Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 µm), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Assessing the Bacterial Ecology of Organohalide Respiration for the Design of Bioremediation Strategies

Noam Shani

Groundwater is essential for human activities and is sometimes referred to as a non-renewable resource in the same way as oil and gas. During the past century, precious groundwater reserves worldwide have been threatened by anthropogenic release of chemical compounds. Among these, chlorinated ethenes (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) belong to the most common class of groundwater contaminants. In oxic conditions, PCE and TCE are recalcitrant to any form of degradation and constitute a long-term source of groundwater contamination. It has been reported that CEs can be degraded biologically in anoxic conditions, serving as electron acceptors of an anaerobic respiratory process called organohalide respiration (OHR). In this process, PCE is sequentially reduced to TCE, cis-dichloroethene (cDCE), vinyl chloride (VC), and finally to harmless ethene (Eth). Engineering strategies based on reductive dechlorination, such as monitored natural attenuation (MNA), have been designed for the bioremediation of aquifers contaminated with CEs. However, stalling of the sequence of dechlorination has often been observed, resulting from incomplete or impeded biodegradation of the highly toxic daughter molecules (cDCE and VC) and leading to their accumulation in situ. It was shown in laboratory experiments that OHR of CEs is more efficient in mixed cultures and that organohalide-respiring bacteria (OHRB) live in association with other microorganisms in microbial consortia. In the aquifer ecosystem, OHRB rely on complex interactions between members of bacterial communities for their electron donors and carbon supplies. On the other hand, they are in competition for these resources with other terminal electron-accepting processes (TEAPs). These complex interactions together with the intrinsic structural heterogeneity of aquifers make it difficult to understand the reasons for lower CEs accumulation, to predict the fate of OHR, and to design bioremediation strategies. This thesis aimed at elucidating the reasons for lower CEs accumulation in situ. An ecological approach considering the aquifer ecosystem in its whole complexity was developed and applied to the specific cases of two contaminated sites showing accumulation of lower CEs. The proposed methodology relies on the precise depiction of both aquifer habitat and microbial communities interacting therein. However, the potential impact of the first technical step, namely the impact of the pumping parameters on groundwater samples used for the description of the bacterial communities, was not known at the start of this thesis work. Results of investigations addressing this topic showed that parameters related to the tubing characteristics did not impact the apparent bacterial community structures (BCS) in a laboratory experiment. However, the study revealed a significant impact of the pumping flow rate on apparent BCS extracted from the aquifer. Terminal-restriction fragment length polymorphism (T-RFLP), coupled with an appropriate groundwater sampling strategy, enabled an accurate profiling of the BCS. However no indication was provided concerning the identities of the community members. A bioinformatics tool called PyroTRF-ID was developed to overcome this obstacle. The software enabled affiliating terminal-restriction fragments (T-RFs) to precise phylotypes by coupling T-RFLP and pyrosequencing data. An additional function enables in silico assessment of restriction enzymes for designing appropriate T-RFLP procedures. The developed methodology was applied all along the thesis work. Optimized tools and procedures were used to investigate the reasons for the accumulation of VC, and to a lesser extent cDCE, in the first aquifer showing relatively homogeneous lithological composition and slow groundwater fluxes. Statistical findings indicated that VC reduction was outcompeted by Fe(III) reduction in some sections of the aquifer. T-RFs showing significant correlations with VC reduction variables were identified by sequencing and with PyroTRF-ID. Results showed sequences closely affiliated to uncultured bacteria of the "Lahn Cluster" (LC) within the class Dehalococcoidetes, previously reported as PCE-to-cDCE dechlorinating microorganisms, and to the genus "Dehalococcoides". According to present knowledge, only members of this genus are able to reduce cDCE and VC. A major T-RF negatively correlated with the LC T-RF affiliated to the genus Rhodoferax, containing iron-reducing bacteria (IRB). Furthermore, indications were obtained of a mutualistic interaction between IRB and sulfate-reducing bacteria, potentially playing an important role by reducing the Fe(III) contents locally. The importance of the aquifer hydrogeological structure and functioning was exemplified in the study of the second aquifer ecosystem, where discrepancies in the fate of OHR were observed. In the immediate vicinity of the source zone, complete OHR was occurring, with production of Eth, whereas cDCE was accumulating further downstream, in apparent similar redox conditions. The profiling and metagenomic techniques revealed drastically different bacterial communities in the zones displaying different OHR fate. The source zone displayed bacterial populations typically found in highly reduced environments and involved in OHR, such as Dehalococcoides sp. In contrast, the downstream zone showing an accumulation of cDCE displayed bacterial populations typically found in more oxidized environments, such as aerobic bacteria, nitrifiers, and denitrifiers. In this zone, the conditions were probably slightly oxidized periodically by the combination of aquifer recharge, a specific lithological configuration, and lower organic matter content. Finally, and based on these considerations, a methodology for the investigation of the reasons for lower CEs accumulation in contaminated aquifers is proposed and discussed. This methodology follows on a multidisciplinary ecological approach for the description of the functioning of the aquifer ecosystem, and enables formulating scenarii of the reasons for lower CEs accumulation.

