This manuscript has been authored by Battelle Memorial Institute under Contract No. DE-AC05-76RL01830 with the U.S. Department of Energy. The U.S Government retains and the publisher, by accepting the article for publication, acknowledges that the U.S. Government retains a non-exclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for U.S. Government purposes.

1. Sample Preparation
<ol>
	<li>Generate a mixed aqueous/organic product stream via continuous flow HTL of algae according to the reactor design and experimental procedure found in the literature10,11.</li>
	<li>Use a gravity separator to separate the product stream into an aqueous phase and organic phase.</li>
	<li>Filter 10 ml of the HTL aqueous phase using a 0.45 &#181;m syringe filter and store in a refrigerator maintained at 4 &#176;C for GC &#215; GC&#8211;TOF-MS analysis.</li>
</ol>
2. Instrument Components
<ol>
	<li>Use a gas chromatograph (GC) equipped with a quad-jet dual stage cooling-based modulator and time-of-flight (TOF) mass spectrometer (MS) for these experiments.</li>
	<li>Configure the auto-sampler to inject 1 &#181;l of each sample or standard into the GC. Use a randomized block design of sample and standard injections for the auto-sampler sequence as described in the literature13. The randomized block design is commonly used in quantitative studies to control for instrument operation. Our laboratory utilizes the design routinely even in comparative studies to verify instrument operation.</li>
	<li>Connect the primary and secondary column using a press-tight connector before the modulator. Ensure that both edges of both primary and secondary columns are cut straight without sharp edges before connecting to the press-tight connector.</li>
	<li>Place ferrule on the GC column and then connect primary column to the GC injector so that 5 mm of column is inside the injector.</li>
	<li>Ensure that glass liner, non-stick liner O-ring and septa for GC injector are new and free of contamination.</li>
	<li>Use 1/16 x 0.5 mm ID transfer line ferrules to connect the secondary column and transfer line. Place a 0.2 m portion of the secondary column in the transfer line.</li>
	<li>Ensure that a 0.1 m portion of the secondary column is in the modulator.</li>
	<li>Use ultrahigh purity helium gas as carrier gas for GC at a flow rate of 1.5 ml min-1.</li>
	<li>Ensure there is sufficient liquid nitrogen in the Dewar which acts as a coolant in the modulator. The level of the liquid nitrogen in the Dewar can be predicted using a pressure gauge attached to its outlet. A 69 kPa reading of the pressure gauge indicates that the Dewar is full, while 0 kPa indicates that it is empty.</li>
</ol>
3. Protocols Before Analyzing Samples
<ol>
	<li>Ensure there are no major leaks in the instrument. If the vacuum gauge reading of the TOF-MS is higher than 2.7 &#215; 10-5 Pa for 1.5 ml min-1 GC column flow rate, this indicates a major leak in the system.</li>
	<li>Set-up the quality control (QC) method and run in-built &#39;acquisition system adjustments&#39; protocol to achieve maximum signal response using manufacturer&#39;s protocol.</li>
	<li>Run in-built &#39;instrument optimization&#39; protocols of QC method, in series - filament focus, ion optic focus and mass calibration tests using manufacturer&#39;s protocol. Ensure that mass calibration test passes. This QC method ensures that all the hardware parameters of the instrument are at optimum level.</li>
	<li>Perform a &#34;leak check&#34; using manufacturer&#39;s protocol. Analyze automatically generates leak check report. Ensure that the relative concentration of 28 (N2), 32 (O2) and 18 (moisture) ions must be below less than 10%, 3% and 5% of internal standard mass spectra of 69 ion, respectively.</li>
	<li>Tune the TOF-MS using manufacturer&#39;s protocol.</li>
	<li>Run quality control method as well as TOF-MS tune protocol before and after leak check and also while analyzing samples and standards.</li>
</ol>
4. Protocol to Determine the Optimum Modulation Period of Modulator
<ol>
	<li>Arbitrarily select a long modulation period (e.g. 10 sec or 13 sec). Inject a sample as described in 2.2.</li>
	<li>Identify the retention time in second dimension of the contour plot after which no peaks elutes. Select identified second dimension retention time as optimum modulation period. Figure 2 clearly elucidate the identification of retention time in second dimension of the contour plot.</li>
	<li>Increase the modulation period used in step 4.1 and perform the analysis again if &#34;wrap around&#34; is observed18. Wrap around phenomena occurs if the peaks in the second dimension elutes below the baseline of first dimension. Example contour plot for &#39;wraparound&#39; is shown in supplementary information Figure 3.</li>
	<li>Repeat steps 4.2 and 4.3 until optimum value is determined.</li>
</ol>
5. Experimental Parameters of Instrument Set-up
<ol>
	<li>Install a polar (60 m x 0.25 mm x 0.5 &#181;m film thickness) capillary column as the primary column and a non-polar (2.3 m x 0.25 mm x 0.5 &#181;m film thickness) capillary column as the secondary column. Bake both the primary and secondary column for at least 2 hr to remove trace amounts of moisture, air and contaminants associated with new GC columns.</li>
	<li>Use ultrahigh purity helium gas as carrier gas for GC at a flow rate of 1.5 ml min-1.</li>
	<li>Set the GC injector to a temperature of 260 &#176;C and a split ratio of 1:250.</li>
	<li>Employ the following temperature program for the primary column: a constant temperature of 40 &#176;C for 0.2 min followed by a temperature ramp to 260 &#176;C at 5 &#176;C min-1, followed by a constant temperature of 260 &#176;C for 5 min.</li>
	<li>Maintain the modulator temperature 5 &#176;C higher than that of the secondary column and the secondary column temperature at 5 &#176;C higher than that of the primary column.</li>
	<li>Use an optimum modulation period of 4 sec with 0.8 sec of hot pulse and 1.2 sec of cold pulse. This value is determined based on the protocol described in section 4.</li>
	<li>Set transfer line temperature to 270 &#176;C.</li>
	<li>Set the acquisition delay or solvent delay to 0 sec.</li>
	<li>Set the lower and higher range of m/z as 35 and 800, respectively.</li>
	<li>Set the MS detector acquisition rate at 400 spectra/sec.</li>
	<li>Maintain the MS detector voltage at 150 V higher than the optimized value.</li>
	<li>Maintain the MS ion source temperature at 225 &#176;C.</li>
</ol>
6. Data Analysis
<ol>
	<li>Perform data processing using the software supplied by the instrument manufacturer.</li>
	<li>Select the following tasks in the data analysis method - Compute baseline, find peaks above the baseline, library search and calculate are/height.</li>
	<li>Track the baseline through the data file. Enter baseline offset as 0.5. 
	Enter expected peak width of 15 sec in the first dimension and 0.15 sec in the second dimension.</li>
	<li>Set signal-to-noise ratio as 5,000 and similarity values of &#62;850 for identification of compounds.</li>
	<li>Select a commercially available mass spectral library to identify chemical compounds present in samples and set the library search mode to forward.</li>
	<li>Process the data files using this data analysis method using manufacturer&#39;s protocol. It requires at least 1 hr to process a data file.</li>
</ol>

