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Enriching Acidophilic Fe(II)-oxidizing Bacteria in No-flow, Fed-batch Systems
非流动、补料系统中嗜酸 Fe(II)氧化细菌的富集   

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Abstract

Low-pH microbial Fe(II) oxidation occurs naturally in some Fe(II)-rich acid mine drainage (AMD) ecosystems across so-called terraced iron formations. Indigenous acidophilic Fe(II)-oxidizing bacterial communities can be incorporated into both passive and active treatments to remove Fe from the AMD solution. Here, we present a protocol of enriching acidophilic Fe(II)-oxidizing bacteria in no-flow, fed-batch systems. Mixed cultures of naturally occurring microbes are enriched from the fresh surface sediments at AMD sites using a chemo-static bioreactor (Eppendorf BioFlo®/Celligen® 115 Fermentor) with respect to constant stirring speed, temperature, pH and unlimited dissolved oxygen. Ferrous sulfate is discontinuously added to the reactor as the primary substrate to enrich for acidophilic Fe(II)-oxidizing bacteria. Successfully and efficiently enriching acidophilic Fe(II)-oxidizing bacteria helps to exploit this biogeochemical process into AMD treatment systems.

Keywords: Acid mine drainage(酸性矿井排水), Enrichment(富集), Bioreactor(生物反应器), Fe(II)-oxidizing bacteria(Fe(II)氧化细菌), Bioremediation(生物治疗)

Background

Low-pH microbial Fe(II) oxidation can be incorporated into AMD passive treatment systems by enhancing the development of terraced iron formations (DeSa et al., 2010; Brown et al., 2011; Larson et al., 2014a and 2014b). For extremely difficult-to-treat AMD (very low pH, very high concentrations of Fe(II) and associated metals), an active treatment bioreactor is required by enriching acidophilic Fe(II)-oxidizing bacterial communities. This process can effectively change a high acidity, high metals discharge into a moderate acidity (still low pH), low metals discharge (Sheng et al., 2016).

Acidophilic aerobic Fe(II) oxidizers Acidithiobacillus spp., Leptospirillum spp., and Ferrovum myxofaciens have all been enriched in both suspended growth and fixed-film laboratory-scale bioreactors for AMD treatment (Hedrich and Johnson, 2012; Heinzel et al., 2009a and 2009b; Janneck et al., 2010; Tischler et al., 2013). For instance, Hedrich and Johnson (2012) designed an AMD remediation system that integrated low-pH Fe(II) oxidation and Fe removal in a multi-reactor system. A pure culture of the Fe(II)-oxidizer Ferrovum myxofaciens was enriched in first suspended-growth reactor. Heinzel et al. (2009a and 2009b), Janneck et al. (2010) and Tischler et al. (2013) all developed a natural mixed community of Fe(II)-oxidizers with porous fixed-film media in a pilot-scale reactor. A protocol of enriching mixed culture acidophilic Fe(II)-oxidizing bacteria in no-flow, fed-batch systems without fixed-film media is suggested here in a chemostatic bioreactor with controlled hydrogeochemical conditions (Sheng et al., 2016).

Materials and Reagents

  1. Sterile plastic containers
  2. 0.45 μm sterile bottle-top filters (Corning, catalog number: 430514 )
  3. Al foil
  4. 50 ml sterile centrifuge tubes (VWR, catalog number: 89039-656 )
  5. 15 ml sterile centrifuge tubes (VWR, catalog number: 89039-664 )
  6. 100% N2
  7. 0.1% (m/v) sodium pyrophosphate (EMD Millipore, catalog number: SX0741 )
  8. 0.1 M sulfuric acid (H2SO4)
  9. 0.2 N sodium hydroxide (NaOH)
  10. FeSO4·7H2O (VWR, catalog number: 97061-538 )
  11. 1 g/L ferrozine (Thermo Fisher Scientific, Fisher Scientific, catalog number: AC410570050 )
  12. 50 mM HEPES (pH = 7.0) (Sigma-Aldrich, catalog number: H3375 )
  13. Hydroxylamine-HCl (VWR, BDH®, catalog number: BDH9236-500G )
  14. Bio-Rad Protein Assay Kit II (Bio-Rad Laboratories, catalog number: 5000002 )
  15. 10% (w/v) oxalic acid (VWR, BDH®, catalog number: BDH7336-1 )

