Microalgal Cell Wall Research Articles

Monitoring Microalgal Neutral Lipid Accumulation with Nile Red.

The widely applied Nile red (NR) method allows near real-time monitoring of microalgal neutral lipid accumulation. When added to a culture sample, optimally, the fluorescent dye NR penetrates the microalgal cell wall staining the intracellular neutral lipids, and the measured fluorescence is linearly correlated to the neutral lipid concentration. Here I describe an optimization protocol for determining the optimal staining parameters for each new microalgal species, followed by a basic NR staining protocol to be applied for monitoring of microalgal neutral lipid accumulation.

Protein and carbohydrate extraction from S. platensis biomass by ultrasound and mechanical agitation

Spirulina platensis is considered an alternative and excellent source of protein [46–63% dry basis (DB)], having protein levels comparable to meat and soybeans. Thus, it can be considered an adequate ingredient to supply the necessity of this compound in the food industry. Its carbohydrates (8–14% DB) may also be a useful food ingredient or a potential source of bioenergy. Thus, extracting these compounds from the microalgae biomass will maximize its exploitation. Sonication can completely or partially degrade the microalgal cell wall, providing a useful technique to extract the protein and carbohydrate. This study used a sequential strategy of experimental design (fractional factorial design and central composite rotatable design) to evaluate the protein and carbohydrate extraction from S. platensis defatted biomass using ultrasonic waves and mechanical agitation, under alkaline conditions. The optimal conditions for protein and carbohydrate co-extraction were established by selecting and maximizing the variables that significantly influenced the extraction. The optimized percentages recovery from the extraction process yielded 75.76% protein and 41.52% carbohydrate at 33–40min sonication and 40–55min agitation. The protein fraction may be further concentrated and purified for use in food formulations, and the carbohydrates may be a useful feedstock for bioethanol production.

Evaluation of Different Solvent Types on the Extraction of Proteins from Microalgae

The commercial production of microalgal proteins at low cost remains challenging. The release of proteins from microalgae is restricted by the presence of rigid thick cell wall that could hinder the extraction process and affect the proteins recovery yield. In view of this, this study aimed to provide some guidance on the selection of the efficient solvent in facilitating the proteins release during the cell disruption process. The effects of different solvent types such as methanol, ethanol, 1-propanol and water in facilitating the process of proteins release from microalgal cell wall were studied in the present study. Based on the results, it was found that water able to release the highest concentration of proteins from microalgae compared to the other solvents.

Hydrothermal acid treatment for sugar extraction from Golenkinia sp.

In this study, hydrothermal acid treatment for efficient recovery of sugar from Golenkinia sp. was investigated. The initial glucose and XMG (xylose, mannose, and galactose) contents of a prepared Golenkinia sp. solution (40g/L) were 15.05 and 5.24g/L, respectively. The microalgal cell walls were hydrolyzed, for sugar recovery, by enzymatic saccharification and/or hydrothermal acid treatment. Among the various hydrothermal acid treatment conditions, the most optimal were the 2.0% H2SO4 concentration at 150°C for 15min, under which the glucose- and XMG-extraction yields were 71.7% and 64.9%, respectively. By pH 4.8, 50°C enzymatic hydrolysis after optimal hydrothermal acid treatment, the glucose- and XMG-extraction yields were additionally increased by 8.3% and 0.8%, respectively. After hydrothermal acid treatment, the combination with the enzymatic hydrolysis process improved the total sugar yield of Golenkinia sp. to 75.4%.

Solid-State NMR Study of Intact Microalgae

The study of intact cells by solid-state NMR is challenging considering their high level of complexity. Therefore interactions with the lipid membranes for example have been traditionally studied using model cell membranes composed of a few representative lipids. Microalgae are excellent examples of the complexity of microorganisms. Their lipid-rich plasma membrane is, in most species, protected by a cell wall, and the cytoplasm contains a variety of organelles including the chloroplast and the nucleus, as well as storage lipids and sugars. Microalgal cell walls are diverse and can be made of cellulose, proteins, polysaccharides, silica or calcium carbonate. The diversity of lipids is also notable in terms of the headgroup variety encountered as well as degrees of unsaturation. Because microalgae are at the basis of the aquatic food chain, we are developing approaches to study the interaction of contaminants with these cells by solid-state NMR using magic-angle spinning (MAS) for maximum resolution. We have first established a protocol of 13C enrichment, enabling the acquisition of signal from all the constituents. Freshwater C. reinhardtii and marine water species P. lutheri and N. galbana have thus been 13C-labeled. The use of dynamic filters to evidence particular constituents will be discussed, such as experiments based on through-space (cross polarization) or through-bond (RINEPT) magnetization transfer to study rigid and dynamic components, respectively. Isotopic labeling strategies will also be discussed as well as the challenges to study interactions with the lipid membranes and cell wall.