The authors declare that they have no competing financial interests.

Results clearly illustrate the ability of the column combination of polar &#215; non-polar to resolve polar compounds and light volatiles present in the aqueous fraction of algae bio-crude without prior sample preparation techniques. Drastic peak tailing was observed for organic acids and N-compounds while using the non-polar&#215; polar column combination. This peak tailing was not observed for the early eluting light organics. This behavior has been reproducible when verifying the instrument is free of leaks (the vacuu...

Steady growth in demand for liquid fuels, finite fossil fuel resources, uncertainty of fossil fuel supplies, and concerns over the increasing concentration of greenhouse gases in the atmosphere have increased global awareness for renewable resources1. Solar energy (including photovoltaics and solar-thermal), wind energy, hydropower, geothermal, and biomass are the primary renewable sources that could potentially replace fossil-derived energy2. Of these, biomass is the only carbon-based alternative energy resource for the production of liquid transportation fuels and high-value chemicals3. Biomass includes any organic material such as forest resources, agricultural residue, algae, oilseeds, municipal solid waste, and carbon-rich industrial wastes (e.g. from pulp and paper industry or from food processing)1. Biomass is classified into two broad categories: lignocellulosic and non-ligneous feedstocks based on compositional characteristics. Lignocellulosic biomass consists of carbohydrates and lignin, while non-ligneous feedstocks have proteins, carbohydrates and lipids/oils4. Lignocellulosic feedstocks, derived from terrestrial plants, can only satisfy 30% of the current liquid fuel (gasoline, jet fuel, and diesel) demand if sustainably cultivated and harvested5,6. Hence, non-ligneous aquatic microorganisms, such as microalgae and fungi, are considered potential feedstocks for the production of renewable liquid fuels to complement lignocellulosic resources.
Microalgae feedstocks have the potential to satisfy current liquid transportation fuels demand7,8. Algae have many advantages: high areal productivity8, the ability to grow in low-quality, brackish, or sea water9, and the ability to accumulate energy-dense triglycerides or hydrocarbons7,8. Hydrothermal liquefaction (HTL) is a viable and scalable conversion process which utilizes water naturally associated with algal or aquatic feedstocks10,11. It is a thermo-chemical process with operating temperatures of 250-400 &#176;C and operating pressures of 10-25 MPa which produces a liquid product, or bio-crude, which can be upgraded into a fuel blend stock. Bio-crude produced from HTL of algae has distinguishable and easily separable organic and aqueous fractions. The organic fraction of bio-crude can be efficiently converted into a refinery ready blend stock via catalytic hydro-treating processes11. The aqueous fraction of bio-crude contains ~30% of the total carbon present in the algal feedstock. Although thousands of compounds have been identified in the HTL aqueous stream, the predominant fractions consist of low molecular weight oxygenates (including acids, alcohols, ketones, and aldehydes) formed by the degradation of carbohydrates and lipids, and nitrogen heterocyclics (including pyrroles, pyridines, pyrazines, and imidazoles) derived from protein decomposition12. Studies on utilizing the aqueous fraction to improve overall process economics as well as sustainability are ongoing. Synthesis gas can be produced from the aqueous fraction of algae bio-crude via catalytic hydrothermal gasification10,13,14. Alternatively, organics in the aqueous fraction can also be catalytically converted to fuel additives and specialty chemicals. Research on optimizing catalytic hydrothermal gasification and catalyst screening studies for conversion of organics in the aqueous liquid phase is currently underway at the Pacific Northwest National Laboratory (PNNL). For this work, qualitative as well as quantitative characterization of the aqueous fraction of algae bio-crude is required. Since the aqueous fraction of algae bio-crude is considered a waste stream, there are very few studies that have analyzed the aqueous fraction of algae bio-crude13,15. Moreover, recent studies concluded that converting this HTL algae water into high-value bio-products would improve the sustainability as well as economics of an HTL-based bio-refinery11. Therefore, this study focused on developing a method for qualitative characterization of the aqueous fraction of bio-crude obtained from HTL of algae by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC &#215; GC&#8211;TOF-MS).