Equipment

  1. Heavy-duty round carboy (VWR, catalog number: 10755-104 )
  2. Standard magnetic stirrer
  3. Fermentor (Eppendorf, BrunswickTM, model: BioFlo®/Celligen® 115 )
  4. pH meter
  5. Plate centrifuge
  6. Autoclave
  7. Freezer
  8. Spectrophotometer

Software

  1. Eppendorf BioFlo®/Celligen® 115 fermentor automatic control software
  2. Adobe® Photoshop® software

Procedure

  1. Sediments are collected from the bottom of pools along the anoxic Fe(II)-rich artesian AMD flow paths. Sediments are collected downstream of the artesian discharges where the AMD have become well aerated (Figure 1A). Sediments are collected by carefully cutting and prying out intact pieces from the top 2 cm of the stream bed (Figure 1B). These samples are preserved in sterile plastic containers closed with lids, transported to the lab with dry ice, and stored in the 4 °C refrigerator for no longer than one week before use.
  2. Water is collected for microbial enrichments (Figure 1C). Water is collected from the anoxic artesian springs into acid washed plastic containers (completely filled with little or no headspace). Polypropylene carboys ranging in size from 12-40 L are used to transport the water to the lab. Immediately upon arrival to the lab, all water is filtered (0.45 μm sterile bottle-top filters) into plastic containers, purged with N2, wrapped in Al foil, and stored at 4 °C. Water is stored for no longer than one week before use.


    Figure 1. Photographs of a typical Fe(II)-rich artesian AMD site (A), sediment collection (B) and water collection (C)

  3. To develop enrichment cultures, 100 g of moist sediment is mixed with 1 L of 0.1% (m/v) sodium pyrophosphate (adjusted to pH of natural AMD water with sulfuric acid) in a sterile bottle (Figure 2A).
  4. For 30 min, this mixture is stirred at 400 rpm on a magnetic stirrer to separate cells from the sediment.
  5. The suspension is allowed to settle for 30 min and then 900 ml of the cell-containing supernatant is poured into a sterile 3 L chemostat reactor vessel (Eppendorf BioFlo®/Celligen® 115 fermentor) (Figure 2B). The reactor vessel is able to accommodate the low pH values (2 < pH < 4).


    Figure 2. Photographs of enriching natural-occurring sediment microbes in a sterile chemostat reactor vessel. A. Cell separation step; B. Bioreactor vessel; C. Bioreactor in use.

  6. The volume of the liquid in the chemostat reactor vessel is increased to 2 L by adding filtered (0.45-μm) site AMD water (Figure 2C).
  7. The chemostat is then operated in a no-flow, fed-batch mode for 4 to 6 weeks. Automated control components of the bioreactors maintained a constant pH, temperature and mixing speed, and continuously recorded dissolved oxygen. Feedback controls between the pH meter in the reactor and two peristaltic pumps delivering either 0.1 M H2SO4 or 0.2 N NaOH are used to maintain any desired pH set-point (Figure 3). A one-pass, tap water-fed, cooling coil within the reactor and a thermal jacket around the reactor are used to maintain the reactor temperature. Air is continuously pumped into the bottom of the reactor vessel to assure unlimited dissolved oxygen. During the enrichment period the pH set-point of the chemostat is same to the field water, the stirring rate is 50 rpm, the temperature is 20 °C and dissolved oxygen is unlimited (e.g., > 3 mg/L) (Sheng et al., 2016). 


    Figure 3. Photograph of bioreactor system including feedback system, peristaltic pumps and stirrer

