搜索

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   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈

本文章节

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

  1. Custom column design and construction (materials needed per column)
    1. Natural organic substrates mixed as described in (Drennan et al., 2016)
      1. Alfalfa hay (Figure 2A)
      2. Wood chips (High Desert Investment Company, Phoenix, AZ) (Figure 2B)
      3. Sawdust (High Desert Investment Company, Phoenix, AZ) (Figure 2C)
      4. 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.

    2. 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.

  2. Anoxic columns sampling of aqueous and solid-phase samples
    1. 10 ml syringes; Luer-Lok® syringes (BD, catalog number: 309604 )
    2. 15 ml tubes; Falcon® centrifuge tubes (Corning, Falcon®, catalog number: 352196 )
    3. 0.45 µm filters (EMD Millipore, catalog number: SLHV033RB )
    4. Plastic funnel (VWR, catalog number: 300009-435 )
    5. Parafilm (Bemis, catalog number: PM992 )
    6. Aluminum foil (VWR, catalog number: 89107-724 )
    7. 20% carbon dioxide balance nitrogen certified standard mixture, size 300 cylinder, CGA-580 (Airgas, catalog number: X02NI80C3003240 )
    8. Nitric acid; 69.0-70.0% (Avantor Performance Materials, J.T. Baker, catalog number: 9598-00 )
    9. Ethanol (VWR, catalog number: 200057-586 )
    10. De-ionized water (DI)
    11. Ice and coolers (for shipping)

  3. Separation of solid-phase substrate for geochemical and microbial analysis
    1. 50 ml tubes; Falcon® centrifuge tubes (Corning, Falcon®, catalog number: 352098 )
    2. 20% carbon dioxide balance nitrogen certified standard mixture, size 300 cylinder, CGA-580 (Airgas, catalog number: X02NI80C3003240 )
    3. Ethanol (VWR, catalog number: 200057-586)

Equipment

  1. Custom column design and construction (materials needed per column)
    1. Clear PVC pipe with a height of 52’’ (1.32 m) inner diameter (ID) 6” (0.15 m) (Figure 1)
    2. Nylon mesh bags (Phifer 48 in x 25 ft. BetterVue Scereen) (Home Depot Product Authority, catalog number: 3027671 ) (Figure 4E)
    3. Impulse heat sealer (Packco, Midwest Pacific, model: MP-12 )
    4. Tygon tubing for influent and effluent; ¼” ID and 3/8”OD (VWR, catalog number: 89403-862 )
    5. Pump tubing for peristaltic pump (4T [size], Blue and White Industry)
    6. Glass marbles (~16 mm diameter) filled the bottom 10 cm as an inert porous bed support. Alternatively large glass beads would also suffice
    7. Fittings (one entry for each type used):
      1. Column bottom fittings
      2. 3-way valve for effluent sample (Figure 4A)
      3. Valve for liquid sampling from side port (Figure 4B) (5 per column)
      4. 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.

    8. 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)
    9. Pump (Flex Flow, max feed 2.3 GFD, Blue and White Industry) (one per column)
    10. Feed tank (250 gal HDPE drum) one tank for all columns.
    11. Effluent collection tanks (30 gal HDPE drum)
    12. 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.

    13. Column head space gas collection system (to scrub released sulfide to prevent unsafe amounts of sulfide accumulating)
      1. 250 ml filter flask stopper No. 6 (Kimax Chase Life Science and Research Products, catalog number: 27060 )
      2. FEP gas bag 6 x 6 on/off (Labpure) (Saint-GoBain, catalog number: D1075002-10 )

