Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes
利用具有水相和固相取样口的中试规模色谱柱对缺氧生物过程进行地球化学和分子微生物学研究
发布: 2017年01月05日第7卷第1期 DOI: 10.21769/BioProtoc.2083 浏览次数: 8037
评审: Disha SrivastavaAlexander Martin RueckerAnonymous reviewer(s)
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参见作者原研究论文
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Jan 2016
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Abstract
Column studies can be employed to query systems that mimic environmentally relevant flow-through processes in natural and built environments. Sampling these systems spatially throughout operation, while maintaining the integrity of aqueous and solid-phase samples for geochemical and microbial analyses, can be challenging particularly when redox conditions within the column differ from ambient conditions. Here we present a pilot-scale column design and sampling protocol that is optimized for long-term spatial and temporal sampling. We utilized this experimental set-up over approximately 2 years to study a biologically active system designed to precipitate zinc-sulfides during sulfate reducing conditions; however, it can be adapted for the study of many flow-through systems where geochemical and/or molecular microbial analyses are desired. Importantly, these columns utilize retrievable solid-phase bags in conjunction with anoxic microbial techniques to harvest substrate samples while minimally disrupting column operation.
Keywords: Environmental engineering (环境工程)
Geochemistry (地球化学)
Microbiology (微生物学)
Pilot-scale (中试规模)
Anaerobic respiration (厌氧呼吸)
Redox (氧化还原)
Bioremediation (生物修复)
Bioreactor (生物反应器)
Background
The following describes an experimental design and sampling protocol that circumvents the obstacle of vertical coring for temporal and spatial resolution of column systems. The system has the further advantage of minimal disruption to physical, chemical and biological processes. The pilot-scale design incorporates vertically spaced sampling ports for collection of liquid and solid-phase substrate at discrete time points (Figure 1). Spatial sampling of solid-phase substrates that reside within these columns enables researchers to observe biologically relevant processes such as discrete zones of metal immobilization and shifts in microbial biofilm communities. The evolution of these vertical biogeochemical profiles can be tracked and related to performance over time. While optimized for anoxic systems as described below, this experimental design, which surmounts obstacles of more conventional flow-through column systems, could be applied for spatial inquiry into other systems that rely on aqueous and solid-phase interactions.
Of particular interest to our research, Sulfate Reducing Bioreactors (SRBRs) have been employed to mitigate the release of Mining Influenced Water (MIW) for approximately two decades (Wildeman et al., 1994). Due to the anoxic nature of these systems and their spatial heterogeneity, sampling SRBRs without operational disruption presents many challenges. Previously these systems were sampled with limited spatial resolution potentially excluding seminal processes occurring in regions within (Neculita et al., 2008). While sacrificial sampling can surmount this obstacle, it does so at the expense of temporal resolution. Furthermore, spatial inquiry into pilot-scale and larger SRBRs is challenging due to difficulty associated with coring saturated, heterogeneous organic materials (woodchips, sawdust, hay) and disruptions that can result from this form of sample collection. Our design and sampling procedure enabled us to examine the performance of sulfate reducing bioreactors that treat mining influenced water as a function of organic substrate, microbial community structure, water quality, and metal-sulfide precipitation yielding novel insights into the operation of these systems (Drennan et al., 2016).
Figure 1. Schematic of vertical down flow biochemical reactor columns. The three ports, indicated by circles on the column, were designed for solid substrate retrieval in conjunction with flow along the z-axis of the columns. The five liquid ports are depicted in blue along the side of the column. For discrete retrieval of solid substrates, columns were temporarily tilted to a horizontal plane using a custom built rack to mitigate water pressure complications and resultant loss (Figures 5 and 6). As labelled, ‘MIW Inf.’ indicates where the mining influenced water is introduced. The effluent from these columns was collected at the bottom liquid port as visualized in Figure 4A.
Materials and Reagents
- Custom column design and construction (materials needed per column)
- Natural organic substrates mixed as described in (Drennan et al., 2016)
- Alfalfa hay (Figure 2A)
- Wood chips (High Desert Investment Company, Phoenix, AZ) (Figure 2B)
- Sawdust (High Desert Investment Company, Phoenix, AZ) (Figure 2C)
- Limestone (Imery’s 3.35-4.95 mm) (Figure 2D)
Figure 2. Examples of solid-phase matrix components deployed within columns. A. Alfalfa hay; B. Woodchips; C. Sawdust; and D. Limestone. Alternative substrates such as sand or other organic solid-phase materials could have relevance to alternative applications such as studying subsurface flow or aquifer recharge.
- A gas collection system linked to the column headspace was constructed using 10% NaOH solution to trap and scrub released sulfide thereby limiting accumulation of this toxin (Figure 3).
Figure 3. Removal of biogenic sulfide. Produced gases traveled from the column headspace to a connected plastic bag. Sulfide was removed by reacting with a 10% NaOH solution.
