Diffusional Constraints Affecting Microbial Distribution and Activity in Unsaturated Porous Media

影响不饱和多孔介质中微生物分布和活动的扩散约束

• 批准号: 0409364
• 负责人: Thomas Torgersen
• 金额: --
• 依托单位: University of Connecticut
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0409364&HistoricalAwards=false
• 关键词: Diffusional; Constraints; Affecting; Microbial; Distribution

0409364ORDespite remarkable progress in modeling of physical processes in unsaturated porous media, the role of microbial processes affecting media properties and regulating fluxes of biogenic and anthropogenic contaminants has largely been ignored by hydrologists. Advances in pore-scale characterization of fluid behavior in unsaturated porous media, and concurrent advances in experimental methods to study microbial behavior at individual cell resolution in situ, offer new opportunities for mechanistic studies of microbial abundance and activity in natural environments. Variations in liquid organization during desaturation of porous media result in confinement and fragmentation of aquatic habitats and alter substrate and gaseous diffusion pathways. These abiotic changes trigger an array of biological responses including enhanced production of extracellular polymeric substances postulated to serve as a protective matrix for embedded bacterial cells. A reduction in contiguous aquatic pathways limits microbial mobility, reduces substrate diffusion, enhances gaseous exchange with atmosphere, and contributes to the high degree of soil microbial diversity observed in unsaturated soils. It has been estimated that globally, the soil contains approximately 2.6 1029 prokaryotic cells (compared to 1.2 1029 in all oceans) concentrated in a relatively small volume on the earth skin making the vadose zone the richest compartment of prokaryal life on earth [Whitman et al., 1998]. Observations in natural habitats have established that bacteria exist primarily attached to solid surfaces as part of colonies or biofilm structures and are not planktonic. In contrast to the wealth of structural, physiological, and genetic details on microbial biofilms in saturated aquatic systems, very little is known on spatial structure and properties of biofilms in unsaturated soils, primarily due to the absence of well-authenticated model systems and of suitable observational techniques. We propose a conceptual framework supported by experiments for quantifying the impact of wetting-drying cycles on microbial activity in unsaturated porous media. An experimental 2-D porous surface model (PSM) containing prescribed geometrical features will serve as observable and tractable analogue of natural 3-D pore spaces. The PSM builds on recent modeling and experimental results regarding liquid behavior in angular pores and on rough surfaces. We plan a series of experiments to manipulate diffusional conditions within the PSM and apply modern microbiological methods to observe, analyze and quantify microbial response. Pseudomonas putida strains KT2440 and PaW85, expressing genes encoding fluorescent proteins under control of constitutive promoters will serve as model bacteria. Microscale estimates of microbial distribution, activity, and structural features will be revealed and quantified via confocal scanning laser microscopy. The proposed porous surface model enables testing of the roles of rough vs. smooth surfaces as microhabitats under variable wetting-drying conditions. It further