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Two green microalgae species Monoraphidium contortum (M. contortum) and Chlamydomonas sp. that were identified to accumulate lipids were subjected to four different nutrient treatments (NP1-NP4), ranging in nitrate (0.05-5 mM N) and phosphate (2.8-264 µM P) concentrations, at a fixed N:P ratio of ∼18. The effect of nutrient variation on lipid productivity in the species was investigated using second derivative (SD) FTIR and Raman spectroscopy of algal biomass. SD spectral analysis revealed high production of lipid in the form of hydrocarbons (CH) (3000-2800 cm-1), triacylglycerides (TAGs)(∼1740 cm-1), saturated (SFA)(∼1440 cm-1), and unsaturated fatty acids (UFA)(∼3010 cm-1) for the nutrient deplete condition (NP1) in both species. Changes in signals attributed to lipids in proportion to other biochemical components were consistent with physiological changes expected from nutrient depletion. Relative signal intensities for lipids showed a significant increase in NP1, in particular, CH, TAGs in relation to protein signals (in SD-FTIR), and SFA, UFA in relation to carotenoid signals (in SD-Raman). PCA performed on the negative spectral values of the SD-FTIR and SD-Raman data for the four NP treatments enabled discrimination not only between the species but also between the NP treatments and the timing of harvest. M. contortum was found to contain a relatively higher proportion of CH, TAGs, SFA, and UFA compared to Chlamydomonas sp. Peak areas from the negative SD spectra, informed by PCA analysis, enabled capturing quantifiable changes in a manner that is consistent with known microalgal physiology. SD-FTIR and SD-Raman spectroscopy have been shown to possess superior potential to capture relevant microalgal physiological changes.

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The application of microbial co-cultures is now recognized in the fields of biotechnology, ecology, and medicine. Understanding the biological interactions that govern the association of microorganisms would shape the way in which artificial/synthetic co-cultures or consortia are developed. The ability to accurately predict and control cell-to-cell interactions fully would be a significant enabler in synthetic biology. Co-culturing method development holds the key to strategically engineer environments in which the co-cultured microorganism can be monitored. Various approaches have been employed which aim to emulate the natural environment and gain access to the untapped natural resources emerging from cross-talk between partners. Amongst these methods are the use of a communal liquid medium for growth, use of a solid-liquid interface, membrane separation, spatial separation, and use of microfluidics systems. Maximizing the information content of interactions monitored is one of the major challenges that needs to be addressed by these designs. This review critically evaluates the significance and drawbacks of the co-culturing approaches used to this day in biotechnological applications, relevant to biomanufacturing. It is recommended that experimental results for a co-cultured species should be validated with different co-culture approaches due to variations in interactions that could exist as a result of the culturing method selected.

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A series of commercial powdered media (Cell-Hi F2P, JWP and WP) and a hydroponics medium (FloraMicroBloom) were investigated for the cultivation of P. tricornutum, and compared with f/2 (a commonly employed laboratory cultivation medium; costlier to scale). Cell-Hi JWP showed good performance characteristics including cost-effectiveness. Outdoor cultivation of P. tricornutum in an airlift photobioreactor, using Cell-Hi JWP in the United Kingdom (UK) during September and October (average daily temperature ranging between 8 and 18 °C and natural sunlight) was comparable to cultivation indoors under controlled temperature and lighting. A strong positive correlation between fucoxanthin and chlorophyll a content, and a weak inverse correlation between eicosapentaenoic (EPA) content and temperature were observed. Commensal bacterial counts revealed a sinusoidal growth profile with a change in community dominance from Halomonas sp. to Marinobacter sp. This investigation reveals for the first time that a multi-product approach can be adopted with P. tricornutum in a UK outdoor environment using commercially viable powdered media.