  8. Ferrous sulfate (FeSO4·7H2O) is discontinuously added to the reactor as the primary substrate to enrich for autotrophic Fe(II)-oxidizing microbes. Ferrous sulfate is added to yield 300 mg L-1 dissolved [Fe(II)] and added whenever the dissolved [Fe(II)] decreases below 30 mg L-1.
  9. After Fe(II) oxidation and total Fe removal rate have reached a pseudo-steady state condition (within ± 5% standard deviation), biofilms containing abundant Fe(II)-oxidizing microbial communities are clearly and evenly attached to the reactor wall (Figure 2C). After less than a month, the enrichment cultures required daily doses of Fe(II) because the microorganisms are oxidizing the Fe(II) at a fast rate.
  10. Sampling
    1. Samples for Fe(II) measurement are collected at the top of the liquid level inside the reactor vessel using sterile pipette. An unused port in the top of the vessel closed with a screw cap can be opened to take the sample. A total of 2 ml water is collected each time for Fe(II) and total Fe measurement. Dissolved Fe(II) and dissolved total Fe(T) (after reduction by hydroxylamine-HCl) are determined using the ferrozine assay (1 g/L ferrozine in 50 mM HEPES [pH 7.0]) with samples preserved with 0.1 N HCl (Stookey, 1970). The frequency of the sampling depends on the rates of Fe(II) oxidation. The bioreactor has to be sampled daily for the first two weeks and less often after.
    2. Biofilm samples are collected from the reactor wall and water samples are collected from the suspension to determine the biomass concentrations. The lid of the reactor vessel is opened and water is poured into a sterile container. After using sterile pipet to scrape the biofilm from the reactor wall, the water is poured back to the reactor vessel with closed lid. Biomass concentrations are determined based on protein (Bio-Rad Protein Assay Kit that uses Coomassie® Brilliant Blue G-250 dye). An area of at least 1 cm2 of biofilm is scraped from the wall of the reactor to use for the protein assay. A ruler is used as reference substance (Figure 4). Photographs of the scraped zone are used with Adobe® Photoshop® software to determine the exact area that is removed each time. 


      Figure 4. Exemplary Photographs of the scraped biofilm using a ruler as reference substance for area calculation

    3. The collected biofilm is dissolved in 3 ml of 10% (w/v) oxalic acid. Once dissolved, the cells are lysed by placing 1 ml of this mix with 2 ml of 0.2 N NaOH, and then performing two freeze-thaw cycles on the sample in a -20 °C freezer for at least 6 h and 37 °C water bath for about 10-15 min. The protein colorimetric assay is then performed using the mixture containing the lysed cells.
    4. For the suspension, 135 ml of reactor liquid is centrifuged (13,000 x g for 10 min) to pelletize the cells. The masses of the Falcon® tubes used for centrifugation are measured before and after to determine the mass of each pellet. The liquid is then decanted and the cell pellet is analyzed exactly as the scraped cells described above.

Data analysis

  1. Assuming that the chemostat operates as a completely-mixed reactor, zero-order rates of Fe(II) oxidation [mol Fe(II) L-1 sec-1] in the chemostat are calculated as:

    Where,
    d[Fe(II)] is decreased dissolved Fe(II) concentration (mol L-1),
    dt is increased incubation time.
  2. For the enrichment experiment, the system reaches a pseudo-steady state with respect to the Fe(II) oxidation rate. In order to deem certain measured points within the pseudo-steady state and others not, a range of plus and minus 5% standard deviation of the last measurement is calculated. Any points that fall within the plus or minus 5% standard deviation range are deemed to be at pseudo-steady state. This statistical method placed emphasis on the last measured point since all other points are compared to it.
  3. To determine the concentration of cells in suspension and in the biofilm, a Bio-Rad protein assay kit is used. Once the protein concentrations are obtained, the derived conversion factor of 0.15 x 10-12 g protein/typical cell is used to convert the mass of protein present to the number of cells.

Notes

  1. Because enriched Fe(II)-oxidizing microbial communities are greatly influenced by pH and Fe(II) concentrations (Sheng et al., 2016), the pH set-point and Fe(II) concentrations of the chemostat can be adjusted according to the pH and Fe(II) concentrations of field AMD.
  2. Sediments and water are collected for microbial enrichments. Sediments are collected from the bottom of pools along the anoxic Fe(II)-rich artesian AMD flow paths. Water is collected from the anoxic artesian springs. To develop enrichment cultures, 100 g of moist sediment is mixed with 1 L of 0.1% (m/v) sodium pyrophosphate (adjusted to pH of natural AMD water with sulfuric acid).
  3. Chemostat bioreactors are operated at pH of the field AMD, temperature 20 °C, stirring rate 50 rpm and unlimited dissolved oxygen (> 3 mg/L) for 4-6 weeks.
  4. Ferrozine assay (1 g/L ferrozine in 50 mM HEPES [pH 7.0]) is used to measure Fe(II) oxidation rate (Stookey, 1970).
  5. Attached-growth and suspended biomass is dissolved in 10% (w/v) oxalic acid, mix with 0.2 N NaOH, and then performing two freeze-thaw cycles to lyse cells. Biomass concentrations are determined based on protein (Bio-Rad Protein Assay Kit that uses Coomassie® Brilliant Blue G-250 dye).