  2. Anoxic columns sampling of aqueous and solid-phase samples 
    1. Heavy-Duty Single-Stage Gas Regulator (VWR, catalog number: 55850-277 )
    2. PVC hose ¾ in ID (VWR, catalog number: 89068-590 )
    3. Write-On bags (Nasco, Whirl-Pak®, catalog number: B01196WA )
    4. Vacuum sealer (Manufacturer Rival, model: FSFGSL0150-015 )
    5. Vacuum bags (Seal-A-Meal [11-Inch by 9-Foot Rolls, 2pk])
    6. Dissecting forceps; VWR® dissecting forceps, fine tip, curved (VWR, catalog number: 82027-406 )
    7. Needle nose multi-tool; Multi-Plier® 600 Needlenose Pliers, Gerber® (Gerber Gear, catalog number: 47550N )
    8. Bic Classic lighters
    9. Sharpie® permanent ink pen (VWR, catalog number: 500020-888 )
    10. Spray bottle to sterilize instruments with 70% ethanol (VWR, catalog number: 23609-182 )

  3. Separation of solid-phase substrate for geochemical and microbial analysis
    1. Anaerobic chamber (Sheldon Manufacturing, model: Bactron Anaerobic/Environmental chamber )
    2. Scale (OHAUS, model: ES 100 L )
    3. Large weigh boats (VWR, catalog number: 10803-168 )
    4. Scissors; VWR® dissecting scissors, sharp tip, 4½" (VWR, catalog number: 82027-578 )
    5. Needle nose multi-tool; Multi-Plier® 600 Needlenose Pliers, Gerber® (Gerber Gear, catalog number: 47550N )
    6. Dissecting forceps; VWR® dissecting forceps, fine tip, curved (VWR, catalog number: 82027-406)
    7. Spray bottle (VWR, catalog number: 23609-182)

Procedure

  1. Sample bag design for non-invasive retrieval of representative solid-phase substrate: Permeable substrate bags were deployed in each of the 3 solid-phase ports (Figure 1 and Figures 4D and 4E) to facilitate spatial and temporal inquiry. After a sample bag was retrieved it was replaced with an identical bag to circumvent any disturbance to column operation between time points.
    1. Determine the mass of material needed for analyses, and ensure sample bags contain at least that amount. In this case 3 inch by 3 inch bags are sufficient for the 1 g of material used for digests and 0.5 g x 3 for DNA extraction. Remaining material was anaerobically archived in Whirl-Paks in the anaerobic chamber, sealed in a secondary vacuum evacuated bag and then stored at -20 °C.
    2. Pack material homogenously in porous nylon bags (i.e., mosquito netting or window screen) and ensure the sample material is consistent with the rest of the column material. Four bags are put in each sample port at the beginning of the experiment.
    3. Record dry mass of each of the sample bags.
    4. Prepare extra bags for replacing harvested samples during experimental operation in order to minimize resultant flow perturbations.
    5. Pre-soak bags in experimentally-relevant water (in this case MIW) before packing them into sample ports to account for expansion, record wet mass.

  2. Liquid sample retrieval for aqueous metal analysis using Inductively Coupled Plasma (ICP-AES) Spectroscopy with a dual view PerkinElmer Optima Model 5300 spectrometer.
    1. At least 2 people are needed for sample retrieval.
    2. Ensure pumps are turned off and liquid is flowing freely to avoid pulling a vacuum.
    3. Harvest liquid samples before substrate to circumvent any flow effects.
    4. Flush a 10 ml syringe with N2/CO2 gas mix 3 times, letting the gas push the plunger up.
    5. Attach funnel to tubing to ensure gas is dispersed over the area of concern.
    6. Continually flush a 15 ml conical with N2/CO2 mix while collecting 10 ml of liquid with flushed syringe.
    7. Attach 0.45 µm filter to the syringe containing the sample and dispense filtered sample into the 15 ml tube while it is being flushed.
    8. For ICP-AES analysis lower the pH of the sample to 2 with nitric acid (in our case, several drops of 70% nitric acid).
    9. Screw on the cap and Parafilm over lid for transportation.

  3. Liquid collection for geochemical analyses 
    1. Flush a syringe three times with the gas mix.
    2. Flush 50 ml tubes while collecting liquid with the flushed syringe.
    3. Fill 50 ml tube allowing no head space.
    4. Screw on lid and Parafilm for transportation.
    5. Wrap in aluminum foil to prevent photooxidation of sulfides if appropriate.
    6. Tubes contained ~3 ml of gases headspace to prevent cracking during freezing.
    7. Run these samples as soon as possible (within 48 h).