allows control of the geometry and rate of nutrient supply ranging from point, to line, and planar sources; and provides direct observability of biological activity on well-defined porous surfaces. The proposed research will contribute to (1) elucidation of pore-scale interactions between diffusion processes and microbial activity, distribution, and coexistence; (2) development of new experimental methods to study biological processes in the vadose zone; (3) improved understanding of microbial response to environmental cycles; and (4) provide new insights into the origins of the unparalleled biodiversity found in soils. (2) Refinements in Response to Key 5ers Suggestions:Since the submission of the proposal we have improved our understanding of some of the factors discussed in the proposal, yielding a considerably improved modeling framework and more "mature" experimental setups. 1. Assess potential problems with the use of glass plates - the proposed unsaturated "Petri dish"is designed around porous ceramics such as Kaolinite, hence closely resembling the makeup of soil mineral surfaces. Issues of microbial adhesion and potential for stripping of microbial colonies from glass plates will be addressed by implementing rigorous experimental protocols for surface pre-treatment and retrieval procedures. The examination of different mineral surfaces is beyond the scope of the proposed research. Moreover, we believe that EPS plays a key role in modifying most primary surfaces for microbial adhesion and function. In other words, the role of EPS as a sticky anchoring and shielding substrate limits direct contact between microbes and mineral surfaces. 2. Treatment the bacterial cells and colonies as additional roughness and extend the coverage - We plan to explicitly consider the role of EPS in modifying micro-hydrology and micro-habitats, because bacteria must have an inevitable reliance on biopolymers to extend hydrated states beyond those supported by pore sizes and roughness. This inclusion will require a special effort towards hydraulic characterization of EPS properties and extension of the conceptual model to consider EPS role on soil and rock pore space. 3. Give more attention to the role of pore space patterns on microbial growth - the role of pore space patterns on microbial growth and shaping of diffusion pathways will be expanded and tested numerically as well as experimentally. We believe the intricate geometry plays a critical role in supporting coexistence of microbial species that would not coexist under homogeneous conditions (Dens, E. J., and Van Impe, J. F., 2000. On the importance of taking space into account when modeling microbial competition in structured food products. Mathematics and Computers in Simulation 53, 443-448).4. Pursue insights into physical properties affecting microbial diversity in natural environments - we plan to pursue this point as stated in items #3 above. We have conducted preliminary numerical experiments that demonstrate that heterogeneity of diffusional pathways shields less competitive microbial species and may, in part, explain the unparalleled microbial diversity found in soils. Curtis et al.