Recipes

  1. Solution for Ferrozine assay (pH 7.0)
    1 g/L ferrozine
    50 mM HEPES
  2. Enrichment of acidophilic bacteria
    100 g of moist sediment is mixed with 1 L of 0.1% (m/v) sodium pyrophosphate
    Adjusted to pH of natural AMD water with sulfuric acid

Acknowledgments

This protocol is adapted from Sheng et al. (2016). This work is partially supported by the US Office of Surface Mining Reclamation and Enforcement under Cooperative Agreement S11AC20005, by the China Scholarship Council (to Y.S.), and by the Appalachian Research Initiative for Environmental Science (ARIES). ARIES is an industrial affiliates program at Virginia Tech, supported by members that include companies in the energy sector. The opinions and recommendations expressed herein are solely those of the authors and do not imply any endorsement by ARIES.

References

  1. Brown, J. F., Jones, D. S., Mills, D. B., Macalady, J. L. and Burgos, W. D. (2011). Application of a depositional facies model to an acid mine drainage site. Appl Environ Microbiol 77(2): 545-554.
  2. DeSa, T., Brown, J. and Burgos, W. (2010). Laboratory and field-scale evaluation of low-pH Fe(II) oxidation at Hughes Borehole, Portage, Pennsylvania. Mine Water Environ 29 (4):239-247.
  3. Hedrich, S. and Johnson, D. B. (2012). A modular continuous flow reactor system for the selective bio-oxidation of iron and precipitation of schwertmannite from mine-impacted waters. Bioresour Technol 106: 44-49.
  4. Heinzel, E., Hedrich, S., Janneck, E., Glombitza, F., Seifert, J. and Schlomann, M. (2009a). Bacterial diversity in a mine water treatment plant. Appl Environ Microbiol 75(3): 858-861.
  5. Heinzel, E., Janneck, E., Glombitza, F., Schlomann, M. and Seifert, J. (2009b). Population dynamics of iron-oxidizing communities in pilot plants for the treatment of acid mine waters. Environ Sci Technol 43(16): 6138-6144
  6. Janneck, E., Arnold, I., Koch, T., Meyer, J., Burghardt, D. and Ehinger, S. (2010). Microbial synthesis of schwertmannite from lignite mine water and its utilization for removal of arsenic from mine waters and for production of iron pigments. In: Wolkersdorfer, C. and Freund, A. (Eds.). Mine water and innovative thinking. Proceedings of the international mine water association symposium 131-134.
  7. Larson, L. N., Sánchez-España, J. and Burgos, W. (2014a). Rates of low-pH biological Fe(II) oxidation in the appalachian bituminous coal basin and the Iberian pyrite belt. Appl Geochem 47:85-98.
  8. Larson, L. N., Sánchez-España, J., Kaley, B., Sheng, Y., Bibby, K. and Burgos, W. D. (2014b). Thermodynamic controls on the kinetics of microbial low-pH Fe(II) oxidation. Environ Sci Technol 48(16): 9246-9254.
  9. Tischler, J. S., Wiacek, C., Janneck, E. and Schlömann, M. (2013). Microbial abundance in the schwertmannite formed in a mine water treatment plant. Mine Water Environ 32:258-265.
  10. Sheng, Y., Bibby, K., Grettenberger, C., Kaley, B., Macalady, J. L., Wang, G. and Burgos, W. D. (2016). Geochemical and temporal influences on the enrichment of acidophilic iron-oxidizing bacterial communities. Appl Environ Microbiol 82(12): 3611-3621.
  11. Stookey, L. L. (1970). Ferrozine--a new spectrophotometric reagent for iron. Anal Chem 42(7):779-781.