  4. Solid substrate retrieval for geochemical and molecular analyses
    1. Ensure influent water flow is turned off.
    2. Tilt the column using the hinged rack assembly so that it is parallel to the ground in order to sample substrate bags without substantial fluid loss (Figure 6).


      Figure 6. Intact columns and supporting hardware in use during sampling process. One of the columns is being tilted horizontally for sample bag retrieval to enable analysis during the experiment.

    3. Have one person hold the gas line over the sample port before it is open.
    4. The second person will open the sample port, and using flame sterilized forceps/pliers retrieve the sample bag.
    5. Forceps, pliers, and other sample handling tools were flame sterilized by being sprayed with 70% ethanol and then lit with a handheld lighter (i.e., Bic) until the alcohol burns off.
    6. Place the sample bag into a pre-flushed Whirl-Pak®.
    7. Push out most of the air and ‘whirl’ the bag around the wire per instructions.
    8. Place Whirl-Pak-ed sample into vacuum bag and seal using vacuum sealer.
    9. Put the anoxically sealed sample on ice immediately for molecular analyses.
    10. Replace the harvested sample with a marked extra sample bag as a place holder.
    11. Replacement bag should not be collected as a sample, as it would not have been in contact with the reactive substrate as long as the other sample bags.

  5. Separation of substrate samples for geochemical and molecular analyses
    1. Bring defrosted solid-phase samples into the anaerobic chamber (95%:5% N2:H2) with ethanol spray bottle, 50 ml tubes, 15 ml tubes, forceps, pliers, scissors, weigh boats, and scale.
    2. Loosen 50 ml and 15 ml tube tops before putting them in the evacuation unit so the oxygen inside the tube is also purged during evacuation.
    3. Use sterilized scissors inside the anaerobic chamber to cut open vacuum seal bags.
    4. Remove sample bag from Whirl-Pak®, keeping the Whirl-Pak® intact.
    5. Cut open nylon sample bag with sterilized scissors.
    6. Put substrate in large disposable weigh boat and homogenize by mixing with forceps.
    7. Retrieve substrate from sample bag for DNA extraction with sterilized forceps/pliers. Place the substrate inside a pre-tared 50 ml tube, until there is 5 g wet weight in the tube. While only 0.5 g x 3 was used for DNA extraction, collecting 5 g allows for redundancy.
    8. Set aside DNA extraction tube, and collect 1 g of material for geochemical extractions and place in pre-tared 15 ml tube.
    9. Seal the 15 ml tube with Parafilm.
    10. Replace the remaining material back in the Whirl-Pak®.
    11. Vacuum seal the Whirl-Pak® after coming out of the anaerobic chamber and archive remaining material at -20 °C for extra analyses etc.
    12. Freeze material for molecular analyses at -20 °C, unless performed immediately.

Data analysis

Data collection and analysis encompassing microbial and geochemical properties was performed as described in (Zhou, 1996; Drennan et al., 2016). Reactors were characterized by a temporally stable community structure (as discerned using the 16S rRNA gene) comprised of a rich organoheterotrophic community that maintained a syntrophic relationship with sulfate reducing bacteria. Columns with greater than 10% alfalfa hay were characterized as having better zinc removal than columns dominated with woodchips. These two substrates selected for significantly different bacterial communities from one another (Drennan et al., 2016).

Notes

  1. Liquid should be sampled before solid substrate is collected to circumvent flow perturbations resulting from pulling reactive material.
  2. We advocate for an excess of 3 pore volumes of flow between sampling events, as this is a potentially disruptive sampling protocol.
  3. Genomic DNA harvested from the solid substrate was extracted and amplified in triplicate to minimize complications associated with sample heterogeneity.
  4. In some cases metal concentrations of interest exceeded the limits of the ICP-AES (particularly for mine water influent). When this occurred, samples were diluted with DI (i.e., 1:10) to bring the sample within detection range. An analogous approach was taken to counter complications with the high sodium concentration from the sodium acetate extraction step on ICP-AES analyses (Carlsson et al., 2002).