Sonication-assisted homogenization system for improved lipid extraction from Chlorella vulgaris

Microalgae, as biodiesel feedstock, are promising renewable energy sources that can be continuously developed into the future. Because recovery of microalgal lipid from dry microalgae incurs high costs, particularly in the dewatering process, wet extraction has been suggested as an attractive alternative approach. In the present study, the feasibility of a sonication-assisted homogenization system for lipid extraction from Chlorella vulgaris was evaluated. To that end, lipid-extraction performance, according to reaction time, cell concentration, and solvent type, was investigated. The initial fatty acid content of the C. vulgaris was 360.2 mg/g cell. The combination of sonication and homogenization broke up the microalgal cell walls, thereby facilitating lipid recovery. By this method, the lipid-recovery yields were increased compared with that by solo use of either homogenization or sonication. Further, chloroform-methanol, as a recovery solvent, significantly enhanced lipid recovery (237.5 mg lipid/g cell at 60 min reaction time) relative to hexane (152.0 mg lipid/g cell at 60 min reaction time).

The effect of ultrasound on the growth and viability of microalgae cells

Ultrasound has shown potential for both increasing microalgal lipid extraction yields and for the control of microalgal blooms through cell disruption. The effect of ultrasound on the viability of microalgae was investigated on the following species: Dunaliella salina, Chlamydomonas concordia and Nannochloropsis oculata. Sonication with a 20 kHz probe (0.086 W cm−3) caused complete cell disruption of D. salina after 4 min. This microalgae species does not have a true cell wall. In the case of C. concordia which has a thin cell wall complete cell disruption under the same conditions took 16 min. Under the same conditions, there was no visible disruption of N. oculata, a species which has a thick cell wall. However spectro-fluorophotometer analysis of the sonicated suspension of N. oculata showed that although the cells were intact, the level of intracellular chlorophyll was reduced by ~10 %. This clearly indicated damage to the microalgal cell wall. After 16 min, treatment cultures of all three species remained viable. Programmed cell death (PCD) has been induced in some microalgal species to terminate algal blooms; ultrasonic application did not induce PCD in any species tested. The supernatant of sonicated D. salina and C. concordia has also been shown to be able to boost the growth of established cultures. These results provide important information concerning the uses of ultrasound in both the microalgal biofuels industry and for the control of microalgal blooms.

Improving microalgal oil collecting efficiency by pretreating the microalgal cell wall with destructive bacteria

Converting microalgae oil into biodiesel is considered a promising route in the field of biofuel production. However, the cost of microalgae-based biodiesel is still too high to be economically feasible. The high cost of microalgae-based biodiesel is mainly a result of downstream processing, in particular from the extraction of oil out of the microalgal biomass. This study proposes a bacterial (enzymatic) destruction pretreatment of the microalgae cell wall to render the microalgal oil extraction more efficient. It has been found that the cell wall of microalgae can be modified if the microalgal biomass is co-cultured with an indigenous bacterial isolate Flammeovirga yaeyamensis in a salt concentration of 3% and a pH of 8.0. Following this treatment, the activities of some hydrolytic enzymes (i.e., amylase, cellulases, and xylanase) have been detected in the co-culture of F. yaeyamensis and the oil-rich microalga (Chlorella vulgaris ESP-1). The SEM micrographs clearly show specific damage to the microalgae cell wall caused by the bacterial treatment. We found that when the microalgae is pretreated with a concentrated co-culture supernatant (containing the hydrolytic enzymes), a nearly 100% increase in lipid extraction efficiency is obtained. The proposed bacterial disruption method seems to be an effective and environmentally friendly way of improving the efficiency of oil extraction and biofuel production from a microalgal biomass.

Dynamic microstructures and fractal characterization of cell wall disruption for microwave irradiation-assisted lipid extraction from wet microalgae

To extract lipids from wet microalgae through cell disruption, the effects of microwave treatment on the dynamic cell wall microstructures were investigated. The fractal dimension of raw, untreated microalgal cells was 1.46. The disruption level of microalgal cell walls was enhanced when microwave treatment temperature increased from 80 to 120°C, resulting in an increase in microalgal cell fractal dimension from 1.61 to 1.91. The cell wall thickness and pore diameters in cell walls increased from 0.11 to 0.59 μm and from 0.005 to 0.18 μm, respectively, when microwave treatment time increased from 0 to 20 min. The outer pectin layers of cell walls gradually detached and the porosity of inner cellulose layers increased when microwave treatment time increased to 26 min. The initial point of disruption appeared at the maximum curvature (approximately 1.01×10(7) m(-1)) of cell walls. Numbers of short-chain and saturated lipids increased because of microwave electromagnetic effect.

Using wet microalgae for direct biodiesel production via microwave irradiation

To address the large energy consumption of microalgae dewatering and to simplify the conventional two-step method (cellular lipid extraction and lipid transesterification) for biodiesel production, a novel process for the direct conversion of wet microalgae biomass into biodiesel by microwave irradiation is proposed. The influences of conventional thermal heating and microwave irradiation on biodiesel production from wet microalgae biomass were investigated. The effects of using the one-step (simultaneous lipid extraction and transesterification) and two-step methods were also studied. Approximately 77.5% of the wet microalgal cell walls were disrupted under microwave irradiation. The biodiesel production rate and yield from wet microalgae biomass obtained through the one-step process using microwave irradiation were 6-fold and 1.3-fold higher than those from wet microalgae obtained through the two-step process using conventional heating.