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This study aimed to evaluate the effect of microplastics on Spirulina sp., the pigment phycocyanin in Spirulina sp., and the effect of Spirulina sp. on the degradation of PE and PP plastic. The interaction of Spirulina sp. with microplstic (PE and PP) was conducted by adding the microplastic (500 mg/500 mL, with a size of 0.5-1 mm2) to microalgae culture. The optical density was measured for 30 days to determine the growth of Spirulina sp. Harvesting was performed to obtain dry Spirulina sp biomass. Phycocyanin was obtained through extraction by mixing 0.1 g dry Spirulina sp. biomass with 25 ml of 1% CaCl2 in an ultrasonic water bath at 50 kHz, 300 W at 30 °C for 15 min. The results showed that the growth rate of Spirulina sp significantly decreased (p < 0.05) with treatment of PE (SP + PE) (0.0228/day) and PP (Sp + PP) (0.0221/day), compared to the control (Sp-Control) (0.0312/day). Scanning electron microscopy and Fourier transform infrared spectroscopy (FTIR) analyses of Spirulina sp. biomass with the addition of PE and PP revealed surface damage of Spirulina sp. cells and loss of carboxyl groups from proteins in Spirulina sp. at wavelengths of 1397-1450 cm-1. In addition, Spirulina sp. had decreased the intensity of amine and amide groups from proteins at wavelengths of 3280, 1637, and 1537 cm-1 in the microplastic treatment. The phycocyanin yield and protein content in Spirulina sp. control were 19.69% and 0.147%, respectively, which decreased by 10.7% and 0.121%, respectively, with PE treatment and by 8.7% and 0.108%, respectively, with PP treatment. Moreover, the investigation of PE and PP treated by Spirulina sp showed more significant changes of functional group indicated by the formation of hydroxyl (3286 cm-1), carbonyl (1700 cm-1), ester (1750 cm-1) and primary alcohol (1085 cm-1). The results of the EDX microplastic analysis showed a decrease in carbon in PE (1.62%) and PP (1.08%). These FTIR and EDX analysis also proved that microplastic has experienced degradation when treated by Spirulina sp cell culture.

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Large-scale algal oil production requires continuous outputs and a trade-off between growth and oil content. Two unrelated marine algae (Nannochloropsis oceanica [CCAP 849/10] and Chlorella vulgaris [CCAP 211/21A]) that showed high oil production under batch culture were studied under controlled semicontinuous cultivation conditions. Three essential attributes maximized oil productivity: (i) downregulation of cell size to maximize light absorption under N limitation; (ii) low nutrient-depletion thresholds to trigger oil induction; (iii) a means of carbohydrate suppression in favor of oil. N. oceanica responded better to input N/P variations and is more suited to continuous oil production. A low N/P ratio was effective in both suppressing carbohydrate and reducing cell size concomitant with oil production. In C. vulgaris, nutrient starvation thresholds for oil were higher and carbohydrate was preferentially induced, which impeded stress-level optimization for oil. These differences, which impact continuous oil production at scale, are driven by species adaptation to specific marine habitats.

