Towards quantitative mass spectrometry based metabolomics in microbial and mammalian systems

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

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

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

Metabolic insights into infochemicals induced colony formation and flocculation in scenedesmus subspicatus unraveled by quantitative proteomics

Microalgae can respond to natural cues from crustacean grazers, such as Daphnia, by forming colonies and aggregations called flocs. Combining microalgal biology, physiological ecology, and quantitative proteomics, we identified how infochemicals from Daphnia trigger physiological and cellular level changes in the microalga Scenedesmus subspicatus, underpinning colony formation and flocculation. We discovered that flocculation occurs at an energy-demanding ‘alarm’ phase, with an important role proposed in cysteine synthesis. Flocculation appeared to be initially stimulated by the production of an extracellular matrix where polysaccharides and fatty acids were present, and later sustained at an ‘acclimation’ stage through mitogen-activated protein kinase (MAPK) signaling cascades. Colony formation required investment into fatty acid metabolism, likely linked to separation of membranes during cell division. Higher energy demands were required at the alarm phase, which subsequently decreased at the acclimation stage, thus suggesting a trade-off between colony formation and flocculation. From an ecological and evolutionary perspective, our findings represent an improved understanding of the effect of infochemicals on microalgae-grazers interactions, and how they can therefore potentially impact on the structure of aquatic communities. Moreover, the mechanisms revealed are of interest in algal biotechnology, for exploitation in low-cost, sustainable microalgal biomass harvesting

Harvesting Environmental Microalgal Blooms for Remediation and Resource Recovery: A Laboratory Scale Investigation with Economic and Microbial Community Impact Assessment

A laboratory based microflotation rig termed efficient FLOtation of Algae Technology (eFLOAT) was used to optimise parameters for harvesting microalgal biomass from eutrophic water systems. This was performed for the dual objectives of remediation (nutrient removal) and resource recovery. Preliminary experiments demonstrated that chitosan was more efficient than alum for flocculation of biomass and the presence of bacteria could play a positive role and reduce flocculant application rates under the natural conditions tested. Maximum biomass removal from a hyper-eutrophic water retention pond sample was achieved with 5 mg·L-1 chitosan (90% Chlorophyll a removal). Harvesting at maximum rates showed that after 10 days, the bacterial diversity is significantly increased with reduced cyanobacteria, indicating improved ecosystem functioning. The resource potential within the biomass was characterized by 9.02 μg phosphate, 0.36 mg protein, and 103.7 μg lipid per mg of biomass. Fatty acid methyl ester composition was comparable to pure cultures of microalgae, dominated by C16 and C18 chain lengths with saturated, monounsaturated, and polyunsaturated fatty acids. Finally, the laboratory data was translated into a full-size and modular eFLOAT system, with estimated costs as a novel eco-technology for efficient algal bloom harvesting

Looking through the FOG: microbiome characterization and lipolytic bacteria isolation from a fatberg site

Sewer systems are complex physical, chemical and microbial ecosystems where fats, oils and grease (FOG) present a major problem for sewer management. Their accumulation can lead to blockages (‘Fatbergs’), sewer overflows and disruption of downstream wastewater treatment. Further advancements of biological FOG treatments need to be tailored to degrade the FOG, and operate successfully within the sewer environment. In this study we developed a pipeline for isolation of lipolytic strains directly from two FOG blockage sites in the UK, and isolated a range of highly lipolytic bacteria. We selected the five most lipolytic strains using Rhodamine B agar plates and pNP-Fatty acid substrates, with two Serratia spp., two Klebsiella spp. and an environmental Acinetobacter strain that all have the capacity to grow on FOG-based carbon sources. Their genome sequences identified the genetic capacity for fatty acid harvesting (lipases), catabolism and utilization (Fad genes). Furthermore, we performed a preliminary molecular characterization of the microbial community at these sites, showing a diverse community of environmental bacteria at each site, but which did include evidence of sequences related to our isolates. This study provides proof of concept to isolation strategies targeting Fatberg sites to yield candidate strains with bioremediation potential for FOG in the wastewater network. Our work sets the foundation for development of novel bioadditions tailored to the environment with non-pathogenic Acinetobacter identified as a candidate for this purpose

The Effect of High-Intensity Ultraviolet Light to Elicit Microalgal Cell Lysis and Enhance Lipid Extraction

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

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

Towards quantitative mass spectrometry-based metabolomics in microbial and mammalian systems

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

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