- Natural organic substrates mixed as described in (Drennan et al., 2016)
- Anoxic columns sampling of aqueous and solid-phase samples
- 10 ml syringes; Luer-Lok® syringes (BD, catalog number: 309604 )
- 15 ml tubes; Falcon® centrifuge tubes (Corning, Falcon®, catalog number: 352196 )
- 0.45 µm filters (EMD Millipore, catalog number: SLHV033RB )
- Plastic funnel (VWR, catalog number: 300009-435 )
- Parafilm (Bemis, catalog number: PM992 )
- Aluminum foil (VWR, catalog number: 89107-724 )
- 20% carbon dioxide balance nitrogen certified standard mixture, size 300 cylinder, CGA-580 (Airgas, catalog number: X02NI80C3003240 )
- Nitric acid; 69.0-70.0% (Avantor Performance Materials, J.T. Baker, catalog number: 9598-00 )
- Ethanol (VWR, catalog number: 200057-586 )
- De-ionized water (DI)
- Ice and coolers (for shipping)
- 10 ml syringes; Luer-Lok® syringes (BD, catalog number: 309604 )
- Separation of solid-phase substrate for geochemical and microbial analysis
- 50 ml tubes; Falcon® centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
- 20% carbon dioxide balance nitrogen certified standard mixture, size 300 cylinder, CGA-580 (Airgas, catalog number: X02NI80C3003240 )
- Ethanol (VWR, catalog number: 200057-586)
- 50 ml tubes; Falcon® centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
Equipment
- Custom column design and construction (materials needed per column)
- Clear PVC pipe with a height of 52’’ (1.32 m) inner diameter (ID) 6” (0.15 m) (Figure 1)
- Nylon mesh bags (Phifer 48 in x 25 ft. BetterVue Screen) (Home Depot Product Authority, catalog number: 3027671 ) (Figure 4E)
- Impulse heat sealer (Packco, Midwest Pacific, model: MP-12 )
- Tygon tubing for influent and effluent; ¼” ID and 3/8”OD (VWR, catalog number: 89403-862 )
- Pump tubing for peristaltic pump (4T [size], Blue and White Industry)
- Glass marbles (~16 mm diameter) filled the bottom 10 cm as an inert porous bed support. Alternatively large glass beads would also suffice
- Fittings (one entry for each type used):
- Column bottom fittings
- 3-way valve for effluent sample (Figure 4A)
- Valve for liquid sampling from side port (Figure 4B) (5 per column)
- Solid sample port (Figures 4C and 4D) (3 per column)
Figure 4. Sample ports for liquid and solid substrate retrieval. A. 3-way valve to sample effluent; B. Intermediate liquid sampling port; C. Position of intermediate solid-phase sampling port in conjunction with liquid sampling ports; D. Side profile of a solid-phase port before substrate was added to columns, bags were lined up in the port adjacent to each other; E. Sacrificial sample bag containing solid-phase substrate utilized in experiments. - PVC sample ports allowing for flow through and sample retrieval (Figure 4D) which can then be packed with sacrificial bags containing solid-phase substrate (Figure 4E)
- Pump (Flex Flow, max feed 2.3 GFD, Blue and White Industry) (one per column)
- Feed tank (250 gal HDPE drum) one tank for all columns.
- Effluent collection tanks (30 gal HDPE drum)
- Custom welded frame (52” height) to hold columns and enable pivoting for sampling (Figures 5A and 4B).
Figure 5. Pilot scale deployment of apparatus detailing. A. Column frame before columns; B. Column frame after columns and plumbing were established. - Column head space gas collection system (to scrub released sulfide to prevent unsafe amounts of sulfide accumulating)
- 250 ml filter flask stopper No. 6 (Kimax Chase Life Science and Research Products, catalog number: 27060 )
- FEP gas bag 6 x 6 on/off (Labpure) (Saint-GoBain, catalog number: D1075002-10 )
- Clear PVC pipe with a height of 52’’ (1.32 m) inner diameter (ID) 6” (0.15 m) (Figure 1)
- Anoxic columns sampling of aqueous and solid-phase samples
- Heavy-Duty Single-Stage Gas Regulator (VWR, catalog number: 55850-277 )
- PVC hose ¾ in ID (VWR, catalog number: 89068-590 )
- Write-On bags (Nasco, Whirl-Pak®, catalog number: B01196WA )
- Vacuum sealer (Manufacturer Rival, model: FSFGSL0150-015 )
- Vacuum bags (Seal-A-Meal [11-Inch by 9-Foot Rolls, 2pk])
- Dissecting forceps; VWR® dissecting forceps, fine tip, curved (VWR, catalog number: 82027-406 )
- Needle nose multi-tool; Multi-Plier® 600 Needlenose Pliers, Gerber® (Gerber Gear, catalog number: 47550N )
- Bic Classic lighters
- Sharpie® permanent ink pen (VWR, catalog number: 500020-888 )
- Spray bottle to sterilize instruments with 70% ethanol (VWR, catalog number: 23609-182 )
- Heavy-Duty Single-Stage Gas Regulator (VWR, catalog number: 55850-277 )
- Separation of solid-phase substrate for geochemical and microbial analysis
- Anaerobic chamber (Sheldon Manufacturing, model: Bactron Anaerobic/Environmental chamber )
- Scale (OHAUS, model: ES 100 L )
- Large weigh boats (VWR, catalog number: 10803-168 )
- Scissors; VWR® dissecting scissors, sharp tip, 4½" (VWR, catalog number: 82027-578 )
- Needle nose multi-tool; Multi-Plier® 600 Needlenose Pliers, Gerber® (Gerber Gear, catalog number: 47550N )
- Dissecting forceps; VWR® dissecting forceps, fine tip, curved (VWR, catalog number: 82027-406)
- Spray bottle (VWR, catalog number: 23609-182)
- Anaerobic chamber (Sheldon Manufacturing, model: Bactron Anaerobic/Environmental chamber )
Procedure
文章信息
版权信息
© 2017 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Drennan, D. M., Almstrand, R., Lee, I., Landkamer, L., Figueroa, L. and Sharp, J. O. (2017). Pilot-scale Columns Equipped with Aqueous and Solid-phase Sampling Ports Enable Geochemical and Molecular Microbial Investigations of Anoxic Biological Processes. Bio-protocol 7(1): e2083. DOI: 10.21769/BioProtoc.2083.
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分类
微生物学 > 微生物生物化学 > 其它化合物
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