Microfluidic devices for high-throughput plant phenotyping and bioenergy harvesting from microbes and living plants

Microfluidics and micro/nanofabrication techniques provide powerful technological platforms to develop miniature bioassay devices for studying cellular and multicellular organisms. Microfluidic devices have many advantages over traditional counterparts, including good throughput due to parallel experiments, low infrastructural cost, fast reaction, reduced consumption of agent and reagent, and avoidance of contamination. This thesis is focused on the development of a microfluidic toolkit with several miniature devices to tackle important problems that the fields of plant phenotyping and bioenergy harvesting are facing. The ultimate goal of this research is to realize high-throughput screening methods for studying environment-genomics of plants through phenomics, and understanding microbial and plant metabolisms that contribute to harvesting bioenergy from microbes and living plants in different environments. First, we develop vertical microfluidic plant chips and miniature greenhouses for high throughput phenotyping of Arabidopsis plants. The vertical design allows for gravitropic growth of multiple plants and continuous monitoring of seed germination and plant development at both the whole-plant and cellular levels. An automatic seed trapping method is developed to facilitate seed loading process. Also, electrospun nanofibrous membranes are incorporated with a seed germination chip to obtain a set of incubation temperatures on the device. Furthermore, miniature greenhouses are designed to house the plant and seed chips and to flexibly change temperature and light conditions for high-throughput plant phenotyping on a multi-scale level. Second, to screen bacteria and mutants for elucidating mechanisms of electricity generation, we develop two types of miniature microbial fuel cells (µMFCs) using conductive poly(3,4-ethylenedioxythiophene) nanofibers and porous graphene foam (GF) as three-dimensional (3D) anode materials. It is demonstrated that in the nanofiber-based µMFC, the nanofibers are suitable for rapid electron transfer and Shewanella oneidensis can fully colonize the interior region of the nanofibers. The GF-based µMFC is featured with a porous anolyte chamber formed by embedding a GF anode inside a microchannel. The interconnected pores of the GF provide 3D scaffolds favorable for cell attachment, inoculation and colonization, and more importantly, allow flowing nutritional and bacterial media throughout the anode with minimal waste. Therefore, the nutrients in bio-convertible substrates can be efficiently used by microbes for sustainable production of electrons. Last, we develop a first miniature plant-MFC or µPMFC device as a technological interface to study bioenergy harvesting from microbes and living plants. A pilot research is conducted to create the µPMFC device by sandwiching a hydrophilic semi-permeable membrane between a µMFC and a plant growth chamber. Mass transport of carbon-containing organic exudates from the plant roots to the µMFC is quantified. This work represents an important step towards screening plants, microbes, and their mutants to maximize energy generation of PMFCs

High-throughput microfluidic assay devices for culturing of soybean and microalgae and microfluidic electrophoretic ion nutrient sensor

In the past decade, there are significant challenges in agriculture because of the rapidly growing global population. Meanwhile, microfluidic devices or lab-on-a-chip devices, which are a set of micro-structure etch or molded into glass, silicon wafer, PDMS, or other materials, have been rapidly developed to achieve features, such as mix, separate, sort, sense, and control biochemical environment. The advantages of microfluidic technologies include high-throughput, low cost, precision control, and highly sensitive. In particular, they have offered promising potential for applications in medical diagnosis, drug discovery, and gene sequencing. However, the potential of microfluidic technologies for application in agriculture is far from being developed. This thesis focuses on the application of microfluidic technologies in agriculture. In this thesis, three different types of microfluidic systems were developed to present three approaches in agriculture investigation. Firstly, this report a high throughput approach to build a steady-state discrete relative humidity gradient using a modified multi-well plate. The customized device was applied to generate a set of humidity conditions to study the plant-pathogen interaction for two types of soybean beans, Williams and Williams 82. Next, a microfluidic microalgal bioreactor is presented to culture and screen microalgae strains growth under a set of CO2 concentration conditions. C. reinhardtii strains CC620 were cultured and screened in the customized bioreactor to validate the workability of the system. Growth rates of the cultured strain cells were analyzed under different CO2 concentrations. In addition, a multi-well-plate-based microalgal bioreactor array was also developed to do long-term culturing and screening. This work showed a promising microfluidic bioreactor for in-line screening based on microalgal culture under different CO2 concentrations. Finally, this report presents a microchip sensor system for ions separation and detection basing electrophoresis. It is a system owning high potential in various ions concentration analysis with high specificity and sensitivity. In addition, a solution sampling system was developed to extract solution from the soil. All those presented technologies not only have advantages including high-throughput, low cost, and highly sensitive but also have good extensibility and robustness. With a simple modification, those technologies can be expanded to different application areas due to experimental purposes. Thus, those presented microfluidic technologies provide new approaches and powerful tools in agriculture investigation. Furthermore, they have great potential to accelerate the development of agriculture

