The authors thank Melanie Frank, Pia Griesheimer, Jessica Henrich, Petra Janke, Jessica Maier, and Norbert Sickinger for technical and analytical support of this work.
Financial support provided within the BioBoost project is greatly acknowledged. BioBoost is a European R&#38;D project co-funded under contract 282873 within the Seventh Framework Programme by the European Commission (www.bioboost.eu).

1. Start-up
<ol>
	<li>Activate the whole pyrolysis and condensation system by starting the auxiliary N2 supply and the pyrolysis gas fan. Flush the pyrolysis test rig with 500 L hr-1 of nitrogen during standby. Regulate the fan by opening the fan&#39;s menu in the process control and adjusting its nominal volumetric flow such that the pressure in the reactor is 3-8 mbar above ambient pressure. 
	Caution: Especially during start-up, there is an increased risk of build-up of explosive atmospheres. The system must be completely inert in order to mitigate this risk.</li>
	<li>Fill the bio-oil cycle (i.e., organic-rich condensate) with an appropriate amount of ethylene glycol as the starting medium for the quenching system to allow safe operation of the pump and homogenizer (e.g., 15 kg in the example given). Record the weight of the starting material.</li>
	<li>Fill the aqueous condensate cycle with an appropriate amount of water to allow safe operation of the pump (e.g., 7 kg in the example given). Record the weight of starting material.</li>
	<li>Heat up the system, including the heat carrier heater and all auxiliary heaters, by opening their menus in the process control and entering the desired values (e.g., around 500 &#176;C). Auxiliary heating is recommended for the reactor itself and the connecting pipes up to the first condenser in order to prevent uncontrolled condensation of vapors.</li>
	<li>Start the cooling cycle for the heat exchangers in both condensation cycles by switching on the cooler.</li>
	<li>Start the pumps of both condensation cycles by opening their menus in the process control and click on activate. Use the same menus to adjust the mass flow to provide enough cooling power. For example, recirculate the bio-oil at a rate of around 350 kg hr-1 and cool it down to 80 &#176;C before spraying it into the quenching vessel. Recirculate the aqueous condensate at a rate of around 600 kg hr-1 and, in addition, supply cooling water at a rate of 300 kg hr-1 at 8 &#176;C.</li>
	<li>Switch on the electrostatic precipitator.</li>
	<li>After both condensation cycles have run for 10-20 min, check the nozzles of the quenching system for blocking and remove any blockage present.</li>
	<li>Start the heat carrier loop by opening the menu of the bucket elevator and the heat carrier feeding screw in the process control and click on activate. Set the heat carrier temperature to a value above the desired reactor temperature in order to allow a smoother start-up by accounting for the heat requirements for the pyrolysis reaction. For example, supply the heat carrier with a mass flow of 1,000 kg hr-1 at a temperature of 520 &#176;C during operation, but heat to 545 &#176;C before starting the biomass feed. 
	Caution: Make sure that the twin-screws of the reactor are started automatically once the heat carrier feeding screw is activated. Otherwise there is the risk of blocking and even damage to the feeding system.</li>
	<li>After the system (i.e., all temperatures) has reached the set values, start feeding biomass by filling the biomass storage with the desired feedstock. Subsequently, open the lock hopper and start the biomass feeding screw by clicking on activate in their menus in the process control. Slowly increase the feed rate in order to prevent excessive pressure fluctuations.</li>
</ol>
2. Steps and Observations Continuously Required during Operation
<ol>
	<li>Record the amount of biomass fed in order to account for balancing and take appropriate samples.</li>
	<li>Check for the desired reactor temperature (exit temperature of the heat carrier) and regulate the heating of the heat carrier loop accordingly.</li>
	<li>Regulate the fan by adjusting its nominal volumetric flow to keep the desired reactor pressure.</li>
	<li>Check for blocking in the nozzles of the quenching system (drop in mass flow and/or increase in quenching temperature).</li>
	<li>Observe the pressure drop across the cyclones and the quenching system in order to detect excessive scaling early enough. Install appropriate measures to be able to remove excessive scaling during operation, especially at the point of the first temperature drop of the pyrolysis vapors (usually the inlet of the quenching system).
	<ol>
		<li>For example, clean the tube&#39;s cross section by using a rod to remove scaling mechanically. Seal the rod with a gasket to prevent intake of air into the quenching system. Install a ball valve at the inlet point of the rod to further decrease air leakage if the cleaning is not in operation. 
		Caution: Cleaning the inlet of the quenching system by inserting a rod leads to temporary blockage of the gas removal from the reactor. Biomass feeding must be stopped if it cannot be assured that the cleaning is performed in &#60;10 sec.</li>
	</ol>
	</li>
	<li>Monitor the condensation temperatures of both condensation cycles and adapt the temperature set-points of the process thermostats if necessary.</li>
	<li>Remove condensate from cycles as soon as 80% of the maximum allowable filling level has been reached (depending on the size of the buffer tanks and the amount and type of biomass fed).</li>
	<li>Conduct measurements of the gas phase. Measure the amount of gas as well as its composition (see details in step 4.5). 
	NOTE: Primary gaseous compounds include N2, CO, CO2, CH4, O2, and H2. Additional compounds are to be expected, such as C2H4, C2H6, and C3H8. An example of a gas measurement system is described below (see step 4.5). 
	Caution: If parts of the pyrolysis unit are operated under pressure, leakage of air may lead to the development of an explosive atmosphere. It is highly recommended to closely observe the amount of oxygen in the pyrolysis gas.</li>
