We acknowledge Ton van der Zalm from Tupola for the development of the irrigation system. Additionally, we thank Jaco Baars from Tupola for the laughs and mental support given during the building of this setup. We thank Peter Garamszegi and &#193;ngel Velasco S&#225;nchez for helping in watering the columns manually when the irrigation system was not functional. We also thank Steven Heesterman, Xuming Li, Karen Mor&#225;n Rivera, Jonna van den Berg and Kangying Xie for the help provided during the sampling. We thank Peggy Bartsch, Tom J&#228;ppinen, Peter Nobels, Brent Rotgans, Andre van Leeuwen and Gerlinde Vink for the assistance in the lab, the analyses of the samples and the fruitful discussions. Finally, we thank Jeroen Zonneveld from Unifarm for the provision and maintenance of the climate chamber. This setup was built as part of the Bio-Accelerated Mineral Weathering (BAM!) project, which is funded by the European Union Horizon 2020 framework program for research and innovation under grant agreement No 964545.

Enhancing CO2 Sequestration: A Soil Biota Approach to Boost Mineral Weathering

<ol>
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The authors have nothing to disclose.

Within the current research context, this setup has been uniquely designed to optimize inorganic carbon sequestration by enhancing mineral weathering through the activity of soil biota, while concurrently manipulating abiotic factors known for stimulating weathering. The possibility in this setup of collecting both the solid processed material and the leachate enables a full characterization of both fractions. Despite the enormous amount of columns, the collection of the samples and the analyses carried out ensure a high-quality data collection. Besides, having a large number of combinations in a single experimental run is very important for analyzing the collected data with modern and advanced statistical methods, such as machine learning. These methods can be used to determine the main variables leading to high weathering rates and further carbon sequestration. Consequently, this setup provides the opportunity to improve the understanding of the effects that soil organisms can have on EW and IC sequestration. This is fundamental to establishing more realistic constraints on the boundaries of EW and its efficiency in reducing atmospheric CO2 concentrations. This setup presents several originalities compared to existing studies investigating EW and the effect of soil organisms.
Regarding the effects of abiotic factors on EW, these have already been investigated in previous studies4,29,30,31,32,33,34. Some of these studies compared different amounts, types, and grain sizes of rocks, but their setup either consisted of a pot experiment32,33 or included mixing rock powder with soil34. Other experiments focused on one rock type with different irrigation rates but did not have the possibility of irrigating frequently with an automated system or focused on multiple irrigation rates and frequencies35. Other studies presented a setup similar to the one presented in the current protocol, with the possibility of adjusting irrigation rates and maintaining temperature constant, besides varying rock grain sizes and types29,30. Furthermore, the design of these setups was comparable to the one proposed in the present manuscript and designed to collect the leachate for further analyses29,30. Additionally, CO2 concentrations were varied in these studies as another factor enhancing weathering29. However, none of these previous studies have focused on the effect of biotic factors on promoting EW. In this setup, the aim is to enhance the weathering process, and further IC sequestration, by inoculating specific bacteria, fungi and earthworms and determining to which extent they can accelerate EW.
In relation to the effect of biotic factors on EW, few studies have not specifically focused on EW but have investigated whether soil organisms can influence mineral weathering. These studies have mainly explored how weathering is affected by soil organisms using culture mediums19,21, Petri dishes36, nylon bags buried in the soil14, or small amounts of rock powder mixed with other substrates36,37. Using such small systems or setups makes it challenging to disentangle the effect of organisms from other variables. Some experiments used a similar setup to the one here proposed but at a smaller scale, with rock powder-filled columns inoculated with soil organisms38,39,40. However, these experiments either concurrently grew plants and did not focus on the exclusive effect of specific soil organisms13,35, or did not collect the leachate36. Besides, most of the studies that showed that bacteria, fungi, and earthworms increase mineral weathering have focused on the effect of these organisms on nutrient release as an indication of weathering rather than on IC sequestration11,13,14,19,36,37,38. Above all, none of these earlier studies aimed at promoting EW or presented the possibility of adjusting and maintaining abiotic factors throughout the experimental period. In this setup, instead of keeping all abiotic factors constant, a multitude of combinations are tested for four abiotic factors, such as water irrigation rates and frequencies, rock powder type, and grain size, with the aim of promoting EW through soil organisms&#39; activity.
