Biofilms, slimy layers formed when bacteria stick together on a surface, allow bacteria to shield themselves from extreme environments. As sources of antibiotic-resistant bacteria, biofilms can cause serious issues in health care and other industries. Conversely, biofilms of harmless bacteria can be used to develop new biomaterials. The power to optically control biofilm formation could allow scientists to harness these microbial layers to develop and enhance bioengineering applications. Researchers at California State University, Northridge investigated the use of optical trapping to regulate bacterial aggregation and biofilm development. In the work, the team used lasers of varying wavelengths and determined which wavelengths support bacterial biofilm formation, and which ones suppress it. Although scientists have shown that synthetic and chemical approaches can be used to activate and control biofilms as well as engineer them into specific spatial structures, physics professor Anna Bezryadina and her team at the Bezryadina Lab aimed to explore the use of optical methods to control biofilm dynamics. The Bezryadina Lab specializes in quantum and nonlinear biophotonics research. “Producing microscopic components usually requires a highly technical fabrication process, but we found that optical tweezers can be used to precisely control the position of individual bacteria or clusters of bacteria,” Bezryadina said. “This allows us to influence the growth patterns of bacterial structures on a microscopic level with high precision.” The team conducted their optical trapping experiments using a 473-nm blue laser and a near-infrared (NIR) Ti:sapphire laser that could be tuned between 700 and 1000 nm. The lasers were used to manipulate the biofilm development of Bacillus subtilis — a nonpathogenic bacterium that naturally forms biofilms. To stimulate the production of a biofilm, the team used a low-nutrient environment hostile to the bacteria. After obtaining small biofilm clusters, they conducted their optical trapping experiments. Researchers used laser-based optical traps to control biofilm formation. They found that lasers at different wavelengths could be used to either stimulate or suppress biofilm growth. Courtesy of Anna Bezryadina/California State University, Northridge. The researchers found that irradiating the bacteria with the NIR laser at a low-absorption wavelength range between 820 and 830 nm enabled prolonged optical trapping of the biofilm clusters and minimized significant photodamage. In contrast, irradiating the bacteria with high optical absorption (i.e., 473 nm) caused the cells to rupture and the biofilm clusters to disintegrate. Using optical tweezers at a wavelength of 820 nm for an hour, the researchers studied the dynamics of biofilm formation in Bacillus subtilis. They observed that bacterial clusters would aggregate near optically trapped clusters, adhere to the surface, and start to form a microcolony. They were able to move optically trapped bacterial clusters to specific positions throughout the sample — a capability that could be useful for building structures out of bacteria. “We can even create a sort of bacterial Lego block that can be moved around, stuck together, and destroyed as needed,” Bezryadina said. Biofilm formation did not appear to be disrupted by the NIR laser, suggesting that NIR wavelengths in the 800- to 850-nm range can be safely used for extended periods of time for optical trapping, manipulation, and pattern formation of bacterial clusters. “Despite the apparent uncontrolled bacterial biofilm formation in nature, our work showed that bacterial biofilm formation can be influenced by light,” Bezryadina said. Overall, the experiments showed that there is some flexibility in the growth conditions, cluster sizes, and wavelengths required to manipulate the biofilms. The researchers said that their methodology has the potential to be used with other types of biofilm-forming microorganisms. The team’s findings could contribute to the advancement of biomaterials composed of bacterial biofilms and increase understanding of the complex interactions that occur inside biofilm communities. Bezryadina believes that the work could lead to the development of new types of biodegradable materials and a new generation of biofilm-based biosensors. In future work, the team will study how to manipulate small clusters to construct intentional structures made of bacterial biofilm and investigate how different types of laser beams can promote or suppress biofilm growth. “This paper represents the first step in the long-term project to create microscopic building materials from readily available resources like bacteria,” Bezryadina said. “In future studies, we are planning to use what we found to develop a process to construct structures from bacterial ‘Lego blocks.’” The research was published in Biomedical Optics Express (www.doi.org/10.1364/BOE.510836).