GC &#215; GC&#8211;TOF-MS is the most promising chromatographic analytical technique to increase resolution (or separation of chemical compounds in a sample), peak capacity (i.e. number of resolved peaks), signal-to-noise ratio (for identification of chemical compounds with high confidence), and to avoid co-elution of chemical compounds16. In order to maximize resolution, peak capacity, and signal-to noise ratio, two GC columns with different stationary phases are connected in series using a press-fit connector or micro-union17(see Figure 1 which is a block diagram of GC &#215; GC&#8211;TOF-MS system used in this study). A modulator is located between the press-fit connector and secondary columns to trap, refocus, and re-inject the effluents from the primary column into the secondary column18. Modulation occurs on the secondary column in the present study as shown in Figure 1. The secondary column is then connected to the TOF-MS via a transfer line assembly.
GC &#215; GC&#8211;TOF-MS was used previously for qualitative as well as quantitative analysis of organic samples such as crude oil16,19, gasoline, jet-fuel, diesel, bio-diesel, and the organic fraction of bio-fuel20-22 produced from thermo-chemical as well as thermo-catalytic conversion processes23,24. For characterization of these organic samples in GC &#215; GC&#8211;TOF-MS instruments, a long non-polar column was used as the primary column, while a short polar column was used as the secondary column. This conventional column configuration resolves chemical compounds based on differences in volatility over the first dimension, followed by polarity in the second dimension18. Aqueous or water samples from biological processes, food processing, and environmental wastes were also characterized using similar primary/secondary column configurations after the sample had been through preparation steps17,25-30. Sample preparation techniques such as derivatization, solid-phase extraction, and organic solvent extraction have all been utilized prior to GC &#215; GC&#8211;TOF-MS analysis17,27-29,31,32. These techniques were aimed at decreasing the polarity of compounds in the sample for analysis using a conventional column configuration33. An alternative strategy was employed in this study based on the nature of the sample (here polar organic compounds in water) utilizing the reverse primary/secondary column configuration for GC &#215; GC&#8211;TOF-MS analysis. Since the aqueous fraction of bio-crude produced from HTL has polar compounds13, a column combination of a primary polar column and a secondary non-polar column was used in the GC &#215; GC&#8211;TOF-MS without any upstream sample preparation. This primary/secondary column combination resolves chemical compounds based on differences in polarity over the first dimension, followed by volatility in the second dimension. Limited analytical methods exist in the literature for characterization of aqueous samples using two-dimensional gas chromatography without prior sample processing15.
The objective of this study was to determine the chemical compounds present in the aqueous fraction of algae bio-crude. To achieve this objective, a GC &#215; GC&#8211;TOF-MS data acquisition method was developed with a column combination of polar column (primary) &#215; non-polar (secondary). Klenn et al. (2015) suggested that increasing the length of the primary column (especially 60 m GC columns) and lowering the offset temperature of the secondary column with respect to the primary column would maximize peak capacity and resolution16-18. Therefore, a 60 m primary column and 5 &#176;C offset temperature of the secondary column with respect to the primary column were used in this study. The optimum modulation period was determined following a protocol described in this study (see section 4). The optimum ramp rate of GC column temperature was determined by a trial and error method and is similar to the value suggested in the literature16-18. To discuss the advantages of this column combination for aqueous samples, we have analyzed HTL algae water samples with the conventional column combination of non-polar &#215; polar. Operating parameters suggested in the literature were employed for analyzing the aqueous fraction of algal bio-crude with a non-polar &#215; polar column combination18.