Acknowledgments

This material was based in part through work supported by the U.S. National Science Foundation (CBET-1055396) and the Office of Biological and Environmental Research in the U.S. Department of Energy (DE-SC0006997 and DE-SC0016451). Additional financial support was provided by Freeport-McMoRan Inc. and the J. Gust. Richert Memorial Fund (PIAH/12:57). RA was supported through a Marie Curie International Outgoing Fellowship (PIOF-GA-2012-328397) within the 7th European Community Framework Programme and The Carl Trygger Foundation for Scientific Research (CTS 12:11).

References

  1. Carlsson, E., Thunberg, J., Öhlander, B. and Holmström, H. (2002). Sequential extraction of sulfide-rich tailings remediated by the application of till cover, Kristineberg mine, northern Sweden. Sci Total Environ 299(1-3): 207-226.
  2. Drennan, D. M., Almstrand, R., Lee, I., Landkamer, L., Figueroa, L. and Sharp, J. O. (2016). Organoheterotrophic bacterial abundance associates with zinc removal in lignocellulose-based sulfate-reducing systems. Environ Sci Technol 50(1): 378-387.
  3. Neculita, C. M., Zagury, G. J. and Bussière, B. (2008). Effectiveness of sulfate-reducing passive bioreactors for treating highly contaminated acid mine drainage: I. Effect of hydraulic retention time. App Geochem 23(12): 3442-3451.
  4. Wildeman, T. R., Updegraff, D. M., Reynolds, J. S. and Bolis, J. L. (1994). Passive bioremediation of metals from water using reactors or constructed wetlands. In: Jeffrey, L. M. and Robert, E. H. (Eds.). Emerging Technology for Bioremediation of Metals. Lewis, pp:13-25.
  5. Zhou, J., Bruns, M. A. and Tiedje, J. M. (1996). DNA recovery from soils of diverse composition. Appl Environ Microbiol 62(2): 316-322.

简介

可以使用列研究来查询在自然和建筑环境中模拟环境相关的流通过程的系统。在整个操作过程中空间采样这些系统,同时保持用于地球化学和微生物分析的水相和固相样品的完整性,特别是当柱内的氧化还原条件不同于环境条件时,可能是具有挑战性的。在这里,我们提出了针对长期空间和时间采样进行优化的中试规模列设计和采样协议。我们利用这个实验装置大约2年来研究设计用于在硫酸盐还原条件下沉淀硫化锌的生物活性系统;然而,它可以适应于需要进行地球化学和/或分子微生物分析的许多流通系统的研究。重要的是,这些色谱柱利用可回收的固相袋结合缺氧微生物技术来收获底物样品,同时最小程度地破坏柱的操作。

背景 以下描述了一种实验设计和采样方案,其规避了垂直取芯的障碍物,用于柱系统的时间和空间分辨率。该系统具有进一步的优点,即对物理,化学和生物过程的破坏最小。中试规模设计采用垂直间隔采样端口,用于在离散时间点收集液相和固相基板(图1)。驻留在这些色谱柱内的固相底物的空间采样使研究人员能够观察生物相关过程,如金属固定的离散区域和微生物生物膜社区的变化。这些垂直生物地球化学特征的演变可以跟踪并与时间相关的性能相关。虽然对如下所述的缺氧系统进行了优化,但该实验设计可以应用于依赖于水相和固相相互作用的其他系统的空间查询。
 对于我们的研究特别感兴趣的是,硫酸盐还原生物反应器(SRBR)已被用于减轻矿井受影响水(MIW)的释放约二十年(Wildeman等人,1994)。由于这些系统的缺点以及它们的空间异质性,采样SRBR没有运行中断,带来了许多挑战。以前,这些系统采用有限的空间分辨率进行采样,可能不包括发生在(Neculita等人,2008年)内的区域内的精细过程。牺牲抽样可以克服这个障碍,但是牺牲了时间分辨率。此外,由于这种形式的样本采集可能导致饱和的,异质的有机材料(木片,锯屑,干草)和中断造成的困难,因此对中试规模和较大的SRBR的空间调查是具有挑战性的。我们的设计和采样程序使我们能够检查硫酸盐还原生物反应器的性能,这些生物反应器可以将受影响的水作为有机底物,微生物群落结构,水质和金属硫化物沉淀的功能进行影响,从而产生对这些系统运行的新见解(Drennan 等,,2016)。