</ol>
3. Shutdown
<ol>
	<li>To stop the experiment, simply turn off the biomass feed and regulate the fan to keep the desired reactor pressure.</li>
	<li>Allow the system (heat carrier loop and condensation cycles) to run for another 30-40 min to ensure that all remainders are pyrolyzed and the products recovered.</li>
	<li>Turn off the heating of the heat carrier loop.</li>
	<li>Turn off the pumps of both condensation cycles and the electrostatic precipitator.</li>
	<li>Empty both condensate cycles and record the weight of each condensate. Subtract the amount of starting material (see steps 1.2 and 1.3) before setting up the balances.</li>
	<li>Allow the containers for char collection to cool down to room temperature in an inert atmosphere. Weigh the amount of char. 
	Caution: The char may exhibit pyrophoric characteristics, and specific care should be taken when handling this material.</li>
	<li>Clean the bio-oil cycle with fresh ethylene glycol and the aqueous condensate cycle with a 1:1 mixture of water and ethanol. Fill with appropriate amounts (see steps 1.2 and 1.3) and allow to run for 30-40 min.</li>
</ol>
4. Required Analyses for Setting up &#39;Dry&#39; and &#39;Elemental Carbon&#39; Balances
<ol>
	<li>Perform the following feedstock analyses (examples for applicable standards are given in parentheses):
	<ol>
		<li>Determine the water content17.</li>
		<li>Determine the ash content18.</li>
		<li>Determine the elemental carbon, hydrogen, and nitrogen contents19. 
		NOTE: It is highly recommended to analyze the water content each experimental day because differences in weather conditions might affect the moisture content of the feedstock. Depending on the size of the lot, several samples are required to reliably characterize the feedstock. Additional analyses such as fiber analysis and higher heating value are recommended but not mandatory for setting up the above-mentioned balances.</li>
	</ol>
	</li>
	<li>Perform the following char powder analyses (examples of applicable standards are given in the references):
	<ol>
		<li>Determine the water content17.</li>
		<li>Determine the ash content18.</li>
		<li>Determine the elemental carbon, hydrogen, and nitrogen contents19. 
		NOTE: It is assumed that char has no moisture content when leaving the process for setting up the balances. Moisture take-up may occur during the course of analyses, and the water content is required for the correction of the other two analyses.</li>
	</ol>
	</li>
	<li>Perform the following bio-oil analyses (examples of applicable standards or other recommended methods are given in parentheses):
	<ol>
		<li>Determine the water content by volumetric Karl-Fischer titration according to standard protocols. Dissolve a sample in dry methanol and titrate it with a mixture of a base, SO2, and a known concentration of I2 (detailed examples of materials are given in the material list). Each mole of water reacts with one mole of I2.</li>
		<li>Determine the solids content by taking a 3,040 g sample of FPBO and dissolve it in methanol to a final solution volume of about 100 ml. Stir the solution for 10 min at room temperature. Filter the solution through cellulose filter at particle retention of 2.5 &#181;m and rinse the residue thoroughly with methanol until a clear filtrate is obtained. Dry the solid residue at 105 &#176;C overnight and determine the residual weight.</li>
		<li>Determine the elemental carbon, hydrogen, and nitrogen contents19.</li>
		<li>Determine the ethylene glycol content by 1H NMR analysis according to standard protocols. Dissolve an FPBO sample in a solution of deuterated methanol with 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (TMSP) as reference material (approximately 0.1 g FPBO in 0.8 g solution). For example, the solution may contain 44 g methanol and 0.1 g TMSP. Centrifuge the dissolved sample in order to remove solids. Analyze the sample by 1H nuclear magnetic resonance spectroscopy (NMR). The hydroxy groups of ethylene glycol show a peak at 3.55&#8211;3.65 ppm. The reference peak of TMSP appears around 0 ppm and is used to quantify the ethylene glycol content. 
		NOTE: Start-up with pure ethylene glycol leads to a dilution of the condensate in the first condenser. This needs to be considered in the calculation of mass and energy balances and for the presentation of the results. It is highly desirable to identify individual chemical compounds. Such an analytical method is very complex due to the vast number of different compounds and the nature of the condensate matrix. A description of such analyses is outside the scope of this paper. It should also be pointed out that the above-mentioned analyses are merely required for setting up balances and are not sufficient for describing the bio-oil as a product. Standards that cover FPBO applications are in preparation.</li>
	</ol>
	</li>
	<li>Perform the following aqueous condensate analyses (examples of applicable standards are given in parentheses):
	<ol>
		<li>Determine the water content by volumetric Karl-Fischer titration (see 4.3.1).</li>
		<li>Determine the total organic carbon as non-purgeable organic carbon20. 
		NOTE: Start-up with pure water leads to a dilution of the condensate in the second condenser. This needs to be considered in the calculation of mass and energy balances and for the presentation of the results.</li>
	</ol>
	</li>
	<li>Monitor the gas composition throughout the experiment because composition varies considerably with time. For example, analyze the product gas during the experiments presented here in a process gas chromatograph every 30-60 min. Analyze the following gas species: Ne, H2, CO, CO2, N2, O2, CH4, and alkane/alkene C2-C5 components.
	<ol>
		<li>Inject a constant gas flow of Ne into the reactor as a reference. Calculate the mass of each gas species based on the reference volumetric flow, the average gas composition ratio, the duration of the experiment, and the density of the species. In order to determine the water content of the pyrolysis gas, assume saturated conditions at the outlet temperature of the last condenser.</li>
	</ol>
	</li>
</ol>