<table><tbody><tr><td>GC &amp;times; GC&amp;#8211;TOF/MS</td><td>Leco</td><td>PEG4D11DLN15</td><td>Commercial Pegasus 4D</td></tr><tr><td>ChromaTOF version 4.50&amp;nbsp;</td><td>Leco</td><td></td><td>Data analysis software</td></tr><tr><td>Rxi-5MS GC column</td><td>Restek</td><td>13420</td><td>2.3 m column was used from this column.</td></tr><tr><td>Stabilwax GC column</td><td>Restek</td><td>10626</td><td></td></tr><tr><td>HP-5 GC column</td><td>Agilent</td><td>19091J-416</td><td></td></tr><tr><td>Stabilwax GC column</td><td>Restek</td><td>15121</td><td></td></tr><tr><td>Presstight Connector</td><td>Restek</td><td>20430</td><td></td></tr><tr><td>GC injector liner</td><td>Restek</td><td>23305.5</td><td></td></tr><tr><td>GC Injector ferrules</td><td>Agilent</td><td>5181-3323</td><td></td></tr><tr><td>Non-stick liner O-rings</td><td>Agilent</td><td>5188-5365</td><td></td></tr><tr><td>Transfer line ferrules</td><td>Restek</td><td>20212</td><td></td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>459844</td><td>Chromatography grade</td></tr><tr><td>Acetone</td><td>Sigma-Aldrich</td><td>414689</td><td>Chromatography grade</td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>320099</td><td>Chromatography grade</td></tr><tr><td>2-butanone</td><td>Sigma-Aldrich</td><td>360473</td><td>Chromatography grade</td></tr><tr><td>Propanoic acid</td><td>Sigma-Aldrich</td><td>402907</td><td>Chromatography grade</td></tr><tr><td>Butanoic acid</td><td>Sigma-Aldrich</td><td>19215</td><td>Chromatography grade</td></tr><tr><td>Pyridine</td><td>Sigma-Aldrich</td><td>270970</td><td>Chromatography grade</td></tr><tr><td>Pyrazine</td><td>Sigma-Aldrich</td><td>65693</td><td>Chromatography grade</td></tr><tr><td>Acetamide</td><td>Sigma-Aldrich</td><td>695122</td><td>Chromatography grade</td></tr><tr><td>2,5-pyrrolididione</td><td>Sigma-Aldrich</td><td>S9381</td><td>Chromatography grade</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-methylsuccinimide</td><td>Sigma-Aldrich</td><td>325384</td><td>Chromatography grade</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(2-hydroxyethyl)succinimide</td><td>Sigma-Aldrich</td><td>444073</td><td>Chromatography grade</td></tr></tbody></table>

