Introduction: In recent years, in the recycling and treatment of scrap metal,biologyDegradation methods have not been effectively developed and applied. Although biodegradation is a generally recognized high-efficiency and environmentally friendly treatment method, the uncontrollability and uncertainty of engineering-grade microbial bacteria have brought great problems to its application, which is also an urgent problem in the current scientific community. .
On October 17, 2021, scientists from Lawrence Livermore National Laboratory (LLNL) developed a controlled mode3D printingThe new method of living microorganisms has expanded the potential of using engineered bacteria to recover rare earth metals, clean wastewater, and detect uranium.
LLNL’s light-curing biodegradable metal method
By using light and bacteria-infused resin to produce 3D patterned microorganisms, the research team at Lawrence Livermore National Laboratory successfully printed a thin layer of artificial biological membrane similar to the survival of microbial communities ubiquitous in the real world. The researchers suspended bacteria in a photosensitive bioresin and used LED light from the Stereolithography Equipment (SLAM) 3D printer developed by LLNL for microbial bioprinting to “capture” the microbes in the 3D structure. The projection stereolithography machine can print at a high resolution of 18 microns-almost as thin as the diameter of a human cell.
Image source: Illustration by Thomas Reason/LLNL
This paper was published online in the journal Nano Letters, and the researchers proved that the technology can be effectively used to design microbial communities with structural definitions. They demonstrated the applicability of this 3D printed biofilm in uranium biosensing and rare earth biomining applications, and showed how geometry affects the performance of printed materials.
Figure 1. Demonstration of stereolithography equipment for microbial (SLAM) bioprinting. Photo of Bio-PμSL printer used for SLAM printing (A). Schematic diagram of SLAM bioprinting process (B). Under a 2x magnifying glass (C) or a 10x magnifying glass (D), encapsulated E. coli expressing GFP (green) was used to demonstrate the engineering biofilm.
William Rick Hynes, lead researcher and LLNL bioengineer, said: “We are working hard to promote the development of 3D microbial culture technology, and believe that this is an area that has not been fully studied, and its importance has not been well understood. We are working hard to develop related With tools and techniques, researchers can use them to better study the behavior of microorganisms under geometrically complex but highly controlled conditions. In the future, by exploring and improving application methods, we can better control the 3D structure of microbial populations, It will be able to directly affect the interaction between them and improve the system performance in the biological manufacturing process.”
Although it may seem simple, Hynes explained that microbial behavior is actually extremely complex and driven by the temporal and spatial characteristics of its environment, including the geometric organization of the members of the microbial community. He also said that the way microbes are organized affects a range of behaviors, such as how and when they grow, what they eat, how they cooperate, how they protect themselves from competitors, and the molecules they produce.
Hines explained that the previous traditional methods of preparing biofilms in the laboratory have made it almost impossible for scientists to control the microbial tissues in the membranes, thus limiting the ability to fully understand the complex interactions of bacterial communities in nature. The ability to bioprint microorganisms in 3D will enable LLNL scientists to better observe the function of bacteria in their natural habitat and study technologies such as microbial electrosynthesis. In microbial electrosynthesis technology, those bacteria that “eat electrons” (electrotrophic bacteria) can convert excess electricity into the production of biofuels and biochemicals during off-peak hours.
Hynes added that at present, microbial electrosynthesis is limited because the interface between the electrode (usually a wire or 2D plane) and the bacteria is inefficient. By 3D printing microorganisms in devices combined with conductive materials, engineers should obtain a highly conductive biomaterial with a greatly expanded and enhanced electrode-microbe interface, resulting in a more efficient electrosynthesis system.
Biofilms are receiving more and more attention from the industry. They are used to repair hydrocarbons, recover key metals, remove barnacles from ships, and serve as biosensors for various natural and man-made chemicals. Based on LLNL’s synthetic biology capabilities, LLNL researchers have explored the influence of bioprinting geometry on microbial functions in the latest paper. Among them, C. crescentus can be genetically modified to extract rare earth metals and detect uranium deposits.