简介

低pH微生物Fe(II)氧化在一些富含Fe(II)的酸性矿井排水(AMD)生态系统中自然发生在所谓的梯田铁层上。土着嗜酸性Fe(II) - 氧化细菌群落可纳入被动和主动处理以从AMD溶液中除去Fe。在这里,我们提出了在无流量,补料分批系统中富含嗜酸性Fe(II) - 氧化细菌的方案。天然存在的微生物的混合培养物使用化学静态生物反应器(Eppendorf BioFlo / Celligen 115发酵罐)从AMD位点的新鲜表面沉积物富集,相对于常数搅拌速度,温度,pH和无限溶解氧。将硫酸亚铁不连续地加入到反应器中作为主要底物,以富集嗜酸性Fe(II) - 氧化细菌。成功和有效地富集嗜酸性Fe(II) - 氧化细菌有助于将这种生物地球化学过程用于AMD治疗系统。

背景 低pH微生物Fe(II)氧化可以通过增强梯田铁地层的发展而被纳入到AMD被动处理系统中(DeSa等人,2010; Brown等人 ,2011; Larson等人,2014a和2014b)。对于非常难治疗的AMD(非常低的pH,非常高浓度的Fe(II)和相关金属),通过富含嗜酸性Fe(II)氧化细菌群落需要主动治疗生物反应器。这个过程可以有效地改变高酸度,高金属排放到中等酸度(仍然很低的pH),低金属排放(Sheng等人,2016)。
&nbsp;嗜酸性好氧Fe(II)氧化剂酸性硫杆菌属,钩端螺旋体属和黑曲霉属Ferrovum myxofaciens都已经丰富了悬浮生长和固定电影实验室规模的用于AMD治疗的生物反应器(Hedrich和Johnson,2012; Heinzel等人,2009a和2009b; Janneck等人,2010; Tischler et al。,2013)。例如,Hedrich和Johnson(2012)设计了一种在多反应器系统中整合低pH Fe(II)氧化和Fe去除的AMD修复系统。第一个悬浮生长反应器富含Fe(II) - 氧化剂Ferrovum myxofaciens的纯培养物。 Heinzel等人。 (2009a和2009b),Janneck等人。 (2010)和Tischler等人。 (2013年)在中试规模的反应堆中开发了具有多孔固定膜介质的Fe(II) - 氧化剂的天然混合物。在具有受控的水文地球化学条件的化学生物反应器中,建议在没有固定膜介质的无流动,补料分批系统中富集混合培养的嗜酸性Fe(II) - 氧化细菌的方案(Sheng et al。 。,2016)。

关键字:酸性矿井排水, 富集, 生物反应器, Fe(II)氧化细菌, 生物治疗

材料和试剂

  1. 无菌塑料容器
  2. 0.45μm无菌瓶顶过滤器(Corning,目录号:430514)
  3. 铝箔
  4. 50ml无菌离心管(VWR,目录号:89039-656)
  5. 15 ml无菌离心管(VWR,目录号:89039-664)
  6. 100%N 2
  7. 0.1%(m/v)焦磷酸钠(EMD Millipore,目录号:SX0741)
  8. 0.1M硫酸(H 2 SO 4 SO 4)
  9. 0.2N氢氧化钠(NaOH)
  10. FeSO 4·7H 2 O(VWR,目录号:97061-538)
  11. 1克/升铁(Thermo Fisher Scientific,Fisher Scientific,目录号:AC410570050)
  12. 50mM HEPES(pH = 7.0)(Sigma-Aldrich,目录号:H3375)
  13. 羟胺-HCl(VWR,BDH ,目录号:BDH9236-500G)
  14. Bio-Rad蛋白测定试剂盒II(Bio-Rad Laboratories,目录号:5000002)
  15. 10%(w/v)草酸(VWR,BDH ,目录号:BDH7336-1)

设备

  1. 重型圆形瓶(VWR,目录号:10755-104)
  2. 标准磁力搅拌器
  3. 发酵罐(Eppendorf,Brunswick TM ,型号:BioFlo /Celligen ® 115)
  4. pH计
  5. 板式离心机
  6. 高压灭菌器
  7. 冰箱
  8. 分光光度计