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Energ. 31, 176-181 (2012).</li><li>Funke, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+pyrolysis+char+-+Assessment+of+alternative+uses+within+the+bioliq+concept.">Fast pyrolysis char - Assessment of alternative uses within the bioliq concept.</a> Bioresour. Technol. 200, 905-913 (2016).</li><li>Lehto, J., Oasmaa, A., Solantausta, Y., Kyt&#246;, M., Chiaramonti, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fuel oil quality and combustion of fast pyrolysis bio-oils. , (2013).</li><li>Fahmi, R., Bridgwater, A. V., Donnison, I., Yates, N., Jones, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+lignin+and+inorganic+species+in+biomass+on+pyrolysis+oil+yields,+quality+and+stability.">The effect of lignin and inorganic species in biomass on pyrolysis oil yields, quality and stability.</a> Fuel. 87, 1230-1240 (2008).</li><li>Oasmaa, A., Solantausta, Y., Arpiainen, V., Kuoppala, E., Sipil&#228;, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+Pyrolysis+Bio-Oils+from+Wood+and+Agricultural+Residues.">Fast Pyrolysis Bio-Oils from Wood and Agricultural Residues.</a> Energ. &amp; Fuels. 24, 1380-1388 (2010).</li><li>DIN German Institute for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DIN EN ISO 18134-3 Solid biofuels - Determination of moisture content - Oven dry method - Part 3: Moisture in general analysis sample. , (2015).</li><li>DIN German Institute for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> DIN EN ISO 18122 Solid biofuels - Determination of ash content. , (2016).</li><li>DIN German Institute for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Institute for Standardization. DIN EN ISO 16948 Solid biofuels - Determination of total content of carbon, hydrogen and nitrogen. , (2015).</li><li>DIN German Institute for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Institute for Standardization. DIN EN 1484 Water analysis - Guidelines for the determination of total organic carbon (TOC) and dissolved organic carbon (DOC). , (1997).</li><li>DIN German Institute for Standardization. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ESO 16993: Solid biofuels - Conversion of analytical results from one basis to another. , (2015).</li><li>Bridgwater, A. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+fast+pyrolysis+of+biomass+and+product+upgrading).">Review of fast pyrolysis of biomass and product upgrading).</a> Biomass Bioenerg. 38, 68-94 (2012).</li><li>Westerhof, R. J. M., Kuipers, N. J. M., Kersten, S. R. A., van Swaaij, W. P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+the+water+content+of+biomass+fast+pyrolysis+oil.">Controlling the water content of biomass fast pyrolysis oil.</a> Ind. Eng. Chem. Res. 46, 9238-9247 (2007).</li></ol>

The authors have nothing to disclose.

