(3w) Rapid Detection and Parallel Screening of Optimized Microbial Communities Using Microwell Recovery Arrays

As a postdoc, I intend to explore plant/organ on a chip systems to rapidly quantify community assembly in a single cell resolution. In addition, I want to develop spatial structures to mimic tumor microenvironments and develop probiotic and biocontrol agents to inhibit the growth of tumors in mammalian cells. I also aim to implement my inter-disciplinary skills in micro and nano-fabrication techniques to investigate diverse applications of microfluidic systems to study contact mediated interactions within a microbiome, drug discovery and drug design.

Teaching interests:

I am interested in teaching undergraduate-level courses of fluid dynamics, transport phenomenon and numerical analysis for chemical engineers as well as graduate-level special topic courses in biotechnology and waste-water treatment. I want to extend my expertise in statistical analysis to develop a graduate level course on the application and concepts of statistical analysis in chemical engineering.

Key Words: Microwell Recovery Arrays, Microbial Interactions

PhD Dissertation: Screening and Discovery of Symbiotic and Antagonistic Microbial Networks Using Extractable Microwell Arrays

Research Experience:

My PhD research was focused on the implementation of microwell recovery arrays (MRA) for simultaneous detection and screening of positive and negative interactions among soil and root microbiomes for the development of improved probiotic and biocontrol agents for plant growth. I have stochastically combined a fluorescently labeled focal species with a controlled number of microbiome isolates and identified those that influence focal species function with off-chip validation. The following projects were completed under the supervision of Dr. Ryan Hansen, Tim Taylor Department of Chemical Engineering, Kansas State University, USA.

(i) Development of Photodegradable membrane for selective extraction of microbes

Despite successful implementation of microwell arrays for understanding microbiome, they are widely limited to on-chip fluorescence-based measurements. Therefore, I collaborated in a project to outline a novel cell retrieval approach by attaching a semipermeable, photodegradable poly(ethylene glycol) (PEG) hydrogel with nitrobenzyl group as a photodegradable membrane on microfabricated silicon microwell recovery arrays (MRA) seeded with motile bacterium Agrobacterium tumefaciens, the causative agent of crown gall disease in plants. This material-based approach affords a high degree spatial control over bacteria retrieval and can be adapted to other applications where screening or studying cell−cell interactions is important (1).

(ii) Exploiting stochastic cellular processes of a model system to generate outlier communities with rare phenotypes in microwell arrays

I successfully demonstrated the proof of concept of microwell arrays to detect rare phenotypes / rare growth outcomes by stochastically confining bulk co-culture of two fluorescently labeled species: Pseudomonas aeruginosa (PAO1-mCherry), a human pathogen and Agrobacterium tumefaciens (C58-GFP), together in MRAs to investigate quorum mediated interactions to prevent A.tumefaciens growth. (2). With the aid of time-lapse fluorescent microscopy (TLFM), I demonstrated that in the majority of the wells, PAO1 suppressed the growth of C58, consistent with the claim that PAO1 can act as an antagonist against C58 in bulk co-culture. I could also identify rare outliers where C58 showed more enhanced growth than PAO1 with an inoculum ratio favorable for C58 growth.

(iii) Application of Microwell Arrays to Enhance Biofuel Production

I extended microwell platform capabilities to screening non-model test species Pantoea sp. YR343-GFP, a plant growth promoting bacteria against unknown isolates from the rhizosphere of P. trichocarpa, a promising biofuel feedstock. I trapped motile bacteria cells from the bulk co-cultures of YR343-GFP and P. trichocarpa root microbiome in microwell arrays using the crosslinked, photo-degradable PEG hydrogel membrane and detected the YR343-GFP growth promoter/antagonist outliers from the fluorescent images. Then we extracted and validated a five member growth promoting consortia and a four member antagonist consortia extracted from Populus trichocarpa root microbiome to enhance and antagonize the growth of Pantoea sp. YR343 and identified them with 16s rRNA sequencing (2).

Future Direction:

Currently I am working on directed evolution of Populus trichocarpa rhizobacteria to arrive at optimal combinations of bacteria that elicit a desired phenotype. My other goal is screening fluorescently labeled pathogenic A. tumefaciens SP.15955 against hundreds of non-pathogenic Agrobacterium isolates collected from Kansas native plant roots to discover multi-membered Agrobacterium consortia capable of suppressing the establishment of this pathogen. In our primary data we discovered few possible A.tumefaciens sp. 15955 growth inhibiting isolates. Discovery of such growth inhibiting isolates will help improve plant productivity by using them as biofertilizers to prevent Crown Gall disease caused by A.tumefaciens sp. 15955. I want to further extend my expertise in microwell arrays to improve the survival and colonization of a commercial nitrogen-fixing PGPB, Azospirillum brasilense, into maize roots to improve crop yield.

Selected publications

1. Van Der Vlies AJ, Barua N, Nieves-Otero PA, Platt TG, Hansen RR. On Demand Release and Retrieval of Bacteria from Microwell Arrays Using Photodegradable Hydrogel Membranes. ACS Appl Bio Mater [Internet]. 2019 Sep 11;2(1):266–76. Available from: http://pubs.acs.org/doi/10.1021/acsabm.8b00592
2. Barua N, Herken AM, Stern KR, Reese S, Powers RL, Morrell-Falvey JL, et al. Simultaneous Discovery of Positive and Negative Interactions Among Root Microbiome Bacteria Using Microwell Recovery Arrays. bioRxiv [Internet]. 2020;2020.01.03.894477. Available from: https://www.biorxiv.org/content/10.1101/2020.01.03.894477v1
3. Dodds WK, Zeglin LH, Ramos RJ, Platt TG, Pandey A, Michaels T, et al. Connections and Feedback: Aquatic, Plant, and Soil Microbiomes in Heterogeneous and Changing Environments. Bioscience. 2020;XX(X):1–15.
4. Masigol M, Fattahi N, Barua N, Lokitz BS, Retterer ST, Platt TG, et al. Identification of Critical Surface Parameters Driving Lectin-Mediated Capture of Bacteria from Solution. Biomacromolecules [Internet]. 2019 Jul 8;20(7):2852–63. Available from: https://doi.org/10.1021/acs.biomac.9b00609
5. Masigol M, Barua N, Lokitz BS, Hansen RR. Fabricating Reactive Surfaces with Brush-like and Crosslinked Films of Azlactone-Functionalized Block Co-Polymers. J Vis Exp [Internet]. 2018 Sep 11;2018(136):1–10. Available from: https://www.jove.com/video/57562/fabricating-reactive-surfaces-with-brus...
6. Masigol M, Barua N, Retterer ST, Lokitz BS, Hansen RR. Chemical copatterning strategies using azlactone-based block copolymers. J Vac Sci Technol B, Nanotechnol Microelectron Mater Process Meas Phenom [Internet]. 2017 Nov 11 [cited 2019 Sep 11];35(6):06GJ01. Available from: http://avs.scitation.org/doi/10.1116/1.4991881