Besides, none of the previous studies that have focused on the effect of either abiotic or biotic factors on EW presented the possibility of having an extremely large number of columns and variables within one experimental run. In this setup, it is possible to test multiple different combinations of various variables during one run of experiments due to the impressive number of columns for which the setup has been designed, while still providing high-quality results. Given the novelty of the setup, below some possible improvements and remaining challenges that could be considered while designing future similar setups are presented.
Homogenous air conditions in the incubation chamber should be ensured. The placement of the setup in a climate chamber ensured constant temperature and relative humidity. Ventilation constraints (e.g., air flow) may have created spatial variability in atmospheric conditions and thus led to disproportional evaporation from the columns at certain locations, which is a common phenomenon in this kind of setup35. To handle this drawback, when replication and randomization are not possible, it is advised to calculate a water balance for columns placed at various locations throughout the chamber.
The columns should be carefully aligned with the funnels once inserted into the acrylic plate to avoid leachate loss. During the experimental period considered, leachate losses occurred from the bottom of the columns due to an incorrect positioning of the funnels or due to the clogging of the meshes. Together with evaporation, this can partly explain why the leachate collected was lower compared to expectations (Figure 13). To minimize these losses, it is important to make sure that the funnels are optimally positioned below the columns. Using wider funnels is also a viable option. In this case, attention should be paid to the diameter of the holes during the construction of the acrylic plates and the distance between acrylic plates.
Slower water flow in soil column experiments where water is applied frequently is a recurrent issue7,30,40. In the experiments carried out with the presented setup, in some cases rather high irrigation rates and very fine mineral grain sizes were used, which initially lack a structure as normally observed in soils. This might have caused the pores of the meshes at the bottom of the columns only containing fine minerals to clog during the run of the experiments. Therefore, water did not flow fast enough through the columns, which resulted both in flooding of the columns, reducing water infiltration and leachate collection, and in anoxic conditions within the columns, impacting biogeochemical processes. To mitigate this issue, it is important to always mix a certain percentage of coarse with finer mineral grain sizes and to avoid 100% very fine mineral grain size mixtures. Another option is to allow is allow the columns to experience a certain number of wetting/drying cycles to initiate soil structure formation, and thus improve water infiltration. Besides, before the start of the experiment, it would be useful to determine basic soil water dynamics, such as saturated and unsaturated flow and water retention curve, in a few mesocosms to better understand gas flow, mineral saturation state and drivers of organisms&#39; activity.
The presented experimental setup is convenient to use, presents a straightforward installation and can be adjusted according to research needs. In the context of mineral weathering, with the necessary adjustments, it can be coupled with a gas chamber in order not only to characterize carbon in the solid and aqueous phase but to look at the dynamics of carbon in the gas phase as well. Besides, this setup can be used to study realistic water infiltration rates with dry-wet sequences, as these temporal dynamics could strongly influence weathering41. The use of this setup is not limited to experiments that focus solely on silicate minerals, but it can be implemented in column experiments that use different substrates. Besides, the length of the experiments can be shortened or extended according to experimental needs, and the number of columns can be changed. The possibility of collecting samples from both the solid processed materials and the leachate allows us to carry out different analyses to focus on one of the two components or both. To present knowledge, this is the only setup that has been built so far with an exceptional number of columns that aims at using soil organisms to enhance mineral weathering while concurrently controlling abiotic conditions in a system made of solely silicate minerals and organic materials.