For all experiments, process conditions such as size of the feedstock material, feed rate, pressure, reaction temperature, condensation temperatures, and flow rates of both the heat carrier and the condensate cycle were the same. Naturally, variations within defined limits cannot be avoided. For a test plant such as the process development unit presented here, the acceptable ranges of fluctuation and required times of operation for reproducible experiments need to be calculated and/or determined by experience. For example, the reactor temperature, which is indicated by the temperature of the heat carrier leaving the reactor, is controlled with a standard deviation of 35 &#176;C over the entire course of the reaction from the start of the reaction at full biomass capacity to the stop of biomass feeding (typically around 4 hr). The pressure in the reactor is controlled with a standard deviation of 300&#8211;500 Pa. Peaks in pressure are likely to occur due to fluctuations in biomass feeding. It is recommended to adjust the feeding screw system to the biomass material under consideration in order to minimize such fluctuations and ensure a constant biomass flow. The condensation temperature in the first and second condensers was maintained at a standard deviation of 3 &#176;C and 1 &#176;C, respectively.
It should be noted at this point that all experiments presented were conducted at the same reactor temperature (500 &#176;C). This temperature does not necessarily reflect the optimum fast pyrolysis temperature which exists for each specific feedstock22. A variation of the reactor temperature could lead to an optimized pyrolysis temperature with even higher organic oil yields.
The choice of balancing method is not trivial for fast pyrolysis of biomass, especially when applying fractionated condensation and when using biomass with high ash content. Three different types of balancing have been presented in the previous section. Reporting the yields of product fractions on an &#39;as received&#39; basis is advantageous for practical considerations such as the design of apparatuses and storage capacities as it reports the actual product distribution to be expected. However, these values are obscured by the water and ash contents of the feedstock. Especially for residual biomass &#8212; e.g., straw, forestry and pruning residues and biogenic &#39;waste&#39; &#8212; this is an issue as these feedstocks have a wide range of water and inorganic contents, see Table 1.
The common balancing method for biomass processes on a &#39;dry basis&#39; is in most cases useful for comparisons between different studies as it eliminates the effect of different moisture contents of the feedstock. However, it should be pointed out that these calculated values from experiments with a specific moist feedstock do not necessarily reflect the behavior and yields of this specific feedstock if it was completely dried by physical means prior to the experiment. It is known that moisture affects the yield distribution of pyrolysis23 and this should be kept in mind when evaluating and comparing &#39;dry&#39; balances.
Furthermore, mass balances on a &#39;dry basis&#39; are inappropriate for feedstocks with high ash content because minerals end up primarily in the char and obscure the results similarly to the initial moisture content. Similarly to water, minerals affect the actual pyrolysis reaction network because they promote secondary pyrolysis reactions, leading to higher char and lower bio-oil yields. Such effects can only be evaluated on a scientific basis if balances are corrected for the ash content. One way to achieve this is by setting up carbon balances. From the comparison of Figure 2 and Figure 4 it can be seen that the increased solids yield observed after pyrolysis of wheat straw as compared to miscanthus is not only due to inorganic material that is recovered with the char, but also due to an increased fraction of organic solids that were formed during the process.
Another advantage of elemental carbon balances is to show the fate of biogenic carbon, i.e., its distribution in the recovered product fractions. This is important for the evaluation of more complex conversion chains &#8212; e.g., pyrolysis, gasification, and synthesis as in the case presented here &#8212; because the biogenic carbon should be used as efficiently as possible. One of the most important roles of biomass in a future bio-based economy is to provide biogenic carbon for a wide range of commodities, thus replacing carbon from fossil resources.
The presented protocol for fast pyrolysis in a twin-screw mixing reactor can be realized on different scales with some adjustments. The presented case of a unit with a feed capacity of 10 kg hr-1 has proven to be a feasible compromise between operational complexity and meaningful results for process behavior. It can be applied both for screening of different types of biomass and optimization of process conditions. Testing a specific biomass feedstock is crucial because certain feedstock characteristics might lead to unfavorable process operation if coarse solid residues accumulate in the heat carrier cycle. Such accumulation was not observed for the biomass presented in the results section, but it has been observed for very hard biogenic material with large particle size (&#62;1 mm) which limits the applicability of the presented process. This problem could be reduced with a different design of the heat carrier loop, e.g., by pneumatic transport of the heat carrier with simultaneous partial combustion.