图1.垂直向下流生化反应器柱的示意图。由柱上的圆圈表示的三个端口设计用于固体衬底检索以及沿着柱的z轴的流动。五个液体端口沿塔的侧面呈蓝色。对于固体基质的离散回收,使用定制的架子将柱临时倾斜到水平面,以减轻水压并发症和产生的损失(图5和图6)。标示为“MIW Inf。”表示采矿对水的引入。如图4A所示,来自这些塔的流出物被收集在底部液体端口处。

关键字:环境工程, 地球化学, 微生物学, 中试规模, 厌氧呼吸, 氧化还原, 生物修复, 生物反应器

材料和试剂

  1. 定制柱设计和施工(每列需要材料)
    1. 如(Drennan等人,2016)中所述混合的天然有机底物
      1. 苜蓿干草(图2A)
      2. 木片(高沙漠投资公司,凤凰城,亚利桑那州)(图2B)
      3. 锯屑(高沙漠投资公司,凤凰城,亚利桑那州)(图2C)
      4. 石灰石(Imery的3.35-4.95 mm)(图2D)


        图2.在柱内部署的固相基质组分的实例。 A.苜蓿干草; B.木片锯末和D.石灰岩。替代的底物如沙子或其他有机固相材料可能与替代应用相关,例如研究地下水或含水层补给。

    2. 使用10%NaOH溶液构建连接到塔顶空间的气体收集系统,以捕集和洗涤释放的硫化物,从而限制该毒素的积累(图3)。


      图3.生物硫化物的去除生产的气体从塔顶空间行进到连接的塑料袋。通过与10%NaOH溶液反应除去硫化物
  2. 水相和固相样品的缺氧柱取样
    1. 10ml注射器; Luer-Lok ®注射器(BD,目录号:309604)
    2. 15 ml管; Falcon ®离心管(Corning,Falcon ®,目录号:352196)
    3. 0.45μm过滤器(EMD Millipore,目录号:SLHV033RB)
    4. 塑料漏斗(VWR,目录号:300009-435)
    5. 石蜡膜(Bemis,目录号:PM992)
    6. 铝箔(VWR,目录号:89107-724)
    7. 20%二氧化碳平衡氮气认证标准混合物,300缸,CGA-580(Airgas,目录号:X02NI80C3003240)
    8. 硝酸; 69.0-70.0%(Avantor Performance Materials,J.T.Baker,目录号:9598-00)
    9. 乙醇(VWR,目录号:200057-586)
    10. 去离子水(DI)
    11. 冰和冷却器(用于运输)

  3. 固相底物分离用于地球化学和微生物分析
    1. 50ml管; Falcon ®离心管(Corning,Falcon ®,目录号:352098)
    2. 20%二氧化碳平衡氮气认证标准混合物,300缸,CGA-580(Airgas,目录号:X02NI80C3003240)
    3. 乙醇(VWR,目录号:200057-586)

设备

  1. 定制柱设计和施工(每列需要材料)
    1. 高度为52"(1.32米)内径(ID)6"(0.15米)的清漆PVC管(图1)
    2. 尼龙网袋(Phacking 48 in x 25 ft。BetterVue Scereen)(Home Depot Product Authority,目录号:3027671)(图4E)
    3. 脉冲热封机(Packco,中西部太平洋,型号:MP-12)
    4. 用于流入和流出的Tygon管; ¼"ID和3/8"外径(VWR,目录号:89403-862)
    5. 蠕动泵用泵管(4T [尺寸],蓝白工业)
    6. 玻璃大理石(约16毫米直径)填充底部10厘米作为惰性多孔床支撑。或者大玻璃珠也足够了
    7. 配件(每种使用一个条目):
      1. 柱底配件
      2. 流出物样品三通阀(图4A)
      3. 从侧面进行液体取样的阀门(图4B)(每列5个)
      4. 固体样品端口(图4C和4D)(每列3个)