qualitative characterization of the aqueous fraction from hydrothermal liquefaction of algae using 2d gas chromatography with time-of-flight mass spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A total ion chromatogram (TIC) obtained for the aqueous fraction of algae bio-crude analyzed with a column combination of polar &#215; non-polar is shown in Figure 4. Retention times and similarity or match factor values of compounds identified by searching against a National Institute of Standards and Technology (NIST) library are tabulated in Table 1. Oxygenates (such as cyclopenatanone, furanic compounds and dianhydromannitol) and organic acids (includ...

Watch this Scientific Journal Video about Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

qualitative-characterization-aqueous-fraction-from-hydrothermal

Research

JoVE Journal

Bioengineering

9.3K Views.  Pacific Northwest National Laboratory. A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating. 

Video: Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Analysis of Fatty Acid Content and Composition in Microalgae

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (&omega;-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes.
With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of.
This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other.
The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.

A method for the determination of fatty acid content and composition in microalgae based on mechanical cell disruption, solvent based lipid extraction, transesterification, and quantification and identification of fatty acids using gas chromatography is described. A tripentadecanoin internal standard is used to compensate for the possible losses during extraction and incomplete transesterification.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of ...

Environment

A Toolkit to Enable Hydrocarbon Conversion in Aqueous Environments

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The toolkit consists of multiple standard interchangeable parts (BioBricks)9 addressing the conversion of alkanes, regulation of gene expression and survival in toxic hydrocarbon-rich environments.
A three-step pathway for alkane degradation was implemented in E. coli to enable the conversion of medium- and long-chain alkanes to their respective alkanols, alkanals and ultimately alkanoic-acids. The latter were metabolized via the native &beta;-oxidation pathway. To facilitate the oxidation of medium-chain alkanes (C5-C13) and cycloalkanes (C5-C8), four genes (alkB2, rubA3, rubA4and rubB) of the alkane hydroxylase system from Gordonia sp. TF68,21 were transformed into E. coli. For the conversion of long-chain alkanes (C15-C36), theladA gene from Geobacillus thermodenitrificans was implemented. For the required further steps of the degradation process, ADH and ALDH (originating from G. thermodenitrificans) were introduced10,11. The activity was measured by resting cell assays. For each oxidative step, enzyme activity was observed.
To optimize the process efficiency, the expression was only induced under low glucose conditions: a substrate-regulated promoter, pCaiF, was used. pCaiF is present in E. coli K12 and regulates the expression of the genes involved in the degradation of non-glucose carbon sources.
The last part of the toolkit - targeting survival - was implemented using solvent tolerance genes, PhPFD&alpha; and &beta;, both from Pyrococcus horikoshii OT3. Organic solvents can induce cell stress and decreased survivability by negatively affecting protein folding. As chaperones, PhPFD&alpha; and &beta; improve the protein folding process e.g. under the presence of alkanes. The expression of these genes led to an improved hydrocarbon tolerance shown by an increased growth rate (up to 50%) in the presences of 10% n-hexane in the culture medium were observed.
Summarizing, the results indicate that the toolkit enables E. coli to convert and tolerate hydrocarbons in aqueous environments. As such, it represents an initial step towards a sustainable solution for oil-remediation using a synthetic biology approach.

