Co-culturing microbial consortia: approaches for applications in biomanufacturing and bioprocessing.

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.

Influence of nutrient status on the biohydrogen and lipid productivity in Parachlorella kessleri: a biorefinery approach.

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.

Microalgae: a robust "green bio-bridge" between energy and environment.

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.

Microbial consortia: a critical look at microalgae co-cultures for enhanced biomanufacturing.

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.

Influence of washing and quenching in profiling the metabolome of adherent mammalian cells: a case study with the metastatic breast cancer cell line MDA-MB-231.

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.

An efficient TOF-SIMS image analysis with spatial correlation and alternating non-negativity-constrained least squares.

MOTIVATION: Advances in analytical instrumentation towards acquiring high-resolution images of mass spectrometry constantly demand efficient approaches for data analysis. This is particularly true of time-of-flight secondary ion mass spectrometry imaging where recent advances enable acquisition of high-resolution data in multiple dimensions. In many applications, the distribution of different species from a sampled surface is spatially continuous in nature and a model that incorporates the spatial correlation across the surface would be preferable to estimations at discrete spatial locations. A key challenge here is the capability to analyse the high-resolution multidimensional data to extract relevant information reliably and efficiently. RESULTS: We propose a framework based on alternating non-negativity-constrained least squares which accounts for the spatial correlation across the sample surface. The proposed method also decouples the computational complexity of the estimation procedure from the image resolution, which significantly reduces the processing time. We evaluate the performance of the algorithm with biochemical image datasets generated from mixture of metabolites.

HILIC- and SCX-based quantitative proteomics of Chlamydomonas reinhardtii during nitrogen starvation induced lipid and carbohydrate accumulation.

Nitrogen starvation induced changes in carbohydrate and lipid content is described in several algal species. Although these phenotypic changes are desirable, such manipulations also significantly deteriorate culture health, ultimately halting growth. To optimize biofuel production from algae, it is desirable to induce lipid accumulation without compromising cell growth and survival. In this study, we utilized an 8-plex iTRAQ-based proteomic approach to assess the model alga Chlamydomonas reinhardtii CCAP 11/32CW15+ under nitrogen starvation. First-dimension fractionation was conducted using HILIC and SCX. A total of 587 proteins were identified (≥3 peptides) of which 71 and 311 were differentially expressed at significant levels (p<0.05), during nitrogen stress induced carbohydrate and lipid production, respectively. Forty-seven percent more changes with significance were observed with HILIC compared to SCX. Several trends were observed including increase in energy metabolism, decrease in translation machinery, increase in cell wall production and a change of balance between photosystems I and II. These findings point to a severely compromised system where lipid is accumulated at the expense of normal functioning of the organism, suggesting that a more informed and controlled method of lipid induction than gross nutrient manipulation would be needed for development of sustainable processes.

A solvation-based screening approach for metabolite arrays.

This paper explores a new method for screening metabolites in an array format based on relative polarity using selective solvent dissolution. A synthetic cocktail of metabolites was spotted onto a hydrophobic silicon surface, and solubilised with solvents of varying polarity. The metabolites retained on the silicon surface after the solvent treatments were detected using time-of-flight static secondary ion mass spectrometry (ToF-sSIMS). Solvent-specific metabolite retention was clearly evident on multivariate analysis of the dataset, using principal component analysis. Selective removal of metabolites was observed when solvents with different polarity were used, with the metabolite retention or removal in most cases correlating to the polarity of the solvent used, although consideration of other forces in operation may be needed to arrive at fully predictable behaviours. This approach provides the basis for development of a technique to separate complex metabolites into simpler constituents in a metabolite array prior to identification and quantification using mass spectrometry. It is an analytical approach that is intermediate between the more rapid but less informative direct analysis methods (such as DIMS) that do not involve any analyte separations and the more comprehensive but time consuming methods (such as GC- and LC-MS) that involve chromatographic or electrophoretic separations. The approach has the potential to be successfully developed for rapid, yet informative screening of metabolomes.

Response to nutrient variation on lipid productivity in green microalgae captured using second derivative FTIR and Raman spectroscopy.

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.

Towards a Phaeodactylum tricornutum biorefinery in an outdoor UK environment.

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.

Interactions between polyethylene and polypropylene microplastics and Spirulina sp. microalgae in aquatic systems.

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.

Enabling large-scale production of algal oil in continuous output mode.

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.

Biomolecular transitions and lipid accumulation in green microalgae monitored by FTIR and Raman analysis.

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.

Phaeodactylum tricornutum: A Diatom Cell Factory.

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.

Diatoms for Carbon Sequestration and Bio-Based Manufacturing.

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.

Explanatory multivariate analysis of ToF-SIMS spectra for the discrimination of bacterial isolates.

Multivariate analysis (PC-CVA and GA-CVA) was carried out on time-of-flight secondary ion mass spectra (ToF-SIMS) derived from 16 bacterial isolates associated with urinary tract infections, with an objective of extracting the spectral information relevant to their species-level discrimination. The use of spectral pre-processing, such as removal of the dominant peaks prior to analysis and analysis of the dominant peaks alone, enabled the identification of 37 peaks contributing to the principal components-canonical variates analysis (PC-CVA) discrimination of the bacterial isolates in the mass range of m/z 1-1000. These included signals at m/z 70, 84, 120, 134, 140, 150, 175 and 200. A univariate statistical analysis (Kruskal-Wallis) of the signal intensities at the identified m/z enabled an understanding of the discriminatory basis, which can be used in the development of robust parsimonious models for predictive purposes. The utility of genetic algorithm (GA)-based feature selection in identifying the discriminatory variables is also demonstrated. A database search of the identified signals enabled the biochemical origins of some these signals to be postulated.

Effects of cryopreservation on viability and functional stability of an industrially relevant alga.

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.

Quenching for Microalgal Metabolomics: A Case Study on the Unicellular Eukaryotic Green Alga Chlamydomonas reinhardtii.

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.