      图4.用于液体和固体基质检索的样品端口。 A.三通阀到样品流出物; B.中间液体采样口; C.中间固相采样口与液体采样口的位置; D.将基材加入到柱中之前的固相端的侧面,将袋子排列在彼此相邻的端口中; E.实验中使用的含有固相底物的牺牲样品袋
    8. PVC样品端口允许流动和样品回收(图4D),然后可以将含有固相底物的牺牲袋(图4E)
    9. 泵(Flex Flow,最大进料2.3 GFD,蓝白工业)(每列一个)
    10. 进料罐(250加仑HDPE鼓)一个罐用于所有柱。
    11. 流出物收集罐(30加仑HDPE桶)
    12. 定制焊接框架(52"高度)以固定柱体并使其能够进行采样(图5A和4B)。


      图5.设备细节的试点规模部署。 A.列之前的列框; B.柱和管道之间的柱架建立。

    13. 柱顶空气采集系统(用于擦洗释放的硫化物以防止不安全量的硫化物积聚)
      1. 250ml过滤瓶塞6号(Kimax Chase Life Science and Research Products,目录号:27060)
      2. FEP气袋6 x 6开/关(Labpure)(Saint-GoBain,目录号:D1075002-10)

  2. 水相和固相样品的缺氧柱取样
    1. 重型单级气体调节器(VWR,目录号:55850-277)
    2. PVC软管¾在ID(VWR,目录号:89068-590)
    3. 写作包(Nasco,Whirl-Pak ®,目录号:B01196WA)
    4. 真空封口机(制造商Rival,型号:FSFGSL0150-015)
    5. 真空袋(Seal-A-Meal [11英寸,9英尺卷,2pk])
    6. 解剖钳VWR ®解剖钳,细尖端,弯曲(VWR,目录号:82027-406)
    7. 针鼻多工具; Multi-Plier ® 600 Needlenose Pliers,Gerber ®(Gerber Gear,目录号:47550N)
    8. Bic经典打火机
    9. Sharpie ®永久墨水笔(VWR,目录号:500020-888)
    10. 喷雾瓶用70%乙醇灭菌仪器(VWR,目录号:23609-182)

  3. 固相底物分离用于地球化学和微生物分析
    1. 厌氧室(Sheldon Manufacturing,型号:Bactron Anaerobic/Environmental chamber)
    2. 量表(OHAUS,型号:ES 100 L)
    3. 大型称重船(VWR,目录号:10803-168)
    4. 剪刀; VWR ®解剖剪刀,锋利尖端,4½"(VWR,目录号:82027-578)
    5. 针鼻多工具; Multi-Plier ® 600 Needlenose Pliers,Gerber ®(Gerber Gear,目录号:47550N)
    6. 解剖钳VWR ®解剖钳,细尖端,弯曲(VWR,目录号:82027-406)
    7. 喷雾瓶(VWR,目录号:23609-182)

程序

  1. 用于代表性固相基底的非侵入性回收的样品袋设计:在3个固相端口(图1和图4D和4E)中的每一个中部署了可渗透的基底袋,以便于空间和时间查询。取出样品袋后,用相同的袋子取代,以规避时间点之间对柱子操作的任何干扰。
    1. 确定分析所需材料的质量,并确保样品袋至少含有该量。在这种情况下,3英寸×3英寸的袋子对于1g用于消化的材料和0.5g×3的DNA提取是足够的。剩余材料在厌氧室的Whirl-Paks中厌氧存放,密封在二次真空抽真空的袋子中,然后储存在-20℃。
    2. 在多孔尼龙袋(即,蚊帐或窗帘)中均匀包装材料,并确保样品材料与柱材料的其余部分一致。实验开始时,每个采样端口都放置四个袋子。
    3. 记录每个样品袋的干质量。
    4. 在实验操作期间准备额外的袋子来取代收获的样品,以减少产生的流动扰动
    5. 在将实验相关的水(在这种情况下为MIW)中预浸泡袋,然后将其包装在样品端口中以解释膨胀,记录湿重。

