Despite 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.