Environments-by-Design: Developing new tools to bring “field conditions” to the laboratory

Maximizing agricultural yields is an urgent priority for our society as the world population will increase to 9 billion by 2050. Additionally, climate change could have detrimental effects on food supply and biomass production. Addressing these problems means considering the relationship the environment has on a plant’s development. The reality is though that there are few tools to plant scientists that can properly control and monitor the growing environment for plant. We believe that as engineers we can provide plant scientists with new tools which we call “environments by design”. These tools can control the environmental stimuli either abiotic (e.g. water availability, nutrient concentration, temperature, relative humidity) or biotic (e.g. microbes or other plants) delivered to plant in time and space. By controlling the environment, hypotheses of how genetic traits interact with environmental factors to yield the plant phenotypes can be addressed inside the laboratory

LEGO® bricks as building blocks for centimeter-scale biological environments

LEGO bricks are commercially available interlocking pieces of plastic that are conventionally used as toys. We describe their use to build engineered environments for cm-scale biological systems, in particular plant roots. Specifically, we take advantage of the unique modularity of these building blocks to create inexpensive, transparent, reconfigurable, and highly scalable environments for plant growth in which structural obstacles and chemical gradients can be precisely engineered to mimic soil

Encapsulation And Culture Of Plant Protoplasts In Hydrogel Microspheres Using Droplet Microfluidics

Plant morphogenesis requires the coordinated growth between cells and their neighbors. This coordination is regulated by internal and external signals that are both physical and chemical. It is understood that both mechanical and molecular signals are involved in the regulation of plant morphogenesis however, how mechanical signals induce molecular signals and vice versa is not. This is due to a lack of research tools for manipulating and measuring the mechanical environment of growing plant cells. Such tools would ideally provide individual plant cells with mechanically tunable physical microenvironments. This may allow changes in the cells physical environment to be linked to molecular changes such as gene expression or cellular architecture. The field of microfluidics has provided biology with a suite of tools for handling cells and tissues precisely at small scales. In this work, droplet microfluidics was used to encapsulate individual plant protoplasts in congealed agarose microspheres. Encapsulated protoplasts were viable and able to regenerate their cell wall and resume growth eventually breaking out of the microsphere. To manipulate the growth of the encapsulated protoplasts agarose microspheres were modified with the addition of pectin or polyelectrolyte coatings. Agarose microspheres were also modified by encapsulating them in a layer of alginate methacrylate creating a two-layer hydrogel microsphere. Alginate methacrylate is capable of dual crosslinking by either ionic or covalent bonding of the polymer chains. Viable protoplasts were successfully encapsulated in each of the described hydrogel microsphere environments. Cell expansion and the orientation of cortical microtubules was compared between cells growing in the agarose, agarose-pectin blend, and agarose-polyelectrolyte coated microspheres. Agarose-polyelectrolyte microspheres decreased anisotropic growth and disrupted the cortical microtubule orientation of encapsulated protoplasts. Alginate methacrylate was shown to be a suitable material for plant cell culture as well as hydrogel microsphere fabrication. Protoplast viability was confirmed within photo crosslinked alginate methacrylate blocks and viable protoplasts were encapsulated within agarosealginate methacrylate two-layer hydrogel microspheres. This work shows that agarose microsphere encapsulation of living plant protoplasts is feasible with high yield and viability using a small microfluidic system. Agarose microspheres provide delicate protoplasts with physical support and a protective layer that is suitable for cell culture. They also may serve as a template that can be modified to create cellular microenvironments tuned for specific applications. This methodology may contribute to studies in plant cell reprogramming and cell differentiation

Lab-On-Chip for Ex-Vivo study of morphogenesis of tip growing cells of pollen tube