Enhanced weathering (EW) is a relatively new and low-tech carbon dioxide removal (CDR) technology with a significant potential to mitigate climate change1,2,3. The principle of this technology relies on accelerating the natural mineral weathering process in soils, leading to the sequestration of carbon dioxide (CO2) as inorganic carbon (IC)3. Enhanced weathering aims at increasing IC sequestration by artificially optimizing the factors governing mineral weathering, thereby enhancing the speed through which weathering occurs to humanly relevant time scales3. For EW to be most effective, fast-weathering silicate minerals are ground into a powder with a grain size distribution in the micrometers to millimeters range to reach a high reactive surface area in the ~1 m2&#183;g-1 range3,4.
So far, knowledge about EW has been mainly provided by experiments that focus on abiotic factors governing the rates at which minerals are dissolved5. These include mineral reactivity and surface area, temperature, solution composition, water residence time, and acidity4,6,7, but research still needs to be done within this context. Besides being influenced by abiotic factors, natural systems, and soils in particular, are shaped by a vast number of organisms, ranging from microbes to macrofauna such as earthworms. Despite some studies having shown little or no influence of the biotic activity of mineral dissolution8,9,10, other studies have provided evidence that soil organisms such as bacteria11,12, fungi13,14, and earthworms15,16 could increase mineral weathering rates. Therefore, biotic components could be key to understanding the actual IC sequestration potential of EW5.
The first common mechanism through which soil organisms could accelerate mineral dissolution is via CO2 release during respiration,which increases soil acidification17. Besides, bacteria and fungi could increase mineral weathering by exuding protons, chelates, organic acids, and enzymes, all of which enhance mineral dissolution18,19,20,21. For example, chelation through carboxyl and hydroxyl groups can create ion imbalances, transporting elements away from the surfaces of minerals and lowering saturation states20,22. This could lead to less secondary mineral formation and higher efficiency of EW. Moreover, by feeding on soil particles, the strong actions of earthworms&#39; body walls could break down mineral grains into finer particles, increasing their available reactive surface area23. Microbes dwelling in earthworms&#39; intestines and fresh droppings could further attack these smaller particles, which further exudate organic acids and enzymes24,25. Through their burrowing activity, in addition to contributing to the mixing of organic and mineral particles, earthworms also create macropores that could allow water flow to bypass saturated pore space17. This could enable the water to interact with different mineral surfaces and enhance the water-rock contact rate.
Until now, no setup has been built to study EW rates and therefore IC sequestration using soil organisms while ensuring the possibility to optimize different relevant abiotic conditions, such as water inputs, temperature, mineral type, and mineral grain size. Here, the design and explanation of the construction steps of an innovative setup that aims at increasing EW rates through the activity of soil organisms in small mesocosms are presented. The experimental setup consists of 203 columns (length 15 cm, diameter 7 cm) placed in a climate chamber (4.54 m x 2.72 m) at 25 &#176;C for 8 weeks. The 203 columns are divided into 10 groups of 18 and 2 groups of 10 to fit in the climate chamber. One of the two groups of 10 columns is used to allow the insertion of three more columns which are used as blanks. Each group is placed above a fridge and is topped by a remotely controllable irrigation system, which allows for variable irrigation rates within and between fridges. The leachate of each column is collected in a jerrycan kept at a constant temperature in the fridge (Figure 1). One fridge collects the leachate of a group of columns, meaning that one fridge can be considered as a single system of either 18 or 10 columns. Therefore, the number of columns in this experimental setup can be adjusted according to experimental requirements with a maximum of 203 columns.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65563/65563fig01.jpg" /> 
Figure 1: Schematic side-view of the setup showing 5 columns but considering a system of 18 columns. The frame holding the columns is made of stainless-steel plates, stainless steel screws and acrylic plates. Columns are positioned in the middle of the frame and are topped by an irrigation system. Below the columns, funnels are connected to jerrycans through pipes to collect the leachate. Jerrycans are in a fridge that holds the whole system. The fridge can be opened by lifting the lid. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In this setup, the use of silicate rock powders of specific grain sizes ensures that high weathering rates can be reached, while the inoculation with specifically selected bacteria, fungi, and earthworms grants the biotic activity in this artificial system. The setup enables concurrent quantification of carbon sequestered in the solid and in the liquid samples by measuring both dissolved and solid IC, as well as total alkalinity (TA). Besides, other parameters such as pH, electrical conductivity (EC), and ions can be measured in the leachate as indicators of weathering. This setup also allows the assessment of the impact of soil organisms&#39; survival and activity. Representative results are shown to prove the suitability of this protocol to build a setup where increases in weathering rates are derived not only from abiotic factors but also from biotic ones.

