FEATURE ARTICLE
Biofilms
A new understanding of these microbial communities is driving a revolution that may transform the science of microbiology
Joe Harrison, Raymond Turner, Lyrium Marques, Howard Ceri
Biofilms Are Everywhere
Most people are familiar with the slippery substance covering the rocks in a river or a stream. This particular slime is an aquatic biofilm made up of bacteria, fungi and algae. It begins to form after bacteria colonize the rock's surface. These microbes produce the extracellular polymeric substance, which is electrostatically charged so that it traps food particles and clay and other minerals. The matter trapped in the slime forms microscopic niches, each with a distinct microenvironment, allowing microorganisms that have different needs to come together to form a diverse microbial consortium.
A biofilm matrix is considered to be a hydrogel, a complex polymer hydrated with many times its dry weight in water. The hydrogel characteristics of the slime confer fluid and elastic properties that allow the biofilm to withstand changes in fluid shear within its environment. So biofilms often form streamers—gooey assemblages of microbes that are tethered to a surface. As running water passes over the biofilm, some pieces may break free and so spread the microbial community downstream. It is believed that bacteria can colonize the lungs of patients on ventilators in this way, causing often-fatal pneumonia in critically ill patients.
A microorganism's extraordinary ability to spread explains how biofilms show up in the unlikeliest of places. The steel hull of a ship at sea can be coated with biofilms that increase the drag on the vessel and so compromise its speed. Other biofilms wreak havoc in the oil industry by facilitating the microscopic corrosion of metals and limiting the lifespan of pipelines. Some biofilms, made up of the ancient lineage of prokaryotes (organisms lacking a nucleus) called archaea, can even survive the hostile hydrothermal environments of hot springs and deep-sea hydrothermal vents. The aptly named archaebacterium Pyrodictium thrives at the bottom of the sea, growing in a moldlike layer on sulfur crystals in the dark, anaerobic environment of a hydrothermal vent, where temperatures may exceed 110 degrees Celsius.
Perhaps one of the most extraordinary environments where one can find a biofilm is in the belly of a dairy cow. Biofilms are part of the normal complement of microbes in many healthy animals, but the presence of these microbial communities in ruminants provides a rich example of the interactions within a biofilm.
We begin with the rumen, the largest compartment of the bovine stomach, which can hold a liquid volume in excess of 150 liters. It is filled with so many microbes that microbiologists refer to cows as mobile fermenters. Bacteria colonize the digestive tract of a calf two days after it is born. Within three weeks the microorganisms have modified the chemistry inside the rumen, which soon becomes home to a reported 30 species of bacteria, 40 species of protozoa and 5 species of yeast. The cells in this biofilm thrive in the mucous layer of the stomach and grow on the food ingested by the animal. Cows, of course, eat grass, which consists largely of cellulose, a complex carbohydrate that cannot be broken down by mammalian digestive enzymes. But cellulose is a perfect fuel for the bacteria in the biofilm, which convert it into a microbial biomass that in turn supplies the proteins, lipids and carbohydrates needed by the cow.
The heart of this process is a microscopic ecosystem that begins when a pioneering planktonic bacterium in the rumen, a species such as Ruminococcus flavefaciens, gains access to the inner parts of a leaf, perhaps one that might have been broken by the cow's chewing. These bacteria attach themselves to the cellulose in the inner layers of the leaf and proliferate to form a rudimentary biofilm. The microbes release cellulose-degrading enzymes, which produce simple sugars and metabolic by-products that attract other bacteria—anaerobic fermenters such as the spiral-shaped Treponema byrantii, which ingest the sugars and produce organic acids, including acetic acid and lactic acid.
The acidic metabolites would normally slow the growth of the bacteria by a process of feedback inhibition, but it so happens that other microorganisms join the biofilm community and eat the organic acids. These are the methanogens, archaea whose actions accelerate the growth of the bacterial community and prevent the inhibitory feedback. As the name suggests, methanogens produce methane—lots of it. Approximately 15 to 25 percent of the global emission of methane, which totals 7.5 billion kilograms per year, is attributable to the flatulence of ruminants. Because methane traps heat in the atmosphere, the biofilm hidden away in a cow's stomach may play a nontrivial role in global climate change.