A sustainable auto regulating bacterial system for the remediation of oil pollutions was designed using standard interchangeable DNA parts (BioBricks). An engineered E. coli strain was used to degrade alkanes via &beta;-oxidation in toxic aqueous environments. The respective enzymes from different species showed alkane degradation activity. Additionally, an increased tolerance to n-hexane was achieved by introducing genes from alkane-tolerant bacteria.

This work puts forward a toolkit that enables the conversion of alkanes by Escherichia coli and presents a proof of principle of its applicability. The ...

Single-throughput Complementary High-resolution Analytical Techniques for Characterizing Complex Natural Organic Matter Mixtures

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to characterize. However, to understand the thermodynamic and kinetic controls on greenhouse gas (carbon dioxide [CO2] and methane [CH4]) production resulting from the decomposition of NOM, a molecular-level characterization coupled with microbial proteome analyses is necessary. Further, climate and environmental changes are expected to perturb natural ecosystems, potentially upsetting complex interactions that influence both the supply of organic matter substrates and the microorganisms performing the transformations. A detailed molecular characterization of the organic matter, microbial proteomics, and the pathways and transformations by which organic matter is decomposed will be necessary to predict the direction and magnitude of the effects of environmental changes. This article describes a methodological throughput for comprehensive metabolite characterization in a single sample by direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), gas chromatography mass spectrometry (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography mass spectrometry (LC-MS), and proteomics analysis. This approach results in a fully-paired dataset which improves statistical confidence for inferring pathways of organic matter decomposition, the resulting CO2 and CH4 production rates, and their responses to environmental perturbation. Herein we present results of applying this method to NOM samples collected from peatlands; however, the protocol is applicable to any NOM sample (e.g., peat, forested soils, marine sediments, etc.).

This protocol describes a single throughput for complementary analytical and omics techniques culminating in a fully-paired characterization of natural organic matter and microbial proteomics in different ecosystems. This approach permits robust comparisons for identifying metabolic pathways and transformations important for describing greenhouse gas production and predicting responses to environmental change.

Natural organic matter (NOM) is composed of a highly complex mixture of thousands of organic compounds which, historically, proved difficult to ...

Coupling Carbon Capture from a Power Plant with Semi-automated Open Raceway Ponds for Microalgae Cultivation

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas electricity generation. Microalgae can biofix CO2 10 to 15 times faster than plants and convert algal biomass to products of interest, such as biofuels. Thus, this study presents a protocol that demonstrates the potential synergies of microalgae cultivation with a natural gas power plant situated in the southwestern United States in a hot semi-arid climate. State-of-the-art technologies are used to enhance carbon capture and utilization via the green algal species Chlorella sorokiniana, which can be further processed into biofuel. We describe a protocol involving a semi-automated open raceway pond and discuss the results of its performance when it was tested at the Tucson Electric Power plant, in Tucson, Arizona. Flue gas was used as the main carbon source to control pH, and Chlorella sorokiniana was cultivated. An optimized medium was used to grow the algae. The amount of CO2 added to the system as a function of time was closely monitored. Additionally, other physicochemical factors affecting algal growth rate, biomass productivity, and carbon fixation were monitored, including optical density, dissolved oxygen (DO), electroconductivity (EC), and air and pond temperatures. The results indicate that a microalgae yield of up to 0.385 g/L ash-free dry weight is attainable, with a lipid content of 24%. Leveraging synergistic opportunities between CO2 emitters and algal farmers can provide the resources required to increase carbon capture while supporting the sustainable production of algal biofuels and bioproducts.

A protocol is described to utilize the carbon dioxide in natural gas power plant flue gas to cultivate microalgae in open raceway ponds. Flue gas injection is controlled with a pH sensor, and microalgae growth is monitored with real time measurements of optical density.

In the United States, 35% of the total carbon dioxide (CO2) emissions come from the electrical power industry, of which 30% represent natural gas ...

