Biofilms consist of a mucous layer (a film) in which mixed populations[1] of microorganisms (e.g. bacteria, algae, fungi, protozoa) in concentrations of 1012 cells per milliliter of biofilm[1] and of multicellular organisms[1] such as Rotifers, nematodes, mites, bristles or insect larvae that feed on the microorganisms are embedded. In everyday life, they are often perceived as a slippery, soft-feeling, water-containing layer of mucus or coating. Other colloquial names are growth, Kahmhaut or Sielhaut.

By Asw-hamburg - Own work, CC BY-SA 4.0, https://commons.wikimedia.org/w/index.php?curid=46898752

Description

Biofilms predominantly form in aqueous systems when microorganisms settle on interfaces. In principle, all surfaces can be covered by biofilms: between gas and liquid phases (e.g. free water level), liquid and solid phases (e.g. gravel on the bottom of the water) or between different liquid phases (e.g. oil droplets in the water ). The interface on which the biofilm forms, or more precisely the phase into which the film does not or hardly grows, forms the substratum (substrate; that which extends underneath).

In the broader sense, biofilm refers to all aggregates of microorganisms that are embedded in a layer of mucus that they form.[2] Suspended solids in water often consist of mineral particles covered by biofilms. The activated sludge in sewage treatment plants also has essential biofilm properties. It consists of flakes that themselves have a surface suitable for colonization. Biofilms can be considered a very primitive form of life because the oldest fossils that have been found so far come from microorganisms in biofilms that lived 3.2 billion years ago. These are stromatolites (biogenic sedimentary rocks) found in Western Australia (Pilbara Craton). Biofilm as a form of life has proven itself so well that it is still widespread today. The vast majority of microorganisms live in nature in the form of biofilms.[4][FSE 1]

Composition

Fig. 2: Macromolecules of a biofilm. (Modified according to Fuchs[FSE 2]) From above:
Cytoplasm (CP) of a spheroplasted bacterium with cytoplasmic membrane (CPM).
Intercellular (IC) glycocalyx with exo-polysaccharides (EPS), DNA (DNA), hydrophobic (HPr) and water-soluble proteins (SPr).
Periplasmic membrane (PPM), cell wall (W), periplasm (PPl), cytoplasmic membrane and cytoplasm of a bacterium.

Apart from the microorganisms, the biofilm mainly contains water. Extracellular polymeric substances (EPS) secreted by the microorganisms combine with water to form hydrogels, creating a mucous-like matrix in which nutrients and other substances are dissolved. Inorganic particles or gas bubbles are often trapped in the matrix. Depending on the type of microorganisms, the gas phase can be enriched with nitrogen, carbon dioxide, methane or hydrogen sulfide.

The EPS consist of biopolymers that are able to form hydrogels and thus give the biofilm a stable shape. This involves a wide spectrum of polysaccharides, proteins, lipids and nucleic acids (extracellular DNA).

Different types of microorganisms usually live together in biofilms. In addition to the original biofilm formers, other single-celled organisms (amoebas, flagellates, etc.) can also be integrated. Aerobic and anaerobic zones can occur a few hundred micrometers apart, allowing aerobic and anaerobic microorganisms to live close together.

Shape

Fluorescence microscopic image of a multi-species biofilm on stainless steel.
In the core area, the biofilm is usually compact (basic biofilm). The edge area (surface biofilm) can either be compact and regularly shaped and form a flat interface to the fluid flowing over it, or it can be blurred and much looser. In the latter case, the surface biofilm can resemble a mountain-and-valley path if, for example, bacterial species grow into the fluid in a thread-like manner (filamentous) or if the substrate is populated with protozoa (e.g. bellworms) or higher types of organisms.

The biofilm matrix is ​​then often permeated by pores, caverns and passages, which enable material exchange between the bacterial cells and a supply of water. Mushroom-shaped or tower-like structures are often found. Convective mass transport processes occur there when liquid flows through them. In the area of ​​the surface of the biofilm, convective mixing processes can also be triggered by the movement of outgrowths protruding into the flow (e.g. “wastewater fungi” such as Sphaerotilus natans). Inside biofilms, dissolved substances are transported primarily through diffusion. Cells or entire parts of the biofilm can repeatedly be released at the boundary layer with the water and be absorbed by the water flowing past.

Formation and maturation of biofilms

Fig. 4: Phases and microscopic images of biofilm development. The emergence and formation of a biofilm can be divided into three phases: the induction phase (Figs. 4 and 6, 1–2), the accumulation phase (3) and the existence phase (4–5).

Colonization of surfaces

According to popular belief, typical microorganisms have flagella (Fig. 6, 1) and move freely in the water column. In fact, such swarmer cells [FSE 3] are usually only the dispersal stage of biofilm inhabitants.