  2. 使用电感耦合等离子体(ICP-AES)光谱进行含水金属分析的液体样品回收,具有双重视图PerkinElmer Optima Model 5300光谱仪。
    1. 需要至少2人进行抽样检索。
    2. 确保泵关闭,液体自由流动,以避免拉动真空。
    3. 在底物之前收集液体样品以避免任何流动效应。
    4. 用N 2/2/CO 2气体混合物冲洗10ml注射器3次,使气体向上推动柱塞。
    5. 将漏斗连接到管道上,以确保气体在所关注的区域分散。
    6. 在用冲洗的注射器收集10ml液体的同时,持续冲洗含有N 2/2/CO 2混合物的15ml锥形物。
    7. 将0.45μm过滤器连接到含有样品的注射器中,并将其过滤的样品在冲洗过程中分配到15 ml管中。
    8. 对于ICP-AES分析,用硝酸(在我们的情况下,几滴70%硝酸)将样品的pH值降至2。
    9. 拧上盖子和石膏膜,盖上盖子运输。

  3. 地球化学分析液体采集
    1. 用气体混合物冲洗注射器三次。
    2. 用冲洗的注射器收集液体冲洗50ml的管子。
    3. 填充50毫升管,不允许头部空间。
    4. 拧上盖子和石蜡膜运输。
    5. 包裹在铝箔中,以防止硫化物的光氧化(如果适用)
    6. 管子含有〜3ml的气体顶部空间,以防止冻结期间开裂。
    7. 尽快(48小时内)运行这些样品。

  4. 固体底物检索用于地球化学和分子分析
    1. 确保流入的水流被关闭。
    2. 使用铰接的机架组件倾斜柱,使其平行于地面,以便对基材袋进行采样而无明显的流体损失(图6)。


      图6.采样过程中正在使用的完整列和支持硬件。其中一列正在水平倾斜,用于样品袋检索,以实现实验期间的分析。

    3. 在打开之前,有一个人在采样端口上持有气体管线。
    4. 第二个人将打开样品端口,并使用火焰灭菌的钳子/钳子取回样品袋。
    5. 镊子,钳子和其他样品处理工具通过用70%乙醇喷雾而进行火焰灭菌,然后用手持式打火机(即,Bic)点燃,直到酒精燃烧。
    6. 将样品袋放入预先冲洗的Whirl-Pak ®中。
    7. 推出大部分的空气,并按照说明将电线"围绕电线"旋转。
    8. 将Whirl-Pak-ed样品放入真空袋中,并使用真空封口机进行密封。
    9. 将无氧密封的样品立即置于冰上进行分子分析。
    10. 将收集的样品用标记的额外样品袋替换为占位符。
    11. 替换袋不应作为样品收集,因为它不会与反应性基底接触,只要其他样品袋。

  5. 分离用于地球化学和分子分析的底物样品
    1. 使用乙醇喷雾瓶,50ml管,15ml管,镊子将解冻的固相样品送入厌氧室(95%:5%N 2:H 2 N 2) ,钳子,剪刀,称重船和秤。
    2. 将50毫升和15毫升的管顶放入撤离单元中,使排气管内的氧气也被清除。
    3. 在厌氧室内使用无菌剪刀切开真空密封袋。
    4. 从Whirl-Pak ®中取出样品袋,保持Whirl-Pak ®完整。
    5. 用无菌剪刀切开尼龙样品袋。
    6. 将基材置于大型一次性称重船中,并与镊子混合均匀
    7. 从样品袋中取出底物,用无菌钳/钳子进行DNA提取。将基材置于预先称量的50ml管中,直至管中有5g湿重。虽然仅使用0.5g x 3进行DNA提取,但是收集5克可以进行冗余。
    8. 放置DNA提取管,并收集1g用于地球化学提取的材料,并置于预处理的15 ml管中。
    9. 用Parafilm密封15 ml管。
    10. 将剩余的材料装回Whirl-Pak ®
    11. 真空密封Whirl-Pak ®,从厌氧室出来,并将剩余物料储存在-20°C以进行额外分析。
    12. 冷冻-20°C分子分析的材料,除非立即执行