The use of biomass as an alternative to fossil carbon sources is becoming increasingly important for reducing the effect of societal activity on the earth&#39;s climate. There exist other renewable energy sources such as wind and solar, but biomass represents the only renewable carbon source to date. Consequently, the most efficient use of biomass is in the production of chemicals and specialized liquid fuels. Residual biomass should be used in order to reduce competition between feed, food, and chemicals/fuel production. These residues often have low bulk density, thus presenting a logistical challenge for industrial scale applications.
To address these challenges, the bioliq concept has been developed at the Karlsruhe Institute of Technology1. It features a decentralized first step to convert residual biomass into an energy dense intermediate (bioslurry), a subsequent conversion in a central gasification unit to synthesis gas and a final synthesis to the desired product(s). The gasification and synthesis unit can be designed on the required industrial scale at the same site to achieve commercial operation. The concept allows for different products, ranging from drop-in fuels to specialized fuel additives and bulk chemicals2-5. This paper presents the first step in which fast pyrolysis is used to convert residual biomass to the intermediate bioslurry. Fast pyrolysis is characterized by rapid heating of biomass in an inert atmosphere to a reaction temperature of typically 450-500 &#176;C with a residence time of the produced pyrolysis vapors of &#60;2 sec6. Most commonly, fluidized bed reactors are used for performing fast pyrolysis but there also exist different reactor designs specifically adapted to optimize reaction conditions7. The work presented in the following has been conducted with a twin-screw mixing reactor. It presents a robust technology that has already been applied on an industrial scale for pyrolysis of coal and on a pilot scale for oil sands8.
The purpose of the twin-screw mixing reactor is to mix the solid biomass feed with a solid, pre-heated heat carrier. Mixing needs to be sufficiently thorough in order to achieve the heating rate that is necessary for converting the biomass under fast pyrolysis conditions. Additionally, the size of both the biomass and heat carrier particles needs to be small to achieve a high heat transfer coefficient and a short particle heating period. At the Institute of Catalysis Research and Technology (IKFT) of the Karlsruhe Institute of Technology (KIT), a process development unit with a biomass input capacity of 10 kg hr-1 has been operational for more than a decade. It uses steel balls as the heat carrier, which is recirculated internally with a bucket elevator and re-heated with an electrical heating system. Its main purpose was the investigation of a unique product recovery technology that was adapted to the use of the product in a gasifier and the validation of its suitability for a broad range of feedstocks9-11. A larger pilot plant was built in parallel to these studies with a biomass input capacity of 500 kg hr-1, which has been operational for five years. It utilizes sand as the heat carrier, which is recirculated pneumatically by a hot lift gas and additionally heated by partial combustion of entrained char particles1,12. The following description of the experimental method is based on the smaller process development unit after its product recovery section was refurbished to better resemble the pilot plant design13. A flow scheme of this experimental setup is illustrated in Figure 1.
It is important to note that product requirements for fast pyrolysis bio-oil (FPBO) for use in gasifiers are different to those developed for conventional FPBO, which is usually intended for direct fuel applications14. Most importantly, the solids content of the FPBO does not have to be very low. In fact, it is desirable to mix the FPBO produced with the char obtained from the conversion process in order to increase the amount of carbon available for gasification and subsequent synthesis of drop-in fuels. These facts are important for understanding the differences in the design of the experimental setup presented here and fast pyrolysis experiments published elsewhere. Another important difference is the fact that the biomass conversion concept under investigation was specifically designed for agricultural residues such as wheat straw. Typically, this kind of feedstock contains a large fraction of ash. Ash is known to significantly influence the product distribution of fast pyrolysis. It leads to a decrease of organic condensate (OC) and an increase in both solid and gaseous products10,15,16. These facts are accounted for both in the design of the experimental setup presented here and the overall process chain. Most industrial installations run on wood with low ash content and simply burn the solids internally. This leads to additional production of heat for external use. When using feedstocks with high ash content, char is a significant by-product that should be used efficiently13.