Animals aren't the only living things that provide a home to biofilms. Microbial colonies have been recognized on tropical plants and grocery-store produce since the 1960s, but it wasn't until the past decade that the term biofilm was used to describe bacterial growth on a plant's surface. In this domain, life in a biofilm confers many advantages to the individual cell, including protection from a number of environmental stresses—ultraviolet radiation, desiccation, rainfall, temperature variations, wind and humidity. The biofilm also enhances a microorganism's resistance to antimicrobial substances produced by competing microorganisms or the host's defenses.
Relations between plants and biofilms can be quite varied. In some instances the plant merely serves as a mechanical support, so the biofilm is simply a harmless epiphyte. In other cases, the plant may provide some nutrients for the microbes, such as the saprophytes that feed on decaying plant matter; these too pose no danger to the plant. But there can be trouble when certain epiphytic populations with the genetic potential to initiate a pathogenic interaction with the host grow large enough to overwhelm the host's defense mechanisms. Then the cells in the biofilm coordinate the release of toxins and enzymes to degrade the plant tissue. What began as an innocuous relationship ends in disease.
Belowground, plants and biofilms may also engage in some fairly elaborate interactions. For example, Pseudomonas fluorescens colonizes roots and protects plants from pathogens by producing antibiotics that exclude fungi and other bacterial colonizers. But fungal biofilms can also be beneficial to the plant. Certain mycorrhizal fungi penetrate a plant's root cells while also forming an extensive network in the soil; thus they provide a drastic increase in the surface area that the plant can use for the absorption of water and nutrients.
On the other hand, bacteria of the genus Rhizobium fix nitrogen from the atmosphere by converting N2 gas into ammonia (NH3). This process can involve some intricate chemical signaling between the plant and the bacteria that results in the formation of nodules within the root where the bacterial aggregates engage in nitrogen fixation. Perhaps the most intricate relation involves an interaction between the rhizobia, the mycorrhizal fungi and a plant host. The bacteria form a biofilm on the surface of the fungus, which in turn makes its connection with the plant, and so creates a tripartite symbiotic system that relies on the formation of biofilms by two microorganisms. (Unless the soil is alkaline, the system requires another player, nitrifying bacteria to oxidize the ammonia; they live not in the nodule but in nearby soil.)
Finally, let us consider the pathogenic interactions of biofilms within the plant's vasculature. Unfortunately, vascular diseases are currently untreatable and tend to be devastating to many economically important crops. A few pathogenic biofilms have been described in the water-carrying xylem of plants, but here we'll merely address Xylella fastidiosa. This pathogen causes Pierce's disease in grapevines and citrus variegated chlorosis in sweet oranges—diseases that have had a major impact on the wine industry in California and the citrus industry in Brazil, with economic losses exceeding $14 billion in the past decade. Pierce's disease also limits the development of a wine industry in Florida because the bacterium is endemic in that region.
X. fastidiosa is transmitted by xylem-feeding insects, called sharpshooters, that acquire the bacteria while feeding from infected plants. The bacteria form a rudimentary biofilm inside the insect's gut, and this allows them to be sloughed off indefinitely in aggregates sufficient to infect another plant when the insect feeds again. In turn, the biofilms clog the plant's xylem and cause symptoms related to water stress. So the biofilm plays a key role in the colonization of the plant vessels, the propagation of the disease and its pathogenicity.
The appreciation of biofilms' importance in plant disease has only just begun, and it will probably take some time for the idea to be applied in plant microbiology. However, the benefits could be significant. A better understanding of the associations between plants and biofilms may lead to more efficacious and environmentally friendly treatments for disease. It may also lead to the development of commercial applications that could improve the beneficial interactions between plants and microorganisms. Indeed, various rhizobia are now being used on commercial farms as a biotic fertilizer.
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