<table><tbody><tr><td>Acryl sheet plates</td><td>WSV kunststoffen BV</td><td>N/A</td><td>Used for holding columns, funnels, irrigation tubes and pipes.</td></tr><tr><td>Adapter ring</td><td>Tameson</td><td>FL2S-FM-B-014G-034G</td><td>Used ot make the system to connect the PU hose to the tap.</td></tr><tr><td>Cable ties</td><td>Gamma</td><td>456196</td><td>Used for holding the mesh system.</td></tr><tr><td>Citric acid</td><td>Nortembio (amazon.nl)</td><td>&amp;nbsp;B01BDLOGW2</td><td>Used for cleaning pipes and funnels.</td></tr><tr><td>CytoFLEX flow cytometer&amp;nbsp;</td><td>Beckam Coulter</td><td>CytoFLEX</td><td></td></tr><tr><td>Dishwasher soap</td><td>BOOM</td><td>77000307.9010</td><td>Used for cleaning the jerrycans.</td></tr><tr><td>Eight relay expansion module</td><td>Control by web</td><td>X-12s</td><td>Used to control the valves of hte irrigation system.&amp;nbsp;</td></tr><tr><td>End cap</td><td>Wildkamp</td><td>819906</td><td>Used to close one end of the main tube of the irrigation system.&amp;nbsp;</td></tr><tr><td>Fridges</td><td>HorecaGemak</td><td>DIA-BVL031/6P</td><td>Used for storing the jerrycans.</td></tr><tr><td>Funnels</td><td>Praxisdienst</td><td>135864</td><td>Used for directing the leachate from the columns to the jerrycans. 75 mm diamater.</td></tr><tr><td>Hand punch</td><td>Wildkamp</td><td>719928</td><td>Used to cut holes for small tubes in the main tube of the irrigation tube.&amp;nbsp;</td></tr><tr><td>HDPE Jerrycan 10 L</td><td>Glas-shop.be</td><td>105157</td><td>Come with lid. Used to collect the leachate.</td></tr><tr><td>HDPE Jerrycan 5 L</td><td>Glas-shop.be</td><td>105156</td><td>Come with lid. Used to collect the leachate.</td></tr><tr><td>Hexagon nut</td><td>Fabory</td><td>51080.100.001</td><td>Used to block acryl sheets on metal screws.&amp;nbsp;</td></tr><tr><td>Label printer</td><td>Brother</td><td>PT-H107B&amp;nbsp;</td><td>Used for printing labels to stick on acryl sheets.</td></tr><tr><td>Ldpe irrigation pipe</td><td>Wildkamp</td><td>15382585</td><td>Used to make main tube of the irrigation system.</td></tr><tr><td>Luggage scale</td><td>United Entertainment</td><td>8718274546996</td><td>Used to weigh jerrycans.</td></tr><tr><td>Mesh 10 &amp;mu;m</td><td>Franz Eckert</td><td>PES-10/2</td><td>Used for the mesh system.</td></tr><tr><td>Mesh 20 &amp;mu;m</td><td>Franz Eckert</td><td>PES-20/13</td><td>Used for the mesh system.</td></tr><tr><td>Metal screws</td><td>Schroeven goothandel.nl</td><td>100975401010</td><td>Used to install acryl sheets.</td></tr><tr><td>Micro hose for drip irrigation</td><td>Wildkamp</td><td>15119128</td><td>Used to make small tubes of the irrigation system.</td></tr><tr><td>Middle ring</td><td>self-made with 3D printer</td><td>self-made with 3D printer</td><td>Used for holding the columns a few centimeters above the funnels.&amp;nbsp;</td></tr><tr><td>Nosepiece</td><td>Wildkamp</td><td>15045986</td><td>Used to connect the solenoid valve to the irrigation pipe.&amp;nbsp;</td></tr><tr><td>Nylon mesh</td><td>Sefar</td><td>N/A</td><td>1 mm mesh used for the top of the columns to prevent earthworms&#039; escape.</td></tr><tr><td>Plastic beads</td><td>lyondelbasell</td><td>TRC 352N C12507</td><td>Used for the mesh system.</td></tr><tr><td>Plug-in fitting with 2 connections</td><td>Tameson</td><td>F24V5</td><td>Used at the end of the system to end the PU hose.&amp;nbsp;</td></tr><tr><td>Polycarbonate enclosure</td><td>RS</td><td>498-5387</td><td>Used to house the electronical compontents of the irrigation system.&amp;nbsp;</td></tr><tr><td>Power cable</td><td>RS</td><td>775-6075</td><td>Used to connect the valves.&amp;nbsp;</td></tr><tr><td>pp coupling</td><td>Wildkamp</td><td>719780</td><td>Used to make the system to connect the PU hose to the tap.&amp;nbsp;</td></tr><tr><td>Pressure regulator</td><td>Wildkamp</td><td>719943</td><td>Used&amp;nbsp; to make sure all small tubes were releasing same amount of water.&amp;nbsp;</td></tr><tr><td>PTFE tape</td><td>GAMMA</td><td>237001</td><td>Used ot wrap the end of hte irrigation pipe.</td></tr><tr><td>PU hose</td><td>Tameson</td><td>PU-8-1198-50-1</td><td>Used to connect all the valves with eath other and to the tap.&amp;nbsp;</td></tr><tr><td>PVC pipes</td><td>Rubbermagazijn</td><td>99001230</td><td>Used for connecting the funnels to the jerrycans.</td></tr><tr><td>PVC tubes</td><td>Wildkamp</td><td>91700</td><td>Used to make the columns.&amp;nbsp;</td></tr><tr><td>Rail power supply</td><td>RS</td><td>145-7873</td><td>Used to supply power to the eight relay expansion module.&amp;nbsp;</td></tr><tr><td>Rubber bands</td><td>PasschierTerpo</td><td>8714603820621</td><td>Used to hold the mesh for earthworms.&amp;nbsp;</td></tr><tr><td>Solenoid valve</td><td>Tameson</td><td>CM-DA014B020E-024DC</td><td>Used for opening and closing of the waterflow.</td></tr><tr><td>Sprinklers</td><td>self-made with 3D printer</td><td>self-made with 3D printer</td><td>Used for evenly distribute the water over the columns.&amp;nbsp;</td></tr><tr><td>Stainless steel plates</td><td>24/7 tailor steel</td><td>N/A</td><td>Used as a frame for the set-up above the fridge.&amp;nbsp;</td></tr><tr><td>T-piece plug in fitting</td><td>Tameson</td><td>F25DT</td><td>Used to connect the solenoid valve to the PU hose.</td></tr><tr><td>TPU 95A material&amp;nbsp;</td><td>MakerPoint</td><td>1756</td><td>Used to print components with 3D printer.&amp;nbsp;</td></tr><tr><td>Washer carriers</td><td>Fabory</td><td>50095.100.001</td><td>Used to put below hexagon nut.</td></tr><tr><td>Web Enabled Controller</td><td>Control by web</td><td>X-400-I(9-28 VDC)</td><td>Used for allowing online control of the irrigation settings.&amp;nbsp;</td></tr></tbody></table>

