Biofilms are aggregates of bacterial cells surrounded by an extracellular matrix. Much progress has been made in studying biofilm growth on solid substrates; however, little is known about the biophysical mechanisms underlying biofilm development in three-dimensional confined environments in which the biofilm-dwelling cells must push against and even damage the surrounding environment to proliferate. Here, combining single-cell imaging, mutagenesis, and rheological measurement, we reveal the key morphogenesis steps of Vibrio cholerae biofilms embedded in hydrogels as they grow by four orders of magnitude from their initial size. We show that the morphodynamics and cell ordering in embedded biofilms are fundamentally different from those of biofilms on flat surfaces. Treating embedded biofilms as inclusions growing in an elastic medium, we quantitatively show that the stiffness contrast between the biofilm and its environment determines biofilm morphology and internal architecture, selecting between spherical biofilms with no cell ordering and oblate ellipsoidal biofilms with high cell ordering. When embedded in stiff gels, cells self-organize into a bipolar structure that resembles the molecular ordering in nematic liquid crystal droplets. In vitro biomechanical analysis shows that cell ordering arises from stress transmission across the biofilm–environment interface, mediated by specific matrix components. Our imaging technique and theoretical approach are generalizable to other biofilm-forming species and potentially to biofilms embedded in mucus or host tissues as during infection. Our results open an avenue to understand how confined cell communities grow by means of a compromise between their inherent developmental program and the mechanical constraints imposed by the environment.

生物膜是由细胞外基质包围的细菌细胞聚集体。在研究固体基质上的生物膜生长方面取得了很大进展；然而，对于在三维受限环境中生物膜发育的生物物理机制知之甚少，在这些环境中，生物膜栖息的细胞必须推动甚至破坏周围环境才能增殖。在这里，结合单细胞成像、诱变和流变测量，我们揭示了霍乱弧菌的关键形态发生步骤当它们从初始尺寸增长四个数量级时嵌入水凝胶中的生物膜。我们表明嵌入式生物膜中的形态动力学和细胞排序与平面上的生物膜根本不同。将嵌入的生物膜视为在弹性介质中生长的夹杂物，我们定量地表明，生物膜与其环境之间的刚度对比决定了生物膜的形态和内部结构，在没有细胞有序性的球形生物膜和具有高细胞有序性的扁椭圆形生物膜之间进行选择。当嵌入坚硬的凝胶中时，细胞自组织成双极结构，类似于向列液晶液滴中的分子排序。体外生物力学分析表明，细胞排序源于生物膜-环境界面的应力传递，由特定的基质成分介导。我们的成像技术和理论方法可推广到其他生物膜形成物种，并可能推广到感染期间嵌入粘液或宿主组织中的生物膜。我们的研究结果为了解受限细胞群落如何通过其固有的发育程序和环境施加的机械约束之间的折衷来生长开辟了一条途径。