数据分析

如(Zhou,1996; Drennan等人,2016)所述进行包含微生物和地球化学性质的数据收集和分析。反应器的特征在于时间上稳定的社区结构(使用16S rRNA基因识别),其由富含有机器官营养的群体组成,其与硫酸盐还原细菌保持了合成关系。具有超过10%苜蓿干草的柱子被表征为具有比以木片为主的列具有更好的锌去除。这两种底物被选择用于彼此显着不同的细菌群落(Drennan等人,2016)。

笔记

  1. 在收集固体基质之前应对液体进行采样,以避免拉动反应性物质导致的流动扰动
  2. 我们主张在采样事件之间超过3孔流量,因为这是潜在的破坏性采样方案。
  3. 从固体底物中收获的基因组DNA被提取并扩增一式三份,以最大限度地减少与样本异质性相关的并发症。
  4. 在某些情况下,感兴趣的金属浓度超出了ICP-AES的限制(特别是对于矿井水流入)。当发生这种情况时,用DI(即,1:10)稀释样品以使样品在检测范围内。采用类似的方法来对抗ICP-AES分析时乙酸钠提取步骤中高钠浓度的并发症(Carlsson et al。,2002)。

致谢

该材料部分基于美国国家科学基金会(CBET-1055396)和美国能源部生物和环境研究办公室(DE-SC0006997和DE-SC0016451)的工作支持。 Freeport-McMoRan Inc.和J. Gust提供额外的财务支持。富克纪念基金(PIAH/12:57)。 RA在第七届欧洲共同体框架计划和卡尔·格瑞格科学研究基金会(CTS 12:11)中通过玛丽居里国际外交奖学金(PIOF-GA-2012-328397)获得支持。

参考文献

  1. Carlsson,E.,Thunberg,J.,Öhlander,B.andHolmström,H。(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/12462586"target ="_ blank">由瑞典北部的克里斯汀贝格矿(Kristineberg mine)采用直接覆盖的硫化物丰富尾矿的顺序提取。 299(1- 3):207-226。
  2. Drennan,DM,Almstrand,R.,Lee,I.,Landkamer,L.,Figueroa,L.and Sharp,JO(2016)。  有机营养细菌丰富与在木质纤维素基硫酸盐还原系统中除去锌有关。环境科学技术 50 (1):378-387。
  3. Neculita,CM,Zagury,GJ和Bussière,B.(2008)。硫酸盐还原被动生物反应器用于处理高度污染的酸性矿井排水的有效性:I.水力停留时间的影响应用Geochem 23(12):3442-3451。 br />
  4. Wildeman,TR,Updegraff,DM,Reynolds,JS和Bolis,JL(1994)。< a class ="ke-insertfile"href ="https://books.google.com.hk/books?id=P1XQAD5oETgC&pg = PA13&lpg = PA13&dq = + Met + + + + + +的+被动+生物修复+ + + + + + + + + +构建+湿地和源= = %20Metals%20 from%20Water%20using%20reactors%20or%20constructed%20wetlands&f = false"target ="_ blank">使用反应堆或建造的湿地从水中对金属的被动生物修复在:Jeffrey,LM和Robert,EH (编)。金属生物治理新兴技术。刘易斯,pp:13-25。
  5. Zhou,J.,Bruns,MA和Tiedje,JM(1996)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/8593035"target = "_blank">从各种组成的土壤中回收DNA。 Appl Environ Microbiol 62(2):316-322。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。