EPFL2012

The Phosphate Specific Transport (Pst) System in Fast- and Slow-Growing Mycobacteria

Cyntia De Piano

Although mycobacteria are known to accumulate polyphosphate (PolyP), a linear polymer of orthophosphate, when subjected to stresses, the exact roles of this molecule are not yet clearly understood. We found that deletion of the pstA gene encoding for a transmembrane domain of the high-affinity phosphate-specific ABC transporter system (Pst system) resulted in accumulation of high amounts of PolyP during growth in phosphate (Pi)-replete conditions in Mycobacterium tuberculosis and Mycobacterium smegmatis. These PolyP stores were gradually used during Pi starvation by the ApstA mutants in order to sustain growth and this allowed the mutant strains to reach a higher cell density than the wild-type (WT) parental strain. Neither the mRNA nor the protein levels of polyphosphate kinase 1 (PPK1) could explain the PolyP accumulation observed in the mutant, suggesting that another regulatory mechanism is involved. The ApstA mutant of M. smegmatis was hypersensitive to several in vitro stresses, including detergent, oxidative stress, and cell wall targeting antibiotics. Deletion of the main enzyme involved in PolyP synthesis, PPK1, in the ApstA mutant background of M. smegmatis completely reversed the PolyP accumulation and partially reversed the stress sensitivity as well as the derepression of PHO regulon genes observed in the ApstA mutant. The mechanism of PolyP accumulation in M. smegmatis was investigated using a genetic approach. We found that this mechanism differs from the (p)ppGpp-mediated PolyP accumulation observed in Escherichia coli, since deletion of the RelA enzyme in a ApstA mutant background did not prevent PolyP accumulation. The mechanism of PolyP degradation during Pi starvation in the M. smegmatis ApstA mutant was also analyzed. Deletion of both polyphosphatase enzymes (PPX1 and PPX2) as well as the polyphosphate kinase 2 (PPK2) enzyme did not have any impact on PolyP degradation. PPK1 is a bifunctional enzyme that can either synthesize or degrade PolyP. Our current hypothesis is that PPK1 might perform the reverse reaction (PolyP degradation) during Pi starvation. The aim of the second part of my project was to make a comparative study of the expression and regulation of the phosphate specific transporter (pst) operon in fast-growing [M. smegmatis] and slow-growing [M. tuberculosis] mycobacteria. To achieve this goal, pst reporter strains of M. smegmatis and M. tuberculosis were constructed by precise insertion of a modified dest_gfp gene (provided by Giulia Manina) encoding a destabilized green fluorescent protein (dest_GFP) into the corresponding pst locus as transcriptional ) fusions. The transcriptional reporter construct [p) was introduced in both the WT and the ApstA mutant backgrounds. The kinetics of induction of the pst-gfp reporters in WT and ApstA mutant strains in response to Pi starvation was analyzed. Population-averaged measurements were made using qRT-PCR and Western blotting. Single-cell measurements using flow cytometry and timelapse fluorescence microscopy were also performed. For the microscopy experiments, the bacteria were cultured in custom-made microfluidic devices that permit rapid switching of the culture medium, e.g., switching between Pi-replete and Pi-depleted media. Because these studies require precise environmental control and resolution of individual and temporal phenotypes, my principal experimental tool was the integrated microfluidics and timelapse fluorescence microscopy systems that have recently been developed in the laboratory. Although there have been many studies published on the population-averaged behavior of bacteria responding to external stresses such as Pi limitation, we are not aware of any studies addressing the individuality of such responses using a real-time single-cell approach. We observed that the kinetics of induction of the Pst operon upon Pi starvation scaled with the growth rate of the bacteria and peak induction occurred within about two generations for both smegmatis and tuberculosis. Interestingly, we found that the basal level of Pst expression and the steady state level reached upon induction by Pi starvation were similar in tuberculosis and M. smegmatis, although the kinetics of induction were much faster in M. smegmatis. These studies contributed to improve the understanding of the regulation and the kinetics of response of the PHO regulon in fast- and slow-growing mycobacteria. The finding that PolyP plays a role in this process could also apply to other organisms where the PHO regulon is regulated in a similar way.