design and construction of an experimental setup to enhance mineral weathering through the activity of soil organisms

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies on accelerating the natural process of mineral weathering in soils by manipulating the abiotic variables that govern this process, in particular mineral grain size and exposure to acids dissolved in water. EW mainly aims at reducing atmospheric CO2 concentrations by enhancing inorganic carbon sequestration. Until now, knowledge of EW has been mainly gained through experiments that focused on the abiotic variables known for stimulating mineral weathering, thereby neglecting the potential influence of biotic components. While bacteria, fungi, and earthworms are known to increase mineral weathering rates, the use of soil organisms in the context of EW remains underexplored. 
 This protocol describes the design and construction of an experimental setup developed to enhance mineral weathering rates through soil organisms while concurrently controlling abiotic conditions. The setup is designed to maximize weathering rates while maintaining soil organisms' activity. It consists of a large number of columns filled with rock powder and organic material, located in a climate chamber and with water applied via a downflow irrigation system. Columns are placed above a fridge containing jerrycans to collect the leachate. Representative results demonstrate that this setup is suitable to ensure the activity of soil organisms and quantify their effect on inorganic carbon sequestration. Challenges remain in minimizing leachate losses, ensuring homogeneous ventilation through the climate chamber, and avoiding flooding of the columns. With this setup, an innovative and promising approach is proposed to enhance mineral weathering rates through the activity of soil biota and disentangle the effect of biotic and abiotic factors as drivers of EW.