There is a compelling reason why the absolute majority of bacteria and archaea are rooted in biofilms: otherwise they would be washed out of their biotope by the water necessary for life. Soil bacteria would end up in the nearest river and from there begin their final journey into the sediment of an ocean. The same would happen to the microorganisms in the activated sludge from sewage treatment plants.

In order to be able to leave the free water at all, microorganisms need water-repellent hydrophobic substances on the surface of their cells. These enable organisms to attach to hydrophobic surfaces based on van der Waals forces. Since almost all areas in aquatic biotopes are covered with biofilms[FSE 4], most swarmer cells associate with existing biofilms.

However, such organisms can also attach themselves directly to unpopulated areas. Smooth hydrophobic surfaces, such as B. polystyrene or the cuticle of many plants can be colonized directly, but only if they can be wetted with water. However, thanks to the lotus effect, many plants avoid the growth of microorganisms on their leaves.

A thin, viscous layer of organic substances initially accumulates on empty hydrophilic surfaces. These biopolymers originate from the mucous membranes that form around bacterial cells (EPS), occasionally detach completely or partially and become adsorptively bound upon contact with interfaces. Such biogenic substances are omnipresent in nature.[FSE 5]

The metamorphosis into a biofilm inhabitant

Fig. 5: Life cycle of Caulobacter. A swarmer cell (1) sheds its flagella and the pili are shortened (2). The resulting stalk cell (3) grows and forms new swarm cells (4)[FSE 6]

If the site of attachment allows the organism in question to grow, it will usually shed its flagellum(s). However, in many organisms a much deeper change occurs.

This is clearly visible in Caulobacter, an aerobic α-Proteobacterium. After losing the flagellum, the swarmer cell retracts its attachment pili and becomes a stalk cell. In contrast to the swarm cell, it is capable of division and immediately begins with an asymmetrical division. This creates a new swarmer cell. After separation, the stalk cell can repeatedly form new swarmer cells under suitable conditions.[FSE 7]

The changes in the soil bacterium Bacillus subtilis are at least as profound (Fig. 6). After attachment and loss of flagellation, filamentous structures arise during subsequent cell divisions because the cell walls of the organisms are not separated. At the same time, polymers are secreted, which give the resulting film lateral strength. Such changes are triggered epigenetically.[6]

As a result of the proliferation of cells that have attached themselves to a surface, the organisms spread. The interface is initially colonized over the surface in the form of a film (biofilm). At the same time or later, the biofilms grow in multiple layers and ultimately form heterogeneous three-dimensional structures. Up to this phase, Bacillus subtilis produces almost exclusively filamentous cell groups.

Avoidance of competition
There is, in principle, competition for nutrients between the cells of a biofilm, with the cells closest to the food source having a clear advantage. In contrast, the cells inside are in danger of starving. If that happens, they will no longer be able to maintain cohesion. In fact, there are mechanisms of cell density regulation and communication between cells (quorum sensing)[FSE 8] that counteract this.

Such a mechanism was elucidated in detail for the first time in 2015 for Bacillus subtilis.[7] For this purpose, a biofilm from a pure culture of these bacteria was examined in a chemostat bioreactor. The biofilm was continuously supplied with nutrients, and yet the cells periodically stopped growing until the cells inside the biofilm stopped starving. This “oscillation” is based on the following process:

Starving cells inside the biofilm send out a pulse of K+ ions. The biofilm cells of B. subtilis have receptors for these ions, which trigger a whole chain of events. 

All cells, including the well-supplied cells, send out a K+ signal immediately after receiving it. Specific K+ channels exist in the biofilm for the propagation of signals. (Normal diffusion through the polymeric biofilm matrix would be too slow.)
The cells, which are still well supplied, immediately stop their growth, but not their metabolic activity. If there is a nitrogen deficiency, they take e.g. B. glutamine from the nutrient medium, but do not use this amino acid for growth, but split off ammonium from it, which they make available to the biofilm.

If the signals diminish, growth will continue together.[8]
K+-based communication between bacterial cells is not the only one. There are a number of pheromones that can be produced and sensed by organisms. This also initiates the next phase in the existence of a biofilm (see Fig. 6.5). Metamorphosis of cells occurs again. In well-supplied cells, flagellate swarm cells are formed again, whose preferred swimming direction is towards the nutrient source. Many bacteria, like B. subtilis, also form spores in this phase. These are carried by the current and are prepared for long-term nutrient shortages.[FSE 9]

This phase of emigration is by no means the end of a biofilm. For the release of the spores and swarmer cells, the extracellular matrix is ​​only actively dissolved in their surroundings. In the old part of the biofilm, life continues with a new phase of growth.