<table border="0" cellpadding="0" cellspacing="0" width="1014">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Wheat straw</td>
			<td style="width:284px;">D&#246;rrmann Kraichtal-M&#252;nzesheim</td>
			<td style="width:141px;">n/a</td>
			<td style="width:357px;">Triticum aestivum L.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Scrap wood</td>
			<td style="width:284px;">Rettenmeier Holding AG</td>
			<td style="width:141px;">n/a</td>
			<td>According to class A2 of the German scrap wood decree (AltholzV &#167;2): glued, coated, painted, or otherwise treated scrap wood without organic halogen compounds and wood preservatives</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Miscanthus</td>
			<td style="width:284px;">Hotel-Heizungsbau Kraichgau-Odenwald</td>
			<td style="width:141px;">n/a</td>
			<td>Miscanthus xGiganteus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Ethylene glycol</td>
			<td style="width:284px;">H&#228;ffner GmbH &#38; Co KG</td>
			<td style="width:141px;">1042090220600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Ethanol</td>
			<td style="width:284px;">H&#228;ffner GmbH &#38; Co KG</td>
			<td style="width:141px;">1026800150600</td>
			<td>Grade 99.9 %</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Nitrogen</td>
			<td style="width:284px;">KIT</td>
			<td style="width:141px;">n/a</td>
			<td>Supplied by internal nitrogen pressure system.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Pyrolysis test rig</td>
			<td style="width:284px;">self-built</td>
			<td style="width:141px;">n/a</td>
			<td>Flow scheme is illustrated in manuscript.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Name</td>
			<td style="width:284px;">Company</td>
			<td style="width:141px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Analyses:</td>
			<td style="width:284px;"></td>
			<td style="width:141px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Gas chromatograph Daniel 700</td>
			<td style="width:284px;">Emerson Process Management</td>
			<td style="width:141px;">n/a</td>
			<td>Designed for this specific application by Emerson; two 20 % SF 96 columns, two HAYESEP N columns, and one MS-5A washed column; carrier gas is helium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Helium</td>
			<td style="width:284px;">Air Liquide</td>
			<td style="width:141px;">P0252L50R2A001</td>
			<td>Grade 6.0</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:232px;">Gas mixture for calibration</td>
			<td style="width:284px;">basi Sch&#246;berl GmbH &#38; Co. KG</td>
			<td style="width:141px;">FG 10002</td>
			<td>Specified gas composition: 5 % Ne, 2 % O2, 20 % CO, 30 % CO2, 5 % CH4, 5 % H2, 2 % C2H6, 0.5 % C3H8, 0.5 % C4H10, 0.5 % C5H12, remainder N2.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Neon</td>
			<td style="width:284px;">Air Liquide</td>
			<td style="width:141px;">P0890S10R2A001</td>
			<td>Grade 4.0; used as fixed reference gas flow; not necessarily required and is only given as an example for quantifying the pyrolysis gas flow.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Elementaranalysator CHN628</td>
			<td style="width:284px;">Leco Instrumente GmbH</td>
			<td style="width:141px;">622-000-000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TGA701</td>
			<td style="width:284px;">Leco Instrumente GmbH</td>
			<td style="width:141px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">DIMATOC 2000</td>
			<td style="width:284px;">Dimatec</td>
			<td style="width:141px;">n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Hydranal methanol dry</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:141px;">34741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Hydranal composite V</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:141px;">34805</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">841 Titrando</td>
			<td style="width:284px;">Deutsche Metrohm GmbH &#38; Co. KG</td>
			<td style="width:141px;">2.841.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">774 Oven Sample Processor</td>
			<td style="width:284px;">Deutsche Metrohm GmbH &#38; Co. KG</td>
			<td style="width:141px;">2.774.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">800 Dosino</td>
			<td style="width:284px;">Deutsche Metrohm GmbH &#38; Co. KG</td>
			<td style="width:141px;">2.800.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">801 Stirrer</td>
			<td style="width:284px;">Deutsche Metrohm GmbH &#38; Co. KG</td>
			<td style="width:141px;">2.801.0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Methanol</td>
			<td style="width:284px;">Carl Roth GmbH &#38; Co KG</td>
			<td style="width:141px;">83884</td>
			<td>99% for synthesis</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:232px;">Whatman cellulose filter grade 42</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td style="width:141px;">WHA1442090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">Methanol-D4</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td align="right" style="width:141px;">151947</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:232px;">3-(Trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt</td>
			<td style="width:284px;">Sigma Aldrich</td>
			<td align="right" style="width:141px;">269913</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:232px;">BZH&#160;250&#160;MHz</td>
			<td style="width:284px;">Bruker</td>
			<td style="width:141px;">n/a</td>
			<td></td>
		</tr>
	</tbody>
</table>