The presented setup consisted of a total of 203 columns located in a climate chamber at 25 &#176;C (Figure 10). The choice of locating the setup in a climate chamber allowed for controlled constant temperature and relative humidity. Placing jerrycans in a fridge at 4 &#176;C ensured that the composition of the leachate was not altered over time because of microbial activity.
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/65563/65563fig10.jpg" /> 
Figure 10: Pictures of the experimental setup in the climate chamber. (A) Overview of a single system. (B) Close-up of a single column. (C) Close-up of jerrycans in the fridge. (D) Overview of all systems in the climate-controlled room. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The use of an advanced automated irrigation system meant that the columns could be watered with varying rates and frequencies using the online control system (Figure 11). The irrigation system allowed to modify the amount of water that the columns received. Validation of the system showed that it led to a minimum difference of 1% and to a maximum difference of 6% in the amount of water given between different columns (Figure 12). Smaller differences were found for lower irrigation rates, while larger differences were found for higher irrigation rates. Overall, the average was lower for irrigation rates of 50 mL&#183;day-1 and 150 mL&#183;day-1, while it was higher for an irrigation rate of 100 mL&#183;day-1 (Figure 12).
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/65563/65563fig11.jpg" /> 
Figure 11: Average amount of water vs. time. Average amount of water measured for an irrigation rate of 50 mL&#183;day-1 distributed over a 24 h period according to three irrigation frequencies of once daily, twice daily, and five times per day for 8 columns. Bars indicate the standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/65563/65563fig12.jpg" /> 
Figure 12:&#160;Average amount of water vs. irrigation rate. Average amount of water measured for an irrigation rate of 50 mL&#183;day-1 over 8 columns and for irrigation rates of 100 mL&#183;day-1 and 150 mL&#183;day-1 over 10 columns. Bars indicate the standard error. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The construction and design of this setup allowed the collection of both the solid content inside the columns, consisting of (processed) rock powder and organic materials, and the total amount of leachate that dripped from the columns over the entire experimental period (Figure 13). Despite being successful in collecting the leachate, the final amount of leachate that was collected was lower than the amount of leachate that was expected to be collected at the end of the experiments according to the irrigation rate (Figure 14). The reduced collected leachate was most likely a result of direct evaporation and leachate spills at the bottom of the columns. This should be taken into account when analyzing the results from the analyses.
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/65563/65563fig13.jpg" /> 
Figure 13: Representative images of the columns and the leachate. Columns filled with rock powder and organic materials at the beginning of the experiments (left side) and leachate collected in the jerrycans at the end of the experiments (right side). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/65563/65563fig14.jpg" /> 
Figure 14: Total liters collected at the end of the experiments per irrigation rate. Dashed lines indicate expected amount of leachate collected according to irrigation rate per experimental period, indicated by the light blue line for 50 mL&#183;day-1, the dark blue line for 100 mL&#183;day-1 and the green line for 150 mL&#183;day-1. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The mix of rock powder and organic material was analyzed to assess the success rate of soil biota in terms of microbial community composition of bacteria and fungi and of survival and activity for earthworms (Figure 15).
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/65563/65563fig15.jpg" /> 
Figure 15: Fungal growth and survival of earthworms. At the end of the experiments and before sampling, visual signs of fungal growth (left side) and earthworms&#39; survival (right side) in the columns filled with rock powder and organic materials. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Besides other parameters, the leachate was analyzed for TA and DIC, as TA and IC are good proxies for mineral weathering rates4,29,30,31. TA was measured with a Metrohm Titrando29,30, while DIC was with a Skalar total organic carbon (TOC) analyzer. By using a TOC analyzer, DIC is calculated from the difference between total dissolved carbon (DC) and dissolved organic carbon (DOC). Figure 16 and Figure 17 show the cumulative distribution for some example values obtained from these analyses for one experimental run. By using this experimental setup, values for TA ranged from 0.019 mol to 0.025 mol, while values for DIC ranged from 7.352 mg C to 259.279 mg C (Figure 16 and Figure&#160;17).
<img alt="Figure 16" class="xfigimg" src="/files/ftp_upload/65563/65563fig16.jpg" /> 
Figure 16: Probability distribution of example values measured for TA in the leachate collected at the end of the experimental period. Treatments where columns flooded, are not displayed. Values are expressed in mol&#160;and are corrected for the total amount of leachate collected at the end of the experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig16large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 17" class="xfigimg" src="/files/ftp_upload/65563/65563fig17.jpg" /> 
Figure 17: Probability distribution of example values measured for DIC in the leachate collected at the end of the experimental period. Treatments where columns flooded, are not displayed. Values are expressed in mg carbon (C) and are corrected for the total amount of leachate collected at the end of the experiments. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65563/65563fig17large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Unraveling the Role of Earthworms in Enhancing Mineral Weathering for CO2 Removal at JoVE.com

Author Spotlight: Unraveling the Role of Earthworms in Enhancing Mineral Weathering for CO2 Removal 

Here we present the construction and operation of an experimental setup to enhance mineral weathering through the activity of soil organisms while concurrently manipulating abiotic variables known to stimulate weathering. Representative results from the functioning of the setup and sample analyses are discussed together with points for improvement.

Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms

Enhanced weathering (EW) is an emerging carbon dioxide (CO2) removal technology that can contribute to climate change mitigation. This technology relies ...

design-construction-an-experimental-setup-to-enhance-mineral

Nanyang Technological University

Research

JoVE Journal

Biochemistry

3.3K Views.  Wageningen University & Research. Verbeterde verwering is een technologie voor het verwijderen van kooldioxide waarbij silicaatgesteente reageert met kooldioxide en water. Microben en andere bodembiota kunnen deze verweringsreactie stimuleren, maar hun potentiële impact op versterkte verwering is nog onbekend. Hier willen we de impact van verschillende bodembiota op verbeterde verwering ontrafelen.Een van de bodembiota waarin we geïnteresseerd zijn, is regenworm. Regenwormen spelen een fundamentele rol in de bodem. Z...