The fact that the depth extent of the biofilm is limited becomes apparent when entire parts of the biofilm are carried away by the current. Due to the formation of gas bubbles (e.g. due to denitrification and carbon dioxide), the cohesion of biofilm parts is lost. The increase in flow resistance with increasing thickness leads to increased erosion if the biofilm has formed on surfaces subject to flow. Life in such biofilm fragments is not fundamentally different from biofilms that are attached somewhere. Such flakes have all the properties needed to attach to a new surface.

Biocorrosion

Biocorrosion is observed in the presence of biofilms. Here, iron oxidizers contained in the oxygen-loving (aerobic) top layer lead to an attack on the passive layer (of metals) - sulfate reducers existing in the anaerobic layer attach to these points and “eat” into the material.

Microbiologically caused corrosion causes considerable economic damage every year. The proportion of total corrosion (ie abiotic and biotic corrosion) is estimated to be at least 20%; According to more recent findings, it is probably significantly higher. Even higher-alloy materials such as V2A and V4A are damaged. Almost all technical systems are affected: including cooling circuits, water treatment and industrial water systems, energy production in power plants, the production of cars, computers, paint, and the oil and gas industry.[27] In contaminated mining sites, biological leaching of minerals through biofilms leads to large-scale environmental damage to soil, water and air through dust pollution and emissions of sulfuric acid, heavy metals, radon and radionuclides.

Biofouling

In water treatment using membrane processes, biofilms are responsible for biofouling, which leads to serious problems with this technology.

Biofouling also includes biofilms that form on underwater bodies. This can cause significant problems. A biofilm of just a tenth of a millimeter reduces the speed of a tanker by 10 to 15 percent due to increased frictional resistance. This results in increased fuel consumption. In the fight against organic growth (including barnacles and mussels), special substances are painted onto ships, platforms and buoys, the active ingredients of which are released into the water and often pose a significant environmental impact. One such substance is highly toxic tributyltin (TBT), which is now banned worldwide. Also affected are sensor systems for research or monitoring purposes in the maritime sector, where fouling can very quickly lead to functional impairments.

Concentration gradients of physical-chemical parameters in biofilms can be determined using high-resolution microsensors (= functional investigation) and correlated with molecular biological data from the depth distribution of the microbial populations present in the biofilm (= structural investigation). The ideal goal is to combine the structure and function of the microbial populations in the biofilm with (damage/corrosion) data from the growth area. This contributes to a better understanding of the interaction between the damage-causing biofilm and the growth area, which is of particular interest in applied systems (e.g. marine biofilms in steel pipes).

Context: 

Sources include: https://de.wikipedia.org/wiki/Biofilm

1.  Karl Höll:  Water.  ISBN 978-3-110-22677-5 , pp. 663–669 ( limited preview  in Google book search).
2. ↑  Michel Vert, Yoshiharu Doi, Karl-Heinz Hellwich, Michael Hess, Philip Hodge, Przemyslaw Kubisa, Marguerite Rinaudo, François Schué:  Terminology for biorelated polymers and applications (IUPAC Recommendations 2012) . In:  Pure and Applied Chemistry . 84th year, No. 2, 2012, pp. 377–410,  doi : 10.1351/PAC-REC-10-12-04  ( Online  ( Memento  from March 19, 2015) [PDF; accessed on February 10, 2016]) .  Info:  The archive link was inserted automatically and has not yet been checked. Please check the original and archive link according to  the instructions  and then remove this notice.
3. ↑  Andreas Schmidt-Wilckerling:  Metabolic activity of freely suspended and  immobilized  cells of ammonia-oxidizing bacteria.  Diploma thesis, Hamburg (1989).
4. ↑  ^{Jump up to: a  b  c}  Luanne Hall-Stoodley, J. William Costerton and others:  Bacterial biofilms: from the natural environment to infectious diseases . In:  Nature Reviews Microbiology . Vol. 2, No. 2, 2004,  ISSN  1740-1526 ,  PMID 15040259 ,  doi:10.1038/nrmicro821 , pp. 95–108  (PDF file; 0.6 MB) .
5. ↑  Hera Vlamakis, Yunrong Chai, Pascale Beauregard, Richard Losick, Roberto Kolter:  Sticking together: building a biofilm the Bacillus subtilis way . In:  Nat Rev Micro . 11th year, No. 3, 2013, pp. 157–168,  doi : 10.1038/nrmicro2960 .
6. ↑  Yunrong Chai, Thomas Norman, Roberto Kolter, Richard Losick:  An epigenetic switch governing daughter cell separation in Bacillus subtilis . In:  Genes & Development . Volume 24, No. 8, 2010, pp. 754–765,  doi : 10.1101/gad.1915010  ( cshlp.org ).
7. ↑  Jintao Liu, Arthur Prindle, Jacqueline Humphries, Marcal Gabalda-Sagarra, Munehiro Asally, Dong-yeon D. Lee, San Ly, Jordi Garcia-Ojalvo, Gurol M. Suel:  Metabolic co-dependence gives rise to collective oscillations within biofilms . In:  Nature . 523rd volume, No. 7562, 2015, pp. 550–554,  doi : 10.1038/nature14660 .
8. ↑  Arthur Prindle, Jintao Liu, Munehiro Asally, San Ly, Jordi Garcia-Ojalvo, Gurol M. Suel:  Ion channels enable electrical communication in bacterial communities . In:  Nature . Volume 527, No. 7576, 2015, pp. 59–63,  doi : 10.1038/nature15709 .
9. ↑  James A Shapiro:  Thinking about bacterial populations as multicellular organisms . In:  Annual Reviews in Microbiology . Volume 51, No. 1, 1998, pp. 81–104,  doi : 10.1146/annurev.micro.52.1.81  ( annualreviews.org  [PDF]).