(2002) estimate prokaryotic diversity at different scales by using the total number of individuals in the community as well as the abundance of the most thriving member. They estimate between 6000 and 38000 different coexisting species in a gram of soil. In the proposed research we will attempt to develop estimates of the numbers of different micro-niches carved out by spatial limitations to diffusion and mobility, heterogeneity and distribution of substrates, and temporal variations in external conditions (water content, temperature). These are likely to provide lower bounds on diversity estimates because they will not consider synergistic microbial associations and manipulation and local modifications of micro-environments through targeted EPS production. 5. Plan ahead to face the many difficulties in excluding unwanted bacteria - Although the PSM models will be subject to repeated microscopic inspections, external contamination will be minimized by setting up the entire assembly (PSM, pumps, liquids, and microscope) in a laminar flow bench (which will provide positive displacement of HEPA-filtered air and minimize ambient air intrusion). Further, external contamination can be checked by staining the PSM with a generic cytological stain (e.g., SYTO-9, DAPI) to detect unwanted bacteria. Finally, both test strains (KT2440 and PaW85 carry antibiotic resistances as part of their chromosomal biomarker modules, enabling us to supplement feed media to further prevent external contamination of the PSMs.

尽管在非饱和多孔介质中物理过程的建模方面取得了显著进展，但微生物过程影响介质性质和调节生物源和人为污染物通量的作用在很大程度上被水文学家所忽视。非饱和多孔介质中流体行为的孔隙尺度表征的进展，以及在单个细胞分辨率下原位研究微生物行为的实验方法的同步进展，为自然环境中微生物丰度和活性的机理研究提供了新的机会。在多孔介质的去饱和过程中，液体组织的变化会导致水生栖息地的限制和破碎，并改变基质和气体扩散途径。这些非生物的变化引发了一系列的生物反应，包括细胞外聚合物物质的增加，这些物质被认为是嵌入细菌细胞的保护基质。连续水生途径的减少限制了微生物的流动性，减少了基质的扩散，增强了气体与大气的交换，并有助于在不饱和土壤中观察到高度的土壤微生物多样性。据估计，在全球范围内，土壤中含有大约2.6 1029个原核细胞（相比之下，所有海洋中含有1.2 1029个原核细胞），这些细胞集中在地球表面相对较小的体积中，使气包带成为地球上原核生命最丰富的区域[Whitman等，1998]。对自然栖息地的观察已经证实，细菌主要作为菌落或生物膜结构的一部分附着在固体表面，而不是浮游的。与饱和水系统中微生物生物膜的丰富的结构、生理和遗传细节相比，对非饱和土壤中生物膜的空间结构和特性知之甚少，这主要是由于缺乏经过良好验证的模型系统和合适的观测技术。我们提出了一个由实验支持的概念框架，用于量化干湿循环对非饱和多孔介质中微生物活性的影响。包含规定几何特征的实验二维多孔表面模型（PSM）将作为自然三维孔隙空间的可观察和可处理的模拟。PSM建立在最近的建模和实验结果关于液体行为在角孔和粗糙表面。我们计划进行一系列实验来控制PSM内的扩散条件，并应用现代微生物学方法来观察、分析和量化微生物的反应。恶臭假单胞菌菌株KT2440和PaW85在组成启动子控制下表达编码荧光蛋白的基因，将作为模式菌。微生物分布、活动和结构特征的微尺度估计将通过共聚焦扫描激光显微镜揭示和量化。所提出的多孔表面模型可以测试在可变干湿条件下粗糙与光滑表面作为微栖息地的作用。它进一步允许控制从点到线和面源的营养供应的几何形状和速率；并提供生物活性在明确的多孔表面上的直接可观察性。该研究将有助于(1)阐明扩散过程与微生物活性、分布和共存之间的孔隙尺度相互作用；(2)开发新的实验方法来研究气膜带的生物过程；(3)提高了对微生物对环境循环反应的认识；(4)为土壤中无与伦比的生物多样性的起源提供新的见解。(2)对关键建议的改进：自提交提案以来，我们提高了对提案中讨论的一些因素的理解，产生了相当改进的建模框架和更“成熟”的实验设置。1. 评估使用玻璃板的潜在问题——拟议的不饱和“培养皿”是围绕多孔陶瓷（如高岭石）设计的，因此与土壤矿物表面的构成非常相似。微生物粘附问题和从玻璃板上剥离微生物菌落的潜力将通过实施严格的表面预处理和回收程序的实验协议来解决。对不同矿物表面的检查超出了本研究的范围。此外，我们认为EPS在修饰微生物粘附和功能的大多数初级表面方面起着关键作用。换句话说，EPS作为粘性锚定和屏蔽底物的作用限制了微生物和矿物表面之间的直接接触。2. 将细菌细胞和菌落处理为额外的粗糙度并扩大覆盖范围-我们计划明确考虑EPS在改变微水文和微栖息地中的作用，因为细菌必须不可避免地依赖生物聚合物来扩展孔隙大小和粗糙度所支持的水合状态。这将需要对EPS特性的水力表征进行特别的努力，并扩展EPS在土壤和岩石孔隙空间中的作用的概念模型。3. 更多地关注孔隙空间模式对微生物生长的作用——孔隙空间模式对微生物生长和扩散路径形成的作用将得到扩展和数值测试以及实验。我们认为，复杂的几何结构在支持微生物物种共存方面起着关键作用，而这些微生物物种在均匀条件下是无法共存的（Dens， E. J., and Van Impe， J. F., 2000）。在对结构化食品中的微生物竞争进行建模时考虑空间因素的重要性。3 .数学与计算机仿真[j]。深入了解自然环境中影响微生物多样性的物理特性——我们计划按照上面第3项的说明来研究这一点。我们已经进行了初步的数值实验，证明扩散途径的异质性屏蔽了竞争较少的微生物物种，并且可能部分解释了土壤中发现的无与伦比的微生物多样性。Curtis et al.（2002）通过使用群落中个体总数以及最活跃成员的丰度来估计不同尺度下的原核生物多样性。他们估计在一克土壤中有6000到38000种不同的共存物种。在拟议的研究中，我们将尝试根据扩散和迁移的空间限制、基质的异质性和分布以及外部条件（含水量、温度）的时间变化，对不同微生态位的数量进行估计。这些方法可能会提供多样性估计值的下限，因为它们没有考虑微生物的协同关联、操纵以及通过定向EPS生产对微环境的局部修改。5. 尽管PSM模型将受到反复的显微镜检查，但通过将整个组件（PSM，泵，液体和显微镜）设置在层流工作台（这将提供hepa过滤空气的正置换并最大限度地减少环境空气侵入），外部污染将最小化。此外，可以通过使用通用细胞学染色剂（例如SYTO-9， DAPI）染色PSM来检查外部污染，以检测不需要的细菌。最后，两种测试菌株（KT2440和PaW85）都携带抗生素耐药性，作为其染色体生物标志物模块的一部分，使我们能够补充饲料培养基，进一步防止psm的外部污染。