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The commercial reality of microalgal biotechnology for the production of individual bioactives is constrained by the high cost of production and requires a biorefinery approach. In this investigation, we examined the influence of different nutrient deprivation (nitrogen (N), phosphorus (P), sulphur (S) and manganese (Mn)) on growth, chlorophyll a (Chl a), biohydrogen (H2) and fatty acid profiles in Parachlorella kessleri EMCCN 3073 under both aerobic and anaerobic conditions. Anaerobic conditions combined with the nutrient deprivation resulted in cell division blockage, reduction in Chl a and remarkable changes in pH, whereas a significant increase in the H2 production was observed after 24 h. The highest cumulative H2 productivity was observed in N-deficient medium (300 µL/L, day 9) followed by Mn-deficient medium (250 µL/L, day 7). The highest H2 production rate (3.37 µL/L/h) was achieved by Mn-deficient medium after 24 h. In terms of fatty acid composition, P. kessleri exhibited a differential response to different nutrient stresses. Under aerobic conditions, N-deficient media resulted in the highest lipid content (119% compared to control, day 7), whereas earlier lipid induction at (1-3 days) was observed with Mn- and S-deficient media with 18-91% and 25-34% increase, respectively, compared with the replete control. Meanwhile, higher lipid content was observed under anaerobic conditions combined with Mn-, N-, P- and S-deprived media (day 1) with 20%, 13%, 8% and 7% increases respectively compared with the control. This investigation, for the first time clearly, highlights the potential of P. kessleri as a sustainable biorefinery platform, for H2 and fatty acid bio-production under anaerobic conditions. KEY POINTS: • Parachlorella kessleri could provide a future sustainable biorefinery platform. • Nutrient-deprived anaerobic conditions blocked cell growth but differentially induced H2 production. • Nutrient status, under both aerobic/anaerobic conditions, alters lipids and fatty acids profile of P. kessleri. • Nutrient-deprived (N- and Mn-) anaerobic conditions: future biorefinery platform.

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Carbon dioxide (CO2) is a major greenhouse gas responsible for climate change. Diatoms, a natural sink of atmospheric CO2, can be cultivated industrially in autotrophic and mixotrophic modes for the purpose of CO2 sequestration. In addition, the metabolic diversity exhibited by this group of photosynthetic organisms provides avenues to redirect the captured carbon into products of value. These include lipids, omega-3 fatty acids, pigments, antioxidants, exopolysaccharides, sulphated polysaccharides, and other valuable metabolites that can be produced in environmentally sustainable bio-manufacturing processes. To realize the potential of diatoms, expansion of our knowledge of carbon supply, CO2 uptake and fixation by these organisms, in conjunction with ways to enhance metabolic routing of the fixed carbon to products of value is required. In this review, current knowledge is explored, with an evaluation of the potential of diatoms for carbon capture and bio-based manufacturing.

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A switch from a petroleum-based to a biobased economy requires the capacity to produce both high-value low-volume and low-value high-volume products. Recent evidence supports the development of microalgae-based microbial cell factories with the objective of establishing environmentally sustainable manufacturing solutions. Diatoms display rich diversity and potential in this regard. We focus on Phaeodactylum tricornutum, a pennate diatom that is commonly found in marine ecosystems, and discuss recent trends in developing the diatom chassis for the production of a suite of natural and genetically engineered products. Both upstream and downstream developments are reviewed for the commercial development of P. tricornutum as a cell factory for a spectrum of marketable products.

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Fourier transform infrared (FTIR) and Raman spectroscopic techniques were employed to analyze the biomolecular transitions and lipid accumulation in three freshwater green microalgal species, Monoraphidium contortum (M. contortum), Pseudomuriella sp. and Chlamydomonas sp. during various phases of their growth. Biomolecular transitions and lipid [hydrocarbons, triacylglycerides (TAGs)] accumulation within the microalgal cells were identified using second derivatives of the FTIR absorption spectroscopy. Second derivative analysis normalized and resolved the original spectra and led to the identification of smaller, overlapping bands. Both relative and absolute content of lipids were determined using the integrated band area. M. contortum exhibited higher accumulation of lipids than the other two species. The integrated band area of the vibrations from saturated (SFA) and unsaturated lipids (UFA) enabled quantification of fatty acids. The percentage of SFA and UFA was determined using GC, FTIR and Raman spectroscopy. From the spectral data, the order of increasing concentration of SFA among the three microalgal species was M. contortum > Chlamydomonas sp. >Pseudomuriella sp. The spectral results on fatty acids were consistent with the separation of lipids by gas chromatography. The results emphasized the significance of FTIR and Raman spectroscopic methods in monitoring the biomolecular transitions and rapid quantification of lipids, without the need for extraction of lipids.