fast pyrolysis of biomass residues in a twin-screw mixing reactor

Fast pyrolysis is being increasingly applied in commercial plants worldwide. They run exclusively on woody biomass, which has favorable properties for conversion with fast pyrolysis. In order to increase the synergies of food production and the energetic and/or material use of biomass, it is desirable to utilize residues from agricultural production, e.g., straw. The presented method is suitable for converting such a material on an industrial scale. The main features are presented and an example of mass balances from the conversion of several biomass residues is given. After conversion, fractionated condensation is applied in order to retrieve two condensates &#8212; an organic-rich and an aqueous-rich one. This design prevents the production of fast pyrolysis bio-oil that exhibits phase separation. A two phase bio-oil is to be expected because of the typically high ash content of straw biomass, which promotes the production of water of reaction during conversion.
Both fractionated condensation and the use of biomass with high ash content demand a careful approach for establishing balances. Not all kind of balances are both meaningful and comparable to other results from the literature. Different balancing methods are presented, and the information that can be derived from them is discussed.

Various types of biomass were successfully pyrolyzed in the pyrolysis unit at IKFT/KIT with the current setup. For example, three different feedstocks (wheat straw, miscanthus, and scrap wood) were compared concerning their properties and yields after pyrolysis following the procedure described. Different kinds of balancing methods are shown and discussed in regard to their applicability towards ash-rich feedstock. It is important to note that the balances have been calculated and summarized according to the state of aggregation of each fraction. The bio-oil recovered in the first condenser still contains solids, which have not been removed by the cyclones. These are marked separately in the balances. For comparison and statistical evaluation, the solids content of the bio-oil was added to the char fraction recovered from the cyclones.
On an &#39;as received&#39; basis, the solids yield, i.e., char recovered via cyclones and char present in the bio-oil, is in the range from 14-25% by weight for the investigated feedstocks. Total condensate yields recovered in the two condensers range from 53-66% by weight, whereas gas yields are relatively similar (around 20%) for all 3 biomasses (see Figure 2). These &#39;as received&#39; values give practical information on the actual amount of product fractions to be expected in fast pyrolysis installations of this kind.
However, total liquid organic yields in the literature are most commonly reported on a dry basis, i.e., excluding moisture and water of reaction in the condensate and in the feed. The advantage of this kind of balance is the fact that initially present moisture of the biomass does not affect the results. This moisture content would artificially increase the condensate yield in an &#39;as received&#39; balance. For reasons of comparability, Figure 3 shows organic oil yield and reaction water on a dry basis. In this study, organic oil yields increase (35 - 46 - 50% by weight) with decreasing ash contents (9.2 - 2.7 - 1.5% by weight) of the feedstocks wheat straw - miscanthus - scrap wood (see Table 1). This is in line with observations from other studies10,15,16. Yields of water of reaction are in a comparatively narrow range from 12-14% by weight.
Mass balances on a dry basis are still directly affected by the ash content of the feedstocks. Minerals contained in the biomass material will artificially increase the yield of solids in both &#39;as received&#39; and &#39;dry&#39; balances. Consequently, elemental carbon balances were determined because they appear to be more suitable for evaluating differences in thermochemical conversion reactions of biomass (see Figure 4). It becomes evident that the larger part of carbon is recovered in the bio-oil (44-54% by weight) and only a mass fraction of 24-32% in solid form as pyrolysis char. About 16-19% by weight of the carbon is not recovered in solid or liquid form and leaves the plant with the pyrolysis gas. In a commercial plant, this gas would be recycled for reasons of energy recovery in an internal combustion device. A mass fraction of only about 3-4% of carbon is recovered in the aqueous condensate, which has a water content of around 80% by weight. This validates the effectiveness of the fractionated condensation setup presented here.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Wheat straw</td>
			<td>Miscanthus</td>
			<td>Scrap wood</td>
		</tr>
		<tr>
			<td>Water, ar</td>
			<td>9.6</td>
			<td>10.1</td>
			<td>15.2</td>
		</tr>
		<tr>
			<td>Ash, d</td>
			<td>9.2</td>
			<td>2.7</td>
			<td>1.5</td>
		</tr>
		<tr>
			<td>Carbon, d</td>
			<td>46.1</td>
			<td>48.6</td>
			<td>49.8</td>
		</tr>
		<tr>
			<td>Hydrogen, d</td>
			<td>5.7</td>
			<td>5.9</td>
			<td>6.1</td>
		</tr>
		<tr>
			<td>Nitrogen, d</td>
			<td>&#60;0.5</td>
			<td>&#60;0.5</td>
			<td>&#60;0.5</td>
		</tr>
		<tr>
			<td colspan="4">ar: as received, d: dry basis21</td>
		</tr>
	</tbody>
</table>
Table 1. Selected properties of the different feedstocks used. All values represent mass fractions (%).
<img alt="Figure 1" src="/files/ftp_upload/54395/54395fig1.jpg" /> 
Figure 1. Flow diagram of the experimental setup. 1) Biomass storage. 2) Lock hopper system. 3) Biomass dosing. 4) Twin-screw mixing reactor. 5) Bucket elevator. 6) Heater for heat carrier. 7) Cyclone for solids removal. 8) Char storage. 9) Spray quenching. 10) Bio-oil storage tank. 11) Homogenizer and pump. 12) Heat exchanger for cooling of recirculated condensate. 13) Electrostatic precipitator. 14) Aqueous condensate storage tank. 15) Pump for recirculating aqueous condensate. 16) Heat exchanger for cooling of recirculated condensate. 17) Condenser for aqueous condensate. 18) Fan for removing gas/vapors. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54395/54395fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54395/54395fig2.jpg" /> 
Figure 2. Mass balances of experiments. Balances are reported on an &#39;as received&#39;21 basis of the feedstock and products. All values are expressed as mass fractions. Three different types of biomass have been used and all experiments were conducted at least in triplicates13. The solids content in the bio-oil is reported separately for illustration purposes. The error bars indicate standard deviations of experiments with one type of feedstock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54395/54395fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54395/54395fig3.jpg" /> 
Figure 3. Total organic oil yields and water of reaction. All values are presented on a dry21 feed basis and are expressed as mass fractions. The solids content of the condensate has been excluded from the organic oil yield13. The error bars indicate standard deviations of experiments with one type of feedstock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54395/54395fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54395/54395fig4.jpg" /> 
Figure 4. Carbon balances. All values are reported as mass fractions of the biomass carbon input. Three different types of biomass have been used and all experiments were conducted at least in triplicates13. The solids content in the bio-oil is reported separately for illustration purposes. The error bars indicate standard deviations of experiments with one type of feedstock. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54395/54395fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Fast Pyrolysis of Biomass Residues in a Twin-screw Mixing Reactor at JoVE.com

Fast Pyrolysis of Biomass Residues in a Twin-screw Mixing Reactor

A procedure for thermochemical conversion of biomass residues is presented that aims at maximizing the yield of liquid products (fast pyrolysis). It is based on a technology proven on an industrial scale and especially suitable for treating a straw type of biomass.

Fast pyrolysis is being increasingly applied in commercial plants worldwide. They run exclusively on woody biomass, which has favorable properties for ...

fast-pyrolysis-of-biomass-residues-in-a-twin-screw-mixing-reactor

Nanyang Technological University

Research

JoVE Journal

Bioengineering

27.5K Views.  Karlsruhe Institute of Technology (KIT). A procedure for thermochemical conversion of biomass residues is presented that aims at maximizing the yield of liquid products (fast pyrolysis). It is based on a technology proven on an industrial scale and especially suitable for treating a straw type of biomass.

Video: Fast Pyrolysis of Biomass Residues in a Twin-screw Mixing Reactor

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Adaptation of a Haptic Robot in a 3T fMRI