Medium Preparation for the Cultivation of Microorganisms under Strictly Anaerobic/Anoxic Conditions

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As oxygen is ubiquitous in air, retaining O2-free conditions during all steps of cultivation is challenging but a prerequisite for anaerobic culturing. The protocol presented here demonstrates the successful cultivation of an anaerobic mixed culture derived from a biogas plant using a simple and inexpensive method. A precise description of the entire anoxic culturing process is given including media preparation, filling of cultivation flasks, supplementation with redox indicator and reducing agents to provide low redox potentials as well as exchanging the headspace to keep media free from oxygen. Furthermore, a detailed overview of aseptically inoculating gas tight serum flasks (by using sterile syringes and needles) and suitable incubation conditions is provided. The present protocol further deals with gas and liquid sampling for subsequent analyses regarding gas composition and volatile fatty acid concentrations using gas chromatography (GC) and high performance liquid chromatography (HPLC), respectively, and the calculation of biogas and methane yield considering the ideal gas law.

As obligate anaerobic organisms are unable to grow upon oxygen exposure, the use of anaerobic culturing techniques is indispensable. Here, we demonstrate a simple and effective method to cultivate a mixed culture derived from a biogas plant from media preparation to gas and volatile fatty acid quantification.

In contrast to aerobic organisms, strictly anaerobic microorganisms require the absence of oxygen and usually a low redox potential to initiate growth. As ...

Biochemistry

Quantitative Analysis by Thermogravimetry-Mass Spectrum Analysis for Reactions with Evolved Gases

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and multi-intermediates. Thermogravimetry-mass spectrum (TG-MS) is seen as a powerful tool to study reaction features. However, reaction details and reaction mechanics have not been effectively obtained directly from the ion current of TG-MS. Here, we provide a method of an equivalent characteristic spectrum analysis (ECSA) for analyzing the mass spectrum and giving the mass flow rate of reaction gases as precise as possible. The ECSA can effectively separate overlapping ion peaks and then eliminate the mass discrimination and temperature-dependent effect. Two example experiments are presented: (1) the decomposition of CaCO3 with evolved gas of CO2 and the decomposition of hydromagnesite with evolved gas of CO2 and H2O, to evaluate the ECSA on single-component system measurement and (2) the thermal pyrolysis of Zhundong coal with evolved gases of inorganic gases CO, H2, and CO2, and organic gases C2H4, C2H6, C3H8, C6H14, etc., to evaluate the ECSA on multi-component system measurement. Based on the successful calibration of the characteristic spectrum and relative sensitivity of specific gas and the ECSA on mass spectrum, we demonstrate that the ECSA accurately gives the mass flow rates of each evolved gas, including organic or inorganic gases, for not only single but multi-component reactions, which cannot be implemented by the traditional measurements.

Precise determination of the evolved gases&#39; flow rate is key to study the details of reactions. We provide a novel quantitative analysis method of equivalent characteristic spectrum analysis for thermogravimetry-mass spectrum analysis by establishing the calibration system of the characteristic spectrum and relative sensitivity, for obtaining the flow rate.

During energy conversion, material production, and metallurgy processes, reactions often have the features of unsteadiness, multistep, and ...

Engineering

Microalgal Cultivation for Biomethane Obtention from Swine Waste Biogas

Biogas Purification through the use of a Microalgae-Bacterial System in Semi-Industrial High Rate Algal Ponds

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of polluting gases such as carbon dioxide and hydrogen sulfide to increase the content of methane. In this study, we used a microalgal cultivation technology to treat and purify biogas produced from organic waste from the swine industry to obtain ready-to-use biomethane. For cultivation and purification, two 22.2 m3 open-pond photobioreactors coupled with an absorption-desorption column system were set up in San Juan de los Lagos, Mexico. Several recirculation liquid/biogas ratios (L/G) were tested to obtain the highest removal efficiencies; other parameters, such as pH, dissolved oxygen (DO), temperature, and biomass growth, were measured. The most efficient L/Gs were 1.6 and 2.5, resulting in a treated biogas effluent with a composition of 6.8%vol and 6.6%vol in CO2, respectively, and removal efficiencies for H2S up to 98.9%, as well as maintaining O2 contamination values of less than 2%vol. We found that pH greatly determines CO2 removal, more so than L/G, during cultivation because of its participation in the photosynthetic process of microalgae and its ability to vary pH when solubilized due to its acidic nature. DO, and temperature oscillated as expected from the light-dark natural cycles of photosynthesis and the time of day, respectively. Biomass growth varied with CO2 and nutrient feeding as well as reactor harvesting; however, the trend remained primed for growth.