Video: Auteur in de schijnwerpers: het ontrafelen van de rol van regenwormen bij het verbeteren van minerale verwering voor CO2-verwijdering 

Experimental Protocol for Manipulating Plant-induced Soil Heterogeneity

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as plant-soil feedbacks (PSF) have large effects on plant performance, and create environmental heterogeneity that depends on the community composition. Understanding the importance of PSF for plant community assembly necessitates understanding of the role of heterogeneity in PSF, in addition to mean PSF effects. Here, we describe a protocol for manipulating plant-induced soil heterogeneity. Two example experiments are presented: (1) a field experiment with a 6-patch grid of soils to measure plant population responses and (2) a greenhouse experiment with 2-patch soils to measure individual plant responses. Soils can be collected from the zone of root influence (soils from the rhizosphere and directly adjacent to the rhizosphere) of plants in the field from conspecific and heterospecific plant species. Replicate collections are used to avoid pseudoreplicating soil samples. These soils are then placed into separate patches for heterogeneous treatments or mixed for a homogenized treatment. Care should be taken to ensure that heterogeneous and homogenized treatments experience the same degree of soil disturbance. Plants can then be placed in these soil treatments to determine the effect of plant-induced soil heterogeneity on plant performance. We demonstrate that plant-induced heterogeneity results in different outcomes than predicted by traditional coexistence models, perhaps because of the dynamic nature of these feedbacks. Theory that incorporates environmental heterogeneity influenced by the assembling community and additional empirical work is needed to determine when heterogeneity intrinsic to the assembling community will result in different assembly outcomes compared with heterogeneity extrinsic to the community composition.

Understanding the role of environmental heterogeneity in species coexistence has typically focused on types of heterogeneity that are extrinsic to the community&#8217;s species composition. We provide novel detailed methods for creating soil heterogeneity treatments using soils subject to plant-soil feedback conditioning, or heterogeneity intrinsic to the community composition.

Coexistence theory has often treated environmental heterogeneity as being independent of the community composition; however biotic feedbacks such as ...

Environment

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Soil Lysimeter Excavation for Coupled Hydrological, Geochemical, and Microbiological Investigations

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled Earth-system processes. Such knowledge is imperative in improving predictions of hydro-biogeochemical cycles, especially under climate change scenarios. We present an experimental method, designed to capture sub-surface heterogeneity of an initially homogeneous soil system. This method is based on destructive sampling of a soil lysimeter designed to simulate a small-scale hillslope. A weighing lysimeter of one cubic meter capacity was divided into sections (voxels) and was excavated layer-by-layer, with sub samples being collected from each voxel. The excavation procedure was aimed at detecting the incipient heterogeneity of the system by focusing on the spatial assessment of hydrological, geochemical, and microbiological properties of the soil. Representative results of a few physicochemical variables tested show the development of heterogeneity. Additional work to test interactions between hydrological, geochemical, and microbiological signatures is planned to interpret the observed patterns. Our study also demonstrates the possibility of carrying out similar excavations in order to observe and quantify different aspects of soil-development under varying environmental conditions and scale.

This study presents an excavation method for investigating subsurface hydrological, geochemical, and microbiological heterogeneity of a soil lysimeter. The lysimeter simulates an artificial hillslope which was initially under homogeneous condition and had been subjected to approximately 5,000 mm of water over eight cycles of irrigation in an 18-month period.

Studying co-evolution of hydrological and biogeochemical processes in the subsurface of natural landscapes can enhance the understanding of coupled ...

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering, which is a fundamental element to the approach of nanosafety-by-design of nanostructured products for their durable development. This approach is basically a preemptive one in which the focus is put on minimizing the emission of engineered nanomaterials' aerosols during the usage phase of the product's life cycle. This can be attained by altering its material properties during its design phase without compromising with any of its added benefits. In this article, an experimental protocol is presented to investigate the nanosafety-by-design of three commercial nanostructured products with respect to their mechanical solicitation and environmental weathering. The means chosen for applying the mechanical solicitation is an abrasion process and for the environmental weathering, it is an accelerated UV exposure in the presence of humidity and heat. The eventual emission of engineered nanomaterials is studied in terms of their number concentration, size distribution, morphology and chemical composition. The purpose of the protocol is to study the emission for test samples and experimental conditions which are corresponding to real life situations. It was found that the application of the mechanical stresses alone emits the engineered nanomaterials' aerosols in which the engineered nanomaterial is always embedded inside the product matrix, thus, a representative product element. In such a case, the emitted aerosols comprise of both nanoparticles as well as microparticles. But if the mechanical stresses are coupled with the environmental weathering, the experimental protocol reveals then the eventual deterioration of the product, after a certain weathering duration, may lead to the emission of the free engineered nanomaterial aerosols too.

In this article, an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental weathering is presented. Results on the emission of engineered nanomaterials, in the form of aerosols are presented. The specific experimental set-up is described in detail.