软件

  1. 发酵罐自动控制软件Eppendorf BioFlo ®/Celligen ®
  2. Adobe ® Photoshop ®软件

程序

  1. 沿着缺氧的富含Fe(II)的自流式AMD流动路径从池底部采集沉积物。沉积物被收集在自发放电的下游,其中AMD已经充分充气(图1A)。通过仔细切割并从流床的顶部2厘米处撬出完整的碎片来收集沉积物(图1B)。将这些样品保存在用盖子封闭的无菌塑料容器中,用干冰运输到实验室,并在使用前不要超过一周储存在4°C冰箱中。
  2. 收集水用于微生物富集(图1C)。水从缺氧自流弹簧收集到酸洗的塑料容器中(完全充满少量或没有顶部空间)。使用范围从12-40升的聚丙烯碳水化合物将水输送到实验室。在实验室抵达实验室后,将所有的水都过滤(0.45μm无菌瓶顶过滤器)放入塑料容器中,用N 2膜吹扫,裹上铝箔,并在4℃下储存。水在使用前不得超过一周。


    图1.典型的富含Fe(II)的自流体AMD站点(A),沉积物收集(B)和集水(C)的照片

  3. 为了培养富集培养物,将100g潮湿沉淀物与无菌瓶中的1L 0.1%(m/v)焦磷酸钠(用硫酸调节至天然AMD水的pH)混合(图2A)。
  4. 30分钟后,将该混合物在磁力搅拌器上以400rpm搅拌,从沉淀物中分离出细胞。
  5. 使悬浮液沉降30分钟,然后将900ml含细胞的上清液倒入无菌的3L恒化器反应器容器中(Eppendorf BioFlo/sup/115发酵罐)(图2B)。反应器容器能够适应低pH值(2

    图2.在无菌恒化器反应器容器中富集天然沉积物微生物的照片。 A.细胞分离步骤; B.生物反应器容器C.使用生物反应器。

  6. 通过加入过滤的(0.45-μm)部位的AMD水(图2C),将恒化器反应器容器中的液体体积增加到2L。
  7. 恒化器然后在无流量,补料分批模式下操作4至6周。生物反应器的自动控制组件保持恒定的pH,温度和混合速度,并连续记录溶解氧。使用反应器中的pH计和两个递送0.1MH 2 SO 4或0.2N NaOH的蠕动泵之间的反馈控制来保持任何所需的pH设定点(图3)。使用反应器内的单程自来水进料冷却盘管和反应器周围的热套管来维持反应器温度。空气被连续地泵送到反应器容器的底部以确保无限的溶解氧。在浓缩期间,恒化器的pH设定点与现场水相同,搅拌速度为50rpm,温度为20℃,溶解氧不受限制(例如<! - SIPO

    图3.包括反馈系统,蠕动泵和搅拌器的生物反应器系统的照片

  8. 将硫酸亚铁(FeSO 4·7H 2 O)不连续地加入到反应器中作为主要底物以富集自养Fe(II)氧化微生物。加入硫酸亚铁,得到溶解的[Fe(II)] 300mg L 1,当溶解的[Fe(II)]降低到低于30mg L -1时, 。
  9. 在Fe(II)氧化和总Fe去除率达到假稳态条件(±5%标准偏差)之后,含有丰富的Fe(II) - 氧化微生物群落的生物膜明显均匀地附着在反应器壁上2C)。不到一个月,富集培养物需要每日剂量的Fe(II),因为微生物以快速的速率氧化Fe(II)。
  10. 抽样
    1. 使用无菌移液管在反应器容器内的液面顶部收集Fe(II)测量样品。用螺丝盖封闭的容器顶部的未使用的口可打开以取样。每次对Fe(II)和总Fe测量每次收集2ml水。用0.1N HCl(Stookey)保存的样品,使用铁氰化物测定(50mM HEPES [pH7.0]中的1g/L铁锌)测定溶解的Fe(II)和溶解的总Fe(T)(通过羟胺-HCl还原后) ,1970)。取样频率取决于Fe(II)氧化速率。生物反应器必须在头两个星期每天进行采样,而不是经常采样。
    2. 从反应器壁收集生物膜样品,并从悬浮液中收集水样品以确定生物质浓度。打开反应器容器的盖子,将水倒入无菌容器中。在使用无菌移液管从反应器壁上刮下生物膜后,将水倒入反应容器中,盖上盖子。基于蛋白质(使用考马斯亮蓝G-250染料的Bio-Rad蛋白质测定试剂盒)测定生物质浓度。将至少1cm 2的生物膜的区域从反应器的壁上刮下来用于蛋白质测定。标尺用作参考物质(图4)。刮刮区域的照片与Adobe ® Photoshop ®软件一起使用,以确定每次删除的确切区域。