Anaerobic granular sludge bed technology refers to a special reactor concept for the anaerobic treatment of wastewater with high throughput. The concept was introduced with the UASB reactor (UASB = upward-flow anaerobic sludge blanket). A schematic of a UASB reactor is shown in the figure.

From a hardware perspective, at first glance, a UASB reactor is nothing more than an empty tank (i.e. an extremely simple and inexpensive design).

The wastewater is fed into the tank via appropriately arranged inlets. The wastewater flows upward through an anaerobic sludge bed where the microorganisms in the sludge come into contact with the wastewater substrates. The sludge bed consists of microorganisms that naturally form granules (pellets) with a diameter of 0.5 to 2 mm, which have a high sedimentation rate and are therefore not washed out of the system even under high hydraulic loads. The resulting anaerobic degradation process is usually responsible for the production of gas (e.g. biogas containing CH4 and CO2). The upward movement of the released gas bubbles causes hydraulic turbulence, which ensures mixing of the reactor without mechanical parts. At the top of the reactor, the water phase is separated from the sludge solids and gas in a three-phase separator (also called a gas-liquid solids separator). The three-phase separator is usually a gas cap with a settler above it. Baffles are used below the gas cap opening to direct the gas to the gas cap opening.

Brief history of UASB

The UASB procedure was developed by Dr. Gatze Lettinga and colleagues developed it in the late 1970s at Wageningen University (Netherlands). Inspired by publications by Dr. Perry McCarty (Stanford, USA), Lettinga's team experimented with an anaerobic filter concept. The Anaerobic Filter (AF) is a high-speed anaerobic reactor in which biomass is immobilized on an inert porous support material. During experiments with the AF, Lettinga observed that, in addition to the biomass fixed to the carrier material, a large part of the biomass developed into free granular aggregates. The UASB concept crystallized during Gatze Lettinga's trip to South Africa, where he observed the sludge developing into compact granules in an anaerobic wine vinasse treatment plant. The reactor design of the plant visited was a "Clarigestor", which can be considered a precursor to the UASB. The upper part of the "Clarigestor" reactor has a clarifier but no gas cap.

The birth of the UASB

The UASB concept emerged from the realization that an inert support material for biomass attachment is not necessary to maintain a high proportion of active sludge in the reactor. Instead, the UASB concept is based on a high degree of biomass retention through the formation of sludge granules. When developing the UASB concept, Lettinga took into account the need to promote the accumulation of granular sludge and prevent the accumulation of disperse sludge in the reactor. The most important features for the development of granular sludge are, firstly, maintaining an upward flow in the reactor that selects microorganisms to aggregate, and secondly, ensuring adequate separation of solids, liquid and gas to prevent leaching of the sludge grains.

First UASB. The UASB reactor concept was quickly developed into technology, with the first pilot plant installed at a beet sugar refinery in the Netherlands (CSM suiker). Afterwards, a large number of large-scale systems were installed in sugar refineries, potato starch processing plants and other food industries as well as in waste paper factories in the Netherlands. The first publications on the UASB concept appeared in Dutch-language journals in the late 1970s, and the first international publication appeared in 1980 (Lettinga et al. 1980).

Grahik: By Tilley, E., Ulrich, L., Lüthi, C., Reymond, Ph., Zurbrügg, C. - Compendium of Sanitation Systems and Technologies - (2nd Revised Edition). Swiss Federal Institute of Aquatic Science and Technology (Eawag), Duebendorf, Switzerland. ISBN 978-3-906484-57-0., CC BY-SA 3.0, https://commons.wikimedia.org/w/index.php?curid=42267210