EPFL2013

Utilization of silicate minerals for pH control during in situ bioremediation of chlorinated solvent

David Andrew Barry, Alessandro Brovelli, Christof Holliger, Elsa Marina Lacroix

Chloroethenes such as tetrachloroethene (PCE) and trichloroethene (TCE) are among the most prevalent contaminants in groundwater due to their extensive use in industrial processes. In situ bioremediation (ISB) is an attractive technology for removal of these compounds. It relies on an anaerobic process in which specialized bacteria obtain energy for growth using chloroethenes as an electron acceptor via organohalide respiration. Engineered bioremediation is achieved by stimulating these microorganisms through the addition of electron donor in the subsurface. This technology has been widely used for bioremediation of chloroethene plumes and recent studies have indicated promising results for bioremediation of chlorinated solvent source zones. However, application of source zone ISB is still a significant technical challenge. One of the main issues is the groundwater acidification due to organohalide respiration and fermentation processes, which can inhibit the activity of dehalogenating micro-organisms. The main objective of this work was to develop an efficient pH control strategy for chloroethene ISB by using the acid neutralizing potential of silicate minerals. To do so, modeling and experimental approaches were combined. A geochemical model, implemented within PHREEQC, was developed to select appropriate buffer candidates and to help determine main parameters influencing mineral buffering capacity. The model included chloroethene microbial degradation kinetics, mineral dissolution kinetics and chemical speciation. Second, anaerobic microcosm experiments were performed to determine the influence of pH on dehalogenation. These microcosms were inoculated with enriched consortia of dehalogenating bacteria and fed with PCE and hydrogen. Another set of microcosm experiments was carried out to compare the buffering capacity of ten silicate minerals and to investigate interactions between minerals and dehalogenating bacteria, e.g., the potential inhibitory effect of minerals on the dehalogenating activity. These microcosms were amended with 5 mmol l-1 of PCE and 4 g l-1 of mineral with grain sizes between 50 and 100 μm. The cultivation medium was modified such that the silicate mineral powder was the sole pH buffer present. Chloroethenes, pH and dissolved cation measurements were conducted to determine the system efficiency. Abiotic dissolution experiments were also performed to determine mineral dissolution rates in the absence of bacteria. The model confirmed that the efficiency of the system is dependent mainly on mineral dissolution kinetic constants, equilibrium constants and reactive surface area. The geochemical model and literature parameter data were used to pre-select minerals with a buffering capacity sufficient to counterbalance acidity produced by dehalogenating bacteria at a rate of 4 mmol l-1.d-1of chloride. Of the 31 silicate minerals for which there were published kinetic data, 10 were identified as suitable candidates. The inhibitory pH for the dehalogenating consortia was found to vary between 5 and 6. The last steps of the dechlorination from DCE to ethene were more sensitive to pH than the first steps from PCE to DCE, as has been noted in other dechlorination studies. Results of microcosm experiments with silicate minerals demonstrated that, under the selected conditions, the pH control behavior and the impact on bacterial activity exhibited strong variations depending on the mineral. Of the ten minerals tested experimentally, three (olivine, fayalite and diopside) maintained the pH in the appropriate range, i.e., between 5.5 and 6.5 and led to complete transformation of PCE. For the other minerals tested, either the acid neutralization capacity was insufficient due to slow dissolution kinetics (glaucophane and staurolite) or dechlorination activity was inhibited by (unmeasured) compounds released during mineral dissolution. Both modeling and experimental results demonstrated the feasibility of using selected silicate minerals as a buffering agent during ISB. However, the experimental results also revealed a mineral-induced potential inhibitory effect that should be investigated prior to application at a contaminated site.

2011