Author Spotlight: Scaling Microalgal Biotechnology for Enhanced Biomethane Production 

Air pollution impacts the quality of life of all organisms. Here, we describe the use of microalgae biotechnology for the treatment of biogas (simultaneous removal of carbon dioxide and hydrogen sulfide) and the production of biomethane through semi-industrial open high-rate algal ponds and subsequent analysis of treatment efficiency, pH, dissolved oxygen, and microalgae growth.

In recent years, a number of technologies have emerged to purify biogas into biomethane. This purification entails a reduction in the concentration of ...

Derivatization and Separation of Barley Flour Metabolites into Four Fractions

Metabolomic Analysis of Barley by Gas Chromatography/Mass Spectrometry

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe and quantify the impacts of drought on grain and other agricultural products. The identified metabolites may allow for the identification of the biochemical response that allows the plant to tolerate stressful environments. The methodology presented herein allowed for the total metabolomic analysis of barley flour using gas chromatography/mass spectrometry (GC/MS). 
Barley flour metabolite extracts were fractionated into four fractions based on polarity. To allow for analysis by GC/MS, metabolites were derivatized to increase volatility and metabolite separation: fatty acids esters were derivatized into fatty acid methyl esters; sugars were oximated into their straight chain form; and metabolites with hydroxyl groups were converted to their corresponding silyl ethers. The derivatized samples were injected into the GC/MS and the generated mass spectra were used for metabolite identification by comparing the generated spectra to the National Institute of Standards and Technology (NIST) Tandem Mass Spectra library. The method described here can also be used to examine the total metabolome for other plants, furthering our understanding of the biochemical responses of stressed plants.

A method for metabolomic analysis of barley is presented. The method entails fractionation and derivatization of metabolites and analysis thereof by gas chromatography/mass spectrometry (GC/MS). Metabolomic analysis can be used to determine the effect of intercropping and drought stress on the grain.

Climate change increases drought risk to agriculture and impacts both food nutrient content and overall food security. Metabolomics is one way to observe ...

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable photosynthetic models facilitating the understanding of the basic cellular processes. System wide studies enable comprehensive and in-depth understanding of molecular functions of the organisms. However, multiple independent samples and protocols are required for proteomics, lipidomics and metabolomics studies introducing higher error and variability. A robust high throughput extraction method for the simultaneous extraction of chlorophyll, lipids, metabolites, proteins and starch from a single sample of the green alga Chlamydomonas reinhardtii is presented here. The illustrated experimental setup is for Chlamydomonas cultures synchronized using 12 h/12 h light/dark conditions. Samples were collected over a 24 h cell cycle to demonstrate that the metabolites, lipids and starch data obtained using various analytical platforms are well conformed. Furthermore, protein samples collected using the same extraction protocol were used to conduct detailed proteomics analysis to evaluate their quality and reproducibility. Based on the data, it can be inferred that the illustrated method provides a robust and reproducible approach to advance understanding of various biochemical pathways and their functions with greater confidence for both basic and applied research.

A Multi-Omics Extraction Method for the In-Depth Analysis of Synchronized Cultures of the Green Alga Chlamydomonas reinhardtii

System-wide analysis of multiple biomolecules is crucial to gain functional and mechanistic insights into biological processes. Hereby, an extensive protocol is described for high throughput extraction of lipids, metabolites, proteins and starch from a single sample harvested from synchronized Chlamydomonas culture.

Microalgae have been the focus of research for their applications in the production of high value compounds, food and fuel. Moreover, they are valuable ...

Developmental Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

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