Effects of enzymatic hydrolysis on lipid extraction from Chlorella vulgaris

In this study, the effects of enzymatic hydrolysis on lipid extraction from microalga (Chlorella vulgaris) were investigated prior to biodiesel production. The initial fatty acids content of C. vulgaris was 87.6 mg/g cell. The microalgal cell walls were hydrolyzed by cellulases and then their lipid fractions were extracted using various organic solvents such as hexane, methanol, and chloroform. Optimal pH and temperature for the enzymatic hydrolysis were pH 4.8 and 50 °C, respectively, and the maximal hydrolysis yield was 85.3%, which was achieved after 72 h. After the enzymatic hydrolysis, the lipid extraction yield by the organic solvents was improved compared to when there was no enzymatic hydrolysis process, by 1.29–1.73-fold depending on the solvents used. The total fatty acid methyl ester (FAME) productivity through the enzymatic hydrolysis was higher than when there was no enzymatic hydrolysis, by 1.10–1.69-fold depending on the solvents used. When lipid was extracted from the C. vulgaris after the enzymatic hydrolysis in chloroform-methanol solution, FAME productivity was 59.4 mg FAME/g cell.

Unravelling the Origin of Ultralaminae in Sedimentary Organic Matter: The Contribution of Bacteria and Photosynthetic Organisms

Conditions for OM preservation depend on both the chemical nature of constituents and environmental factors. Among known preservation pathways, the selective preservation is based on refractory molecules such as algaenans, which are algal components. So far, ultralaminae have been considered as remains of microalgal cell walls on the basis of microscopic and organic geochemical similarities between fossil kerogen and recent algae. This high-resolution microscopic study focuses on organic matter (OM) from different fossil environments, i.e., from shallow to deep water. This OM is compared with recent organic analogs, i.e., microbial mats and biofilm, using transmission electron microscopy. Four possible origins of ultralaminae can be interpreted: algal and bacterial cell walls, thylakoids, and filamentous organisms. This highlights the importance of ultralaminae as a major indicator for paleoenvironmental interpretation. The multiple origin of ultralaminae implies new considerations about OM preservation and depositional conditions. In this way, the role of bacteria in OM accumulations can be reevaluated and the presence of ultralaminae such as thylakoids has a potential to become a new indicator of photosynthesis in fossil sediments.

Conceptual Bases for Prey Biorecognition and Feeding Selectivity in the Microplanktonic Marine Phagotroph Oxyrrhis marina

It is suspected that phagotrophic marine protozoa might possess feeding receptors that enable them to discern the nutritional quality of individual prey items (during prey-handling) on the basis of their cell-surface biochemistry. This article reviews advances in our understanding of the molecular mechanisms that mediate the biorecognition and selection of nonself (microalgal) prey items by the microplanktonic marine phagotroph Oxyrrhis marina. The potential importance of lectin-glycan interactions is first considered in view of findings which demonstrate that O. marina possesses lectin-like feeding receptors specific for prey-surface (mannose) glycoconjugates. Secondly, some conceptual bases for indirect or 'opsonic' modes of prey biorecognition mediated by soluble prey-labelling proteins are presented. Finally, the possibility that some accounts of selective feeding in O. marina might result from the noxious effects of prey-associated chemicals rather than active 'distaste' by phagotrophic cells is discussed. Recent evidence for toxic superoxide (O(2)(-)) production by marine microalgae is afforded particular attention given that release of O(2)(-) anions can be exacerbated by the binding of mannose-specific lectins to the microalgal cell wall; a novel model for grazing-activated chemical defence is proposed.

Revisiting amorphous organic matter in Kimmeridgian laminites: what is the role of the vulcanization process in the amorphization of organic matter?

AbstractKimmeridgian bituminous laminites from Orbagnoux (France) contain abundant amorphous organic matter (AOM). Previous studies have shown that the vulcanization pathway was the dominant preservation mechanism of AOM in these laminites, and led to its structureless aspect (a process called amorphization) at the nanoscale. In contrast, new observations in scanning electron microscopy and transmission electron microscopy demonstrate that this AOM exhibits typical cyanobacterial structures (exopolymeric substances, filamentous and coccoid bacteria) and ultralaminae. This identification is supported by a comparison with a recent cyanobacterial biofilm considered as an analogue. Moreover, this comparison demonstrates that ultralaminae in the Orbagnoux environment cannot solely be attributed to microalgal cell walls, but also to constituents of cyanobacteria. The microscopic identification of a ubiquitous cyanobacterial imprint demonstrates that the selective preservation pathway has been largely underestimated in Orbagnoux AOM and/or that the vulcanization process does not lead to the amorphization of organic matter automatically.