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As algal biotechnology develops, there is an increasing requirement to conserve cultures without the cost, time and genetic stability implications of conventional serial transfers, including issues regarding potential loss by failure to regrow, contamination on transfer, mix up and/or errors in the documentation on transfer. Furthermore, it is crucial to ensure both viability and functionality are retained by stored stock-cultures. Low temperature storage, ranging from the use of domestic freezers to storage under liquid nitrogen, is widely being used, but the implication to stability and function rarely investigated. We report for the first time, retention of functionality in the maintenance of master stock-cultures of an industrially relevant, lipid-producing alga, under a variety of cryopreservation regimes. Storage in domestic (-15 °C), or conventional -80 °C freezers was suboptimal, with a rapid reduction in viability observed for samples at -15 °C and a >50% loss of viability, within one month, for samples stored at -80 °C. No reduction in viability occurred at -196 °C. Post-thaw culture functional performance was also influenced by the cryopreservation approach employed. Only samples held at -196 °C responded to nitrogen limitation in terms of growth characteristics and biochemical profiles (lipid production and chlorophyll a) comparable to the untreated control, cultured prior to cryopreservation. These results have important implications in microbial biotechnology, especially for those responsible for the conservation of genetic resources.

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Capturing a valid snapshot of the metabolome requires rapid quenching of enzyme activities. This is a crucial step in order to halt the constant flux of metabolism and high turnover rate of metabolites. Quenching with cold aqueous methanol is treated as a gold standard so far, however, reliability of metabolomics data obtained is in question due to potential problems connected to leakage of intracellular metabolites. Therefore, we investigated the influence of various parameters such as quenching solvents, methanol concentration, inclusion of buffer additives, quenching time and solvent to sample ratio on intracellular metabolite leakage from Chlamydomonas reinhardtii. We measured the recovery of twelve metabolite classes using gas chromatography mass spectrometry (GC-MS) in all possible fractions and established mass balance to trace the fate of metabolites during quenching treatments. Our data demonstrate significant loss of intracellular metabolites with the use of the conventional 60% methanol, and that an increase in methanol concentration or quenching time also resulted in higher leakage. Inclusion of various buffer additives showed 70 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) to be suitable. In summary, we recommend quenching with 60% aqueous methanol supplemented with 70 mM HEPES (-40 °C) at 1:1 sample to quenching solvent ratio, as it resulted in higher recoveries for intracellular metabolites with subsequent reduction in the metabolite leakage for all metabolite classes.

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Currently, the energy required to produce biofuel from algae is 1.38 times the energy available from the fuel. Current methods do not deliver scalable, commercially viable cell wall disruption, which creates a bottleneck on downstream processing. This is primarily due to the methods depositing energy within the water as opposed to within the algae. This study investigates ultraviolet B (UVB) as a disruption method for the green algae Chlamydomonas reinhardtii, Dunaliella salina and Micractinium inermum to enhance solvent lipid extraction. After 232 seconds of UVB exposure at 1.5 W/cm², cultures of C. reinhardtii (culture density 0.7 mg/mL) showed 90% disruption, measured using cell counting, correlating to an energy consumption of 5.6 MJ/L algae. Small-scale laboratory tests on C. reinhardtii showed bead beating achieving 45.3 mg/L fatty acid methyl esters (FAME) and UV irradiation achieving 79.9 mg/L (lipids solvent extracted and converted to FAME for measurement). The alga M. inermum required a larger dosage of UVB due to its thicker cell wall, achieving a FAME yield of 226 mg/L, compared with 208 mg/L for bead beating. This indicates that UV disruption had a higher efficiency when used for solvent lipid extraction. This study serves as a proof of concept for UV irradiation as a method for algal cell disruption.