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing radiation, millimeter spatial accuracy of anatomical and functional data 2, and nearly real-time analyses 3. Haptic robots provide precise measurement and control of position and force of a cursor in a reasonably confined space. Here we combine these two technologies to allow precision experiments involving motor control with haptic/tactile environment interaction such as reaching or grasping. The basic idea is to attach an 8 foot end effecter supported in the center to the robot 4 allowing the subject to use the robot, but shielding it and keeping it out of the most extreme part of the magnetic field from the fMRI machine (Figure 1).
The Phantom Premium 3.0, 6DoF, high-force robot (SensAble Technologies, Inc.) is an excellent choice for providing force-feedback in virtual reality experiments 5, 6, but it is inherently non-MR safe, introduces significant noise to the sensitive fMRI equipment, and its electric motors may be affected by the fMRI's strongly varying magnetic field. We have constructed a table and shielding system that allows the robot to be safely introduced into the fMRI environment and limits both the degradation of the fMRI signal by the electrically noisy motors and the degradation of the electric motor performance by the strongly varying magnetic field of the fMRI. With the shield, the signal to noise ratio (SNR: mean signal/noise standard deviation) of the fMRI goes from a baseline of &tilde;380 to &tilde;330, and &tilde;250 without the shielding. The remaining noise appears to be uncorrelated and does not add artifacts to the fMRI of a test sphere (Figure 2). The long, stiff handle allows placement of the robot out of range of the most strongly varying parts of the magnetic field so there is no significant effect of the fMRI on the robot. The effect of the handle on the robot's kinematics is minimal since it is lightweight (&tilde;2.6 lbs) but extremely stiff 3/4" graphite and well balanced on the 3DoF joint in the middle. The end result is an fMRI compatible, haptic system with about 1 cubic foot of working space, and, when combined with virtual reality, it allows for a new set of experiments to be performed in the fMRI environment including naturalistic reaching, passive displacement of the limb and haptic perception, adaptation learning in varying force fields, or texture identification 5, 6.

The adaptation and use of a haptic robot in a 3T fMRI is described.

Functional magnetic resonance imaging (fMRI) provides excellent functional brain imaging via the BOLD signal 1 with advantages including non-ionizing ...

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

From Fast Fluorescence Imaging to Molecular Diffusion Law on Live Cell Membranes in a Commercial Microscope

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn&#8217;t need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the spatial autocorrelation function increases at increasing time delay as a function of particle movement due to diffusion. Therefore, fitting of the series of autocorrelation functions enables to extract the actual protein mean square displacement from imaging (iMSD), here presented in the form of apparent diffusivity vs average displacement. This yields a quantitative view of the average dynamics of single molecules with nanometer accuracy. By using a GFP-tagged variant of the Transferrin Receptor (TfR) and an ATTO488 labeled 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphoethanolamine (PPE) it is possible to observe the spatiotemporal regulation of protein and lipid diffusion on &#181;m-sized membrane regions in the micro-to-milli-second time range.

Spatial distribution and temporal dynamics of plasma membrane proteins and lipids is a hot topic in biology. Here this issue is addressed by a spatio-temporal image fluctuation analysis that provides conceptually the same physical quantities of single particle tracking, but it uses small molecular labels and standard microscopy setups.

It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Microwave-driven Synthesis of Iron Oxide Nanoparticles for Fast Detection of Atherosclerosis

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the synthesis of hydrophobic nanoparticles, our method is based on an adaptation from thermal decomposition method to microwave driven synthesis. The new methodology produces a decrease in the reaction times in comparison with traditional procedures. Moreover, the use of the microwave technology increases the reproducibility of the reactions, something important from the point of view of clinical applications. The novelty of this iron oxide nanoparticle is the attachment of Neridronate. The use of this molecule leads a bisphosphonate moiety towards the outside of the nanoparticle that provides Ca2+ binding properties in vitro and selective accumulation in vivo in the atheroma plaque. The protocol allows the synthesis and plaque detection in about 3 hr since the initial synthesis from organic precursors. Their accumulation in the atherosclerotic area in less than 1 hr provides a contrast agent particularly suitable for clinical applications.

Microwave technology enables extremely fast synthesis of iron oxide nanoparticles for atherosclerosis plaque characterization. The use of an aminobisphosphonate in the external side of the nanoparticle provides a fast accumulation in the atherosclerotic area.

A fast and reproducible microwave-driven protocol has been developed for the synthesis of neridronate-functionalized nanoparticles. Starting from the ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

Synthesis of Cationized Magnetoferritin for Ultra-fast Magnetization of Cells

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide nanoparticles (SPIONs). Achieving sufficient cellular uptake of SPIONs is a challenge that has traditionally been met by exposing cells to elevated concentrations of SPIONs or by prolonging exposure times (up to 72 hr). However, these strategies are likely to mediate toxicity. Here, we present the synthesis of the protein-based SPION magnetoferritin as well as a facile surface functionalization protocol that enables rapid cell magnetization using low exposure concentrations. The SPION core of magnetoferritin consists of cobalt-doped iron oxide with an average particle diameter of 8.2 nm mineralized inside the cavity of horse spleen apo-ferritin. Chemical cationization of magnetoferritin produced a novel, highly membrane-active SPION that magnetized human mesenchymal stem cells (hMSCs) using incubation times as short as one minute and iron concentrations as lows as 0.2 mM.

A protocol for the synthesis and cationization of cobalt-doped magnetoferritin is presented, as well as a method to rapidly magnetize stem cells with cationized magnetoferritin.

Many important biomedical applications, such as cell imaging and remote manipulation, can be achieved by labeling cells with superparamagnetic iron oxide ...