In a set of experiments, the researchers compared the recovery rates of rare earth metals in different bioprinted patterns. The results show that the cells printed in the 3D grid can absorb metal ions faster than traditional bulk hydrogels. The team also printed a live uranium sensor and observed an increase in the fluorescence of the engineered bacteria compared to the control printing.
Co-author and LLNL microbiologist Yongqin Jiao said: “The development of these effective biomaterials with enhanced microbial function and mass transfer characteristics is of great significance for many biological applications. The new bioprinting platform not only improves system performance and Scalability, and maintains cell viability and achieves long-term storage.”
LLNL researchers are continuing to work on the development of more complex 3D grid structures and the creation of new bio-resins with better printing and biological properties. They are evaluating conductive materials, such as carbon nanotubes and hydrogels, to transport electrons and feed bioprinted electrotrophic bacteria to improve the production efficiency of microbial electrosynthesis applications. The team is also determining how to best optimize the geometry of bioprinting electrodes to maximize the delivery of nutrients and products through the system.
LLNL bioengineer and co-author Monica Moya said: “We are just beginning to understand how structures control microbial behavior. This technology is a step in this direction. Manipulating microbes and their physical and chemical environments to achieve more complex functions has A wide range of applications, including biomanufacturing, repair, biosensing/detection, and even the development of engineered biomaterials-these materials have autonomous modes that can repair themselves or perceive/respond to their environment.”
Figure 2. The characteristics of the resin. The diffusibility of 3 kDaFITC-dextran in LOW-MW and BLEND-MW resins (A). Ultraviolet rheology shows the mechanical properties when exposed to 405nm light (B). Characteristics of resin cured in LB medium after 60 minutes. The side view of the confocal Z-stack of the printed structure after incubation (C), as well as the characteristics of the initial printing height (D) and the average height change after incubation (E).
Figure 3. The accumulation of viable biomass over time. The intensity of GFP changes with time (A) and the images of microbes in the biofilm at 0 and 72 hours are printed (B).
Figure 4. Shows the engineering complexity of SLAM printed biofilms. Top-down (A) and side view (B), confocal z-stack images of the grid geometry of encapsulated E. coli expressing GFP (green), showing the increase in printing complexity, rotating view (C), and Top-down view and side view insert (D), a dual-species printed confocal z-stack of encapsulated E. coli expressing GFP (green) or mCherry (red).
Figure 5. SLAM bioprinted biofilms can be used for functional applications, including microbial adsorption and sensing. In order to absorb rare earth elements, engineered crescents are encapsulated in two different geometries to absorb neodymium. A macroscopic image (A) of the geometry of the SLAM printed cube (left) and grid (right) loaded with cells. The neodymium concentration in the cell-filled construct shows an increase in the absorption rate of the cells in the grid geometry compared to the cells encapsulated in the cube (B). Each sample was reused 3 times to create sample copies. In terms of microbial sensing, the crescents designed for uranium sensing are encapsulated in BLEND-MW resin and exposed to three concentrations of uranium solution. The resulting fluorescence intensity shows an increase in the fluorescence of 2.5 and 10 μM uranium compared to the control without uranium exposure (C). A printed LLNL logo with encapsulated crescents that are not in contact with uranium (D) or in contact with 10 mM uranium (E). ** means P value<0.01.
Reference: “Projection Microstereolithographic Microbial Bioprinting for Engineered Biofilms”, KarenDubbin, Ziye Dong, Dan M. Park, Javier Alvarado, Jimmy Su, Elisa Wasson, ClaireRobertson, Julie Jackson, Arpita Bose, Monica L. Moya, Yongqin Jiao and WilliamF. Hynes , 28 January 2021, Nano Letters.
Paper DOI: 10.1021/acs.nanolett.0c04100
Co-authors include LLNL scientists and engineers Karen Dubbin, Ziye Dong, Dan Park, Javier Alvarado, Jimmy Su, Elisa Wasson, Claire Robertson, and Julie Jackson, and Arpita Bose from Washington University in St. Louis.
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