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Engineering

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

An Experimental Protocol for Studying Mineral Effects on Organic Hydrothermal Transformations

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the deep ocean. Roles of minerals are critical in many hydrothermal organic geochemical processes. Traditional hydrothermal methodology, which includes using reactors made of gold, titanium, platinum, or stainless-steel, is usually associated with the high cost or undesired metal catalytic effects. Recently, there is a growing tendency for using the cost-effective and inert quartz or fused silica glass tubes in hydrothermal experiments. Here, we provide a protocol for carrying out organic-mineral hydrothermal experiments in silica tubes, and we describe the essential steps in the sample preparation, experimental setup, products separation, and quantitative analysis. We also demonstrate an experiment using a model organic compound, nitrobenzene, to show the effect of an iron-containing mineral, magnetite, on its degradation under a specific hydrothermal condition. This technique can be applied to study complex organic-mineral hydrothermal interactions in a relatively simple laboratory system.

Earth-abundant minerals play important roles in the natural hydrothermal systems. Here, we describe a reliable and cost-effective method for the experimental investigation of organic-mineral interactions under hydrothermal conditions.

Organic-mineral interactions are widely occurring in hydrothermal environments, such as hot springs, geysers on land, and the hydrothermal vents in the ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Monitoring Pedogenic Inorganic Carbon Accumulation Due to Weathering of Amended Silicate Minerals in Agricultural Soils.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in agricultural soils. To this end, the core soil samples taken at different depth (including 0-15 cm, 15-30 cm, and 30-60 cm profiles) are collected from an agriculture field, the topsoil of which has already been enriched with an alkaline earth metal silicate containing mineral (such as wollastonite). After transporting to the laboratory, the soil samples are air-dried and sieved. Then, the inorganic carbon content of the samples is determined by a volumetric method called calcimetry. The representative results presented herein showed five folded increments of inorganic carbon content in the soils amended with the Ca-silicate compared to control soils. This compositional change was accompanied by more than 1 unit of pH increase in the amended soils, implying high dissolution of the silicate. Mineralogical and morphological analyses, as well as elemental composition, further corroborate the increase in the inorganic carbon content of silicate-amended soils. The sampling and analysis methods presented in this study can be adopted by researchers and professionals looking to trace pedogenic inorganic carbon changes in soils and subsoils, including those amended with other suitable silicate rocks such as basalt and olivine. These methods can also be exploited as tools for verifying soil inorganic carbon sequestration by private and governmental entities to certify and award carbon credits.

The verification method described here is adaptable for monitoring pedogenic inorganic carbon sequestration in various agricultural soils amended with alkaline earth metal silicate-containing rocks, such as wollastonite, basalt, and olivine. This type of validation is essential for carbon credit programs, which can benefit farmers that sequester carbon in their fields.

The present study aims to demonstrate a systematic procedure for monitoring inorganic carbon induced by enhanced weathering of comminuted rocks in ...

Simulating Temperature in a Soil Incubation Experiment

The study of warming impact on soils requires a realistic and accurate representation of temperature. In laboratory incubation studies, a widely adopted method has been to render constant temperatures in multiple chambers, and via comparisons of soil responses between low- and high-temperature chambers, to derive the warming impact on soil changes. However, this commonly used method failed to imitate both the magnitude and amplitude of actual temperatures as observed in field conditions, thus potentially undermining the validity of such studies. With sophisticated environmental chambers becoming increasingly available, it is imperative to examine alternative methods of temperature control for soil incubation research. This protocol will introduce a state-of-the-art environmental chamber and demonstrate both conventional and new methods of temperature control to improve the experimental design of soil incubation. The protocol mainly comprises four steps: temperature monitoring and programming, soil collection, laboratory incubation, and warming effect comparison. One example will be presented to demonstrate different methods of temperature control and the resultant contrasting warming scenarios; that is, a constant temperature design referred to as stepwise warming (SW) and simulated in situ temperature design as gradual warming (GW), as well as their effects on soil respiration, microbial biomass, and extracellular enzyme activities. In addition, we present a strategy to diversify temperature change scenarios to meet specific climate change research needs (e.g., extreme heat). The temperature control protocol and the recommended well-tailored and diversified temperature change scenarios will assist researchers in establishing reliable and realistic soil incubation experiments in the laboratory.

Laboratory soil warming experiments usually employ two or more constant temperatures in multiple chambers. By presenting a sophisticated environmental chamber, we provide an accurate temperature control method to imitate the magnitude and amplitude of in situ soil temperature and improve the experimental design of soil incubation studies.

The study of warming impact on soils requires a realistic and accurate representation of temperature. In laboratory incubation studies, a widely adopted ...

Design and Construction of an Experimental Setup to Enhance Mineral Weathering through the Activity of Soil Organisms

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