolecules effectively. Green synthesis of silver nanoparticles was performed using a novel bacterial isolate of Bacillus subtilis. Characterisation of nanosilver and its effect on cell wall lysis of microalgae were extensively analysed. Cell wall damage was confirmed by lactate dehydrogenase assay and visually by SEM analysis. This first piece of research work on direct use of nanoparticles for cell wall lysis would potentially be advantageous over its conventional approaches and a greener, cost effective and non laborious method for the production of biodiesel.

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Bee pollen possesses a broad range of potential biological activities, but nutrient absorption and biological activities of bee pollen may be restricted due to complex pollen wall. This study aimed to investigate wall-disruption variations of Brassica campestris L. (rape) bee pollen treated with protamex hydrolysis, ultrasonication, and combination of protamex hydrolysis and ultrasonication. Pollen sample treated with these three treatments had higher specific surface area values and smaller particle sizes than the untreated sample. Protamex hydrolysis degraded the pollen coat and disintegrated the intine at the germinal apertures. Ultrasonication treatment cracked the pollen exine into fragments, but seemed to have little effect on the intine. The combination of protamex hydrolysis and ultrasonication can degrade pollen coat and entirely disrupt both the exine and the intine. The exine of rape bee pollen was disrupted into three fragments along germinal apertures.

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Microalgae can produce and contain lipids, proteins and carbohydrates, which can be extracted and marketed as potential novel added-value bio-products. However, microalgae cell wall disruption is one of the most important challenges involved while processing this type of biomass. In this context, white-rot fungi, responsible for the biodegradation of lignin present in wood due to non-specific extracellular enzymes, could be applied for promoting microalgae cell wall degradation. Therefore, the aim of this study was to evaluate the use of an enzymatic extract produced by the white-rot fungi Anthracophyllum discolor as a biotechnological tool for Botryococcus braunii cell wall disruption. The fungus was inoculated in wheat grains and manganese peroxidase (MnP) activity was monitored while obtaining the enzymatic extract. Then, cell wall disruption trials with different MnP activity were evaluated by the biochemical methane potential (BMP). In relation to cell wall disruption, it was observed that the optimal value was obtained with enzymatic concentration of 1000 U/L with a BMP of 521 mL CH4/g VS. Under these conditions almost 90% of biomass biodegradability was observed, increasing in 62% compared to the microalgae without treatment. Therefore, the results indicate that enzymes secreted by A. discolor promoted the attack of the different cell wall components finally weakening it. Therefore, the application of this treatment could be a promissory biotechnological approach to decrease the energetic input required for the cell wall disruption step.

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We tested the hypothesis that alkamides from Echinacea exert antifungal activity by disrupting the fungal cell wall/membrane complex. Saccharomyces cerevisiae cells were treated separately with each of seven synthetic alkamides found in Echinacea extracts. The resulting cell wall damage and cell viability were assessed by fluorescence microscopy after mild sonication. Membrane disrupting properties of test compounds were studied using liposomes encapsulating carboxyfluorescein. Negative controls included hygromycin and nourseothricin (aminoglycosides that inhibit protein synthesis), and the positive control used was caspofungin (an echinocandin that disrupts fungal cell walls). The results show that yeast cells exposed to sub-inhibitory concentrations of each of the seven alkamides and Echinacea extract exhibit increased frequencies of cell wall damage and death that were comparable to caspofungin and significantly greater than negative controls. Consistent with effects of cell wall damaging agents, the growth inhibition by three representative alkamides tested and caspofungin, but not hygromycin B, were partially reversed in sorbitol protection assays. Membrane disruption assays showed that the Echinacea extract and alkamides have pronounced membrane disruption activity, in contrast to caspofungin and other controls that all had little effect on membrane stability. A Quantitative Structure-Activity Relationship (QSAR) analysis was performed to study the effect of structural substituents on the antifungal activity of the alkamides. Among the set studied, diynoic alkamides showed the greatest antifungal and cell wall disruption activities while an opposite trend was observed in the membrane disruption assay where the dienoic group was more effective. We propose that alkamides found in Echinacea act synergistically to disrupt the fungal cell wall/membrane complex, an excellent target for specific inhibition of fungal pathogens. Structure-function relationships provide opportunities for synthesis of alkamide analogs with improved antifungal activities.

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