      图4.使用标尺作为面积计算的参考物质的刮片生物膜的示例性照片

    3. 将收集的生物膜溶于3ml 10%(w/v)草酸中。一旦溶解,通过将1ml该混合物与2ml 0.2N NaOH一起溶解,然后在-20℃冷冻器中对样品进行两次冷冻 - 融化循环至少6小时和37℃水洗澡约10-15分钟。然后使用含裂解细胞的混合物进行蛋白质比色测定。
    4. 对于悬浮液,将135ml反应器液体离心(13,000×g 10分钟)以造粒细胞。测量用于离心的Falcon ®管的质量,以确定每个颗粒的质量。然后将液体倾析,细胞沉淀物与上述刮除细胞完全一致。

数据分析

  1. 假设恒化器作为完全混合的反应器运行,Fe(II)氧化[mol Fe(II)L -1 sec -1]的零级速率在恒电位计算如下:

    哪里,
    d [Fe(II)]降低溶解的Fe(II)浓度(mol L -1 ),
    dt是培养时间的增加
  2. 对于浓缩实验,该系统相对于Fe(II)氧化速率达到假稳态。为了将伪稳定状态下的某些测量点和其他测量点不相等,计算最后一次测量的正负5%标准偏差范围。任何落在正或负5%标准偏差范围内的任何点都被认为处于伪稳态。这个统计方法强调了最后一个测量点,因为所有其他点都与它进行比较。
  3. 为了确定悬浮液和生物膜中细胞的浓度,使用Bio-Rad蛋白测定试剂盒。一旦获得了蛋白质浓度,则使用衍生的0.15×10 12个蛋白质/典型细胞的转化因子将存在的蛋白质质量转换成细胞数量。

笔记

  1. 由于富含Fe(II)的氧化微生物群落受pH和Fe(II)浓度的影响很大(Sheng等人,2016),pH设定点和Fe(II)浓度恒温器可以根据现场AMD的pH和Fe(II)浓度进行调整。
  2. 收集沉淀物和水以进行微生物富集。沿着缺氧的富含Fe(II)的自流式AMD流动路径从池底部采集沉积物。水从无氧自流泉收集。为了开发浓缩培养物,将100克潮湿的沉淀物与1升0.1%(m/v)焦磷酸钠(用硫酸调节至天然AMD水的pH)混合。
  3. Chemostat生物反应器在现场AMD的pH值,温度20℃,搅拌速率50rpm和无限溶解氧(> 3mg/L)下操作4-6周。
  4. Ferrozine测定(1μg/L铁锰在50mM HEPES [pH7.0]中)用于测量Fe(II)氧化速率(Stookey,1970)。
  5. 将附生生长和悬浮的生物质溶解在10%(w/v)草酸中,与0.2N NaOH混合,然后进行两次冻融循环以裂解细胞。基于蛋白质(使用考马斯亮蓝色G-250染料的Bio-Rad蛋白质测定试剂盒)确定生物质浓度。

食谱

  1. Ferrozine测定溶液(pH 7.0)
    1克/升铁锌素 50 mM HEPES
  2. 嗜酸细菌的丰富
    将100克潮湿的沉淀物与1升0.1%(m/v)焦磷酸钠混合 用硫酸调节天然AMD水的pH值

致谢

该协议由Sheng等人改编。 (2016)。这项工作得到了美国中国学术委员会(以Y.S.)和阿巴拉契亚环境科学研究计划(ARIES)合作协议S11AC20005的地表采矿开垦和执法办公室的部分支持。 ARIES是弗吉尼亚理工学院的一个工业附属计划,由包括能源部门公司在内的成员支持。本文所表达的意见和建议只是作者的意见和建议,并不意味着ARIES的任何认可。

参考文献

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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Sheng, Y., Kaley, B. and Burgos, W. D. (2017). Enriching Acidophilic Fe(II)-oxidizing Bacteria in No-flow, Fed-batch Systems. Bio-protocol 7(3): e2130. DOI: 10.21769/BioProtoc.2130.
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