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BACKGROUND: Microalgae accumulate lipids when exposed to stressful conditions such as nutrient limitation that can be used to generate biofuels. Nitrogen limitation or deprivation is a strategy widely employed to elicit this response. However, this strategy is associated with a reduction in the microalgal growth, leading to overall poor lipid productivities. Here, we investigated the combined effect of a reduced source of nitrogen (ammonium) and super-saturating light intensities on the growth and induction of lipid accumulation in two model but diverse microalgal species, Phaeodactylum tricornutum and Nannochloropsis oceanica. We hypothesized that the lower energy cost of assimilating ammonium would allow the organisms to use more reductant power for lipid biosynthesis without compromising growth and that this would be further stimulated by the effect of high light (1000 µmol m-2 s-1) stress. We studied the changes in growth and physiology of both species when grown in culture media that either contained nitrate or ammonium as the nitrogen source, and an additional medium that contained ammonium with tungsten in place of molybdenum and compared this with growth in media without nitrogen. We focused our investigation on the early stages of exposure to the treatments to correspond to events relevant to induction of lipid accumulation in these two species. RESULTS: At super-saturating light intensities, lipid productivity in P. tricornutum increased twofold when grown in ammonium compared to nitrogen free medium that increased further when tungsten was present in the medium in place of molybdenum. Conversely, N. oceanica growth and physiology was not compromised by the high light intensities used, and the use of ammonium had a negative effect on the lipid productivity, which was even more marked when tungsten was present. CONCLUSIONS: Whilst the use of ammonium and super-saturating light intensities in P. tricornutum was revealed to be a good strategy for increasing lipid biosynthesis, no changes in the lipid productivity of N. oceanica were observed, under these conditions. Both results provide relevant direction for the better design of processes to produce biofuels in microalgae by manipulating growth conditions without the need to subject them to genetic engineering manipulation.

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The commercial reality of bioactive compounds and oil production from microalgal species is constrained by the high cost of production. Downstream processing, which includes harvesting and extraction, can account for 70-80% of the total cost of production. Consequently, from an economic perspective extraction technologies need to be improved. Microalgal cells are difficult to disrupt due to polymers within their cell wall such as algaenan and sporopollenin. Consequently, solvents and disruption devices are required to obtain products of interest from within the cells. Conventional techniques used for cell disruption and extraction are expensive and are often hindered by low efficiencies. Microwave-assisted extraction offers a possibility for extraction of biochemical components including lipids, pigments, carbohydrates, vitamins and proteins, individually and as part of a biorefinery. Microwave technology has advanced since its use in the 1970s. It can cut down working times and result in higher yields and purity of products. In this review, the ability and challenges in using microwave technology are discussed for the extraction of bioactive products individually and as part of a biorefinery approach.

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Monocultures have been the preferred production route in the bio-industry, where contamination has been a major bottleneck. In nature, microorganisms usually exist as part of organized communities and consortia, gaining benefits from co-habitation, keeping invaders at bay. There is increasing interest in the use of co-cultures to tackle contamination issues, and simultaneously increase productivity and product diversity. The feasibility of extending the natural phenomenon of co-habitation to the biomanufacturing industry in the form of co-cultures requires careful and systematic consideration of several aspects. This article will critically examine and review current work on microbial co-cultures, with the intent of examining the concept and proposing a design pipeline that can be developed in a biomanufacturing context.

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Microalgae are a potential candidate for biofuel production and environmental treatment because of their specific characteristics (e.g. fast growth, carbon neutral, and rich lipid accumulations). However, several primary bottlenecks still exist in current technologies, including low biomass conversion efficiency, bio-invasion from the external environment, limited or costly nutrient sources, and high energy and capital input for harvest, and stalling its industrial progression. Coupling biofuel production with environmental treatment renders microalgae a more feasible feedstock. This review focuses on microalgae biotechnologies for both bioenergy generation and environmental treatment (e.g. CO2 sequestration and wastewater reclamation). Different intelligent technologies have been developed, especially during the last decade, to eliminate the bottlenecks, including mixotrophic/heterotrophic cultivation, immobilization, and co-cultivation. It has been realized that any single purpose for the cultivation of microalgae is not an economically feasible option. Combinations of applications in biorefineries are gradually reckoned to be necessary as it provides more economically feasible and environmentally sustainable operations. This presents microalgae as a special niche occupier linking the fields of energy and environmental sciences and technologies. The integrated application of microalgae is also proven by most of the life-cycle analysis studies. This study summarizes the latest development of primary microalgal biotechnologies in the two areas that will bring researchers a comprehensive view towards industrialization with an economic perspective.