The purpose of the thesis is to develop a microfluidic based lab-on-chip (LOC) platform providing an Ex-Vivotesting environment that is able to mimic certain aspects of the in vivo growth conditions of the pollen tube, a cellular protuberance formed by the male gametophyte in the flowering plants. The thesis focuses on design, fabrication, modeling and testing of various LOC devices for the study of static and dynamic behavior of pollen tubes in response to mechanical stimulation. TipChip, an LOC platform, was developed to advance both experimentation and phenotyping in cell tip growth research. The platform enabled simultaneous testing of multiple pollen tubes. Using TipChip, we were able to answer several outstanding questions regarding pollen tube biology. We found that contrary to other types of tip growing cells such as root hairs and fungal hyphae, pollen tubes do not have a directional memory. Furthermore, we explored the effect of geometry of the microfluidic cell culture on pollen tube growth. We found that changing the width of the microfluidic channels does not have a significant effect on the pollen tube growth rate, while the growth rate was increased by increasing microchannel depth. We modified the original TipChip design to ascertain identical growth conditions for sequentiallyarranged pollen tubes and to ensure even distribution of entrapment probabilities for all microchannels. The effect of different dimensions of the microfluidic network on cell trapping probability was assessed using computational fluid dynamics and verified by experimental testing. The design was optimized based on trapping probability and uniformity of fluid flow conditions within the microchannels. This thesis also presents a novel method of fabricating a high aspect ratio horizontal PDMS microcantilever-based flow sensor integrated into a microfluidic device. The performance of the flow sensor was tested by introducing various flow rates into the microfluidic device and measuring the deflection of the cantilever’s tip using an optical microscope. The thesis addresses the quantification of cellular growth force of Camellia pollen tip growing cells using FlexChip, a flexure integrated LOC on polymer. We quantified the force that pollen tube is able to exert using a microfluidic lab-on-a-chip device integrated with flexural structure. The pollen grain is trapped in the microfluidic network and the growing tube is guided against a flexible microstructure that is monolithically integrated within the microfluidic chip. The invasive growth force of growing pollen tube was calculated from the maximal bending of microstructure modelled by Finite Element Analysis (FEA). Furthermore, the effect of the mechanical obstacle on the pollen tube's growth dynamics was assessed by quantifying the shift in the peak frequency characterizing the oscillatory behavior of the pollen tube growth rate. Our detailed analysis of the pollen tube growth dynamic before and during the contact with microcantilever revealed that pollen tube growth rate was reduced by 44% during the contact with the microcantilever. Moreover, the peak of oscillation frequency of pollen tube growth rate was reduced more dramatically by 70-75%. This suggests that the pollen tube actively changes its growth pattern to cope with the mechanical obstacle. Our findings in this thesis are novel in terms of pollen biology, and we believe insights from this research will lead to a better understanding of morphogenesis of a kind of tip growing cells, namely, pollen tube

Towards In-situ Based Printed Sensor Systems for Real-Time Soil-Root Nutrient Monitoring and Prediction with Polynomial Regression

This dissertation explores how to increase sensor density in the agricultural framework using low-cost sensors, while also managing major bottlenecks preventing their full commercial adoption for agriculture, accuracy and drift. It also investigated whether low-cost biodegradable printed sensor sheets can result in improved stability, accuracy or drift for use in precision agriculture. In this dissertation, multiple electrode systems were investigated with much of the work focused on printed carbon graphene electrodes (with and without nanoparticles). The sensors were used in two configurations: 1) in varying soil to understand sensor degradation and the effect of environment on sensors, and 2) in plant pod systems to understand growth. It was established that 3) the sensor drift can be controlled and predicted 2) the fabricated low-cost sensors work as well as commercial sensors, and 3) these sensors were then successfully validated in the pod platform. A standardized testing system was developed to investigate soil physicochemical effects on the modified nutrient sensors through a series of controlled experiments. The construct was theoretically modeled and the sensor data was matched to the models. Supervised machine learning algorithms were used to predict sensor responses. Further models produced actionable insight which allowed us to identify a) the minimal amounts of irrigation required and b) optimal time after applying irrigation or rainfall event before achieving accurate sensor readings, both with respect to sensor depth placement within the soil matrix. The pore-scale behavior of solute transport through different depths within the sandy soil matrix was further simulated using COMSOL Multi-physics. This work leads to promising disposable printed systems for precision agriculture

Brachypodium distachyon seedling growth visualization under osmotic stress and overexpression of MIR7757 to increase drought tolerance

Brachypodium distachyon a monocot model plant has facilitated the downscaling for studying the most important cereal crops of the world both genetically and phenetically. This owes to its dwarf stature, small genome size and rapid life cycle which was utilized in our research for analysing its morphological features under osmotic stress. The purpose of this study was to visualize Brachypodium seedlings under osmotic pressure to observe morphological adaptation under drought-like conditions. It was found that Brachypodium displays the typical adaptive mechanisms of cereal plants mainly root apical meristem showing lateral hair growth and stunted growth. The root cells also displayed change in single cell morphology by swelling into compartment like structures as compared to non-stressed cells. This observation was made in the elongation and maturation zones of the root. Lateral hair growth was observed from the root apical meristem after 18 hours of PEG-mediated osmotic stress. Brachypodium not only manifests physiological adaptations to drought stress but also elicits molecular adaptation to counter it. To explore the genetic basis of drought tolerance the microRNAs involved in water deficit were traced out through a reverse genetics approach. The T-DNA mutant library of Brachypodium distachyon allowed for the investigation of a newly discovered microRNA miR7757 involved in water deficit to be overexpressed in Brachypodium to rapidly produce drought tolerant varieties bypassing conventional breeding techniques