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Metabolome characterisation is a powerful tool in oncology. To obtain a valid description of the intracellular metabolome, two of the preparatory steps are crucial, namely washing and quenching. Washing must effectively remove the extracellular media components and quenching should stop the metabolic activities within the cell, without altering the membrane integrity of the cell. Therefore, it is important to evaluate the efficiency of the washing and quenching solvents. In this study, we employed two previously optimised protocols for simultaneous quenching and extraction, and investigated the effects of a number of washing steps/solvents and quenching solvent additives, on metabolite leakage from the adherent metastatic breast cancer cell line MDA-MB-231. We explored five washing protocols and five quenching protocols (including a control for each), and assessed for effectiveness by detecting ATP in the medium and cell morphology changes through scanning electron microscopy (SEM) analyses. Furthermore, we studied the overall recovery of eleven different metabolite classes using the GC-MS technique and compared the results with those obtained from the ATP assay and SEM analysis. Our data demonstrate that a single washing step with PBS and quenching with 60% methanol supplemented with 70 mM HEPES (-50 °C) results in minimum leakage of intracellular metabolites. Little or no interference of PBS (used in washing) and methanol/HEPES (used in quenching) on the subsequent GC-MS analysis step was noted. Together, these findings provide for the first time a systematic study into the washing and quenching steps of the metabolomics workflow for studying adherent mammalian cells, which we believe will improve reliability in the application of metabolomics technology to study adherent mammalian cell metabolism.

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Nitrogen stress is a common strategy employed to stimulate lipid accumulation in microalgae, a biofuel feedstock of topical interest. Although widely investigated, the underlying mechanism of this strategy is still poorly understood. We examined the proteome response of lipid accumulation in the model diatom, Phaeodactylum tricornutum (CCAP 1055/1), at an earlier stage of exposure to selective nitrogen exclusion than previously investigated, and at a time point when changes would reflect lipid accumulation more than carbohydrate accumulation. In total 1043 proteins were confidently identified (≥ 2 unique peptides) with 645 significant (p < 0.05) changes observed, in the LC-MS/MS based iTRAQ investigation. Analysis of significant changes in KEGG pathways and individual proteins showed that under nitrogen starvation P. tricornutum reorganizes its proteome in favour of nitrogen scavenging and reduced lipid degradation whilst rearranging the central energy metabolism that deprioritizes photosynthetic pathways. By doing this, this species appears to increase nitrogen availability inside the cell and limit its use to the pathways where it is needed most. Compared to previously published proteomic analysis of nitrogen starvation in Chlamydomonas reinhardtii, central energy metabolism and photosynthesis appear to be affected more in the diatom, whilst the green algae appears to invest its energy in reorganizing respiration and the cellular organization pathways.

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Metabolome analyses are a suite of analytical approaches that enable us to capture changes in the metabolome (small molecular weight components, typically less than 1500 Da) in biological systems. Mass spectrometry (MS) has been widely used for this purpose. The key challenge here is to be able to capture changes in a reproducible and reliant manner that is representative of the events that take place in vivo Typically, the analysis is carried out in vitro, by isolating the system and extracting the metabolome. MS-based approaches enable us to capture metabolomic changes with high sensitivity and resolution. When developing the technique for different biological systems, there are similarities in challenges and differences that are specific to the system under investigation. Here, we review some of the challenges in capturing quantitative changes in the metabolome with MS based approaches, primarily in microbial and mammalian systems.This article is part of the themed issue 'Quantitative mass spectrometry'.

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