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Biofilm – an instrument
for wastewater treatment
technology
Edyta Łaskawiec
Introduction
Wastewater treatment and water treatment tech-nologies increasingly use the biochemical ability of microorganisms to degrade contaminants. Biological treatment methods owe their popularity primarily to the significant reduction in chemicals consumed. These processes can be differentiated, both using the active sludge method, in which the mass of microorganisms is in suspension and biodegradation by biofilm micro-organisms in reactors and biofilters (Donlan, 2002). The foundations for development of the latter were natural processes of soil self-purification and observations from river debris (Suschka, 2000).
The presence of a film called “biofilm” or “attached film” is now better understood, owing to the develop-ment of scanning electron microscopy and the inven-tion of the confocal laser microscope (Andersson, 2009). The presence of bacteria, fungi, algae and other species in one structure endows it with the property of high resistance to chlorination, toxic substances and antibiotics, improving its effectiveness for biodegrada-tion. Specific metabolic cooperation and effective gene transfer confers improved physical resistance of the biofilm. Micro-organisms forming its structure can
survive nutrient deprivation, changes in pH or attack from oxygen radicals (Jefferson, 2004; Nawrocki, 2010). Species diversity and dynamic adaptation were found not only in the equipment used for purification of waste water or water treatment, but also in soil matrix. In addition to their environmental purification capacity, biofilms can have negative effects. Biofilms, which are common in water networks can contribute to distribu-tion losses. They also account for infecdistribu-tions in living organisms or formation of plaque. This demonstrates the complexity and multiplicity of such structures (Kołwzan, 2011).
Biofilm formation
Biofilm – depending on the environment in which it develops – can include mineral materials, crystals, particles formed as a result of corrosion and sludge in its structure. However, the basis of any biofilm is in the microorganisms with extracellular matrices of poly-meric substances, including exopolysaccharides (EPS – Extracellular polymeric substances) (Donlan, 2002). The structure of the polymer gel is variably composed of bacterial micro-colonies enclosed in the EPS matrix and separated by interstitial voids, so-called “water
Summary:
The article discusses the detailed issues related to the for-mation of biofilms, a multi-step process exploiting physi-cal and chemiphysi-cal phenomena. The main components of the biofilm matrix are described with the functions they perform. The prevalence of this technology makes it valu-able to make detailed investigations of the formation of biofilms. This is of particular relevance to wastewater treatment technologies, which are increasingly adopting these popular biological methods for sewage processing.
Key words: biofilm, wastewater treatment, biofilters,
extracel-lular polymeric substances
Edyta Łaskawiec: Environmental Protection, Faculty of Soil and Water Protection Systems, Department of Energy and Environmental Engineering, Silesian University of Technology
received: 15.10.2014; accepted: 19.02.2015; published: 27.03.2015
Fig. 1. Steps to Biofilm formation http://www.emeryp-harmaservices.com/ blog/eps-offers- quantitative-biofilm- models-for-todays-research-needs coherence with the Curriculum – see. p. 26
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channels” (Jefferson, 2004; Suschka, 2000). Biofilm for-mation on solid surfaces is possible owing to its adhe-sive properties (Kołwzan, 2011).
Biofilm can form on the majority of biotic and abi-otic surfaces. An aquatic environment is particularly favourable for its formation (Donlan, 2002; Fleming, 2010). Formation is a multistep process. The gradual migration of cells towards the potentially populated
surface begins with physical interactions: gravitation-al, hydrodynamic and van der Waals forces, Brownian motion and diffusion. To colonise the surface, bacteria use their external structures: pili, cilia, flagella or fim-briae (Kołwzan, 2011). The presence of proteins such as fibronectin, fibrinogen, vitronectin and elastin, com-monly referred to by the acronym MSCRAMM (Mi-crobial surface component recognizing adhesive matrix
molecules), play a significant role in this process (Flem-ming, 2010 Królasik 2005). The irreversible attachment of microorganisms takes place when exopolysaccha-rides are synthesised on a solid surface by molecular bonding. The mucous coating produced at this stage facilitates adhesion of other microorganisms (Czaczyk et al., 2007; Fleming, 2008).
During maturation of the biofilm, differentiation of the structure occurs, in addition to development of specialised properties, depending on the environment. Gene expression leads to phenotypic traits that allow survival in unfavourable conditions (Kołwzan, 2011). The final stage is the gradual peeling of the structure, its dispersion. The breakdown of biofilm is influenced by physical, biological, and chemical factors. Lack of ac-cess to nutrients and oxygen, erosion, peeling or attenu-ation of the production of auto-inductors responsible for intercellular communication (i.e., quorum sensing) can be identified in EPS degradation (Kołwzan, 2011; Nawrocki, 2010). However, the main factor responsible for biofilm dispersion is the critical thickness when self-maintenance starts to fail. Figure 1 shows the process of biofilm growth from occupation to dispersal and mi-gration.
Characteristics and the basic functions of biofilm
components
The matrix of the biofilm is highly hydrated, con-taining 97% water, 2-5% are cellular bacteria and 3-6% EPS and ions. The following can be identified in the EPS composition: polysaccharides (40-95%), pro-tein (1-60%), nucleic acids (1-10%) and lipids (1-40%) (Donlan, 2002; Nawrocki, 2010). The content of these components is strongly dependent on the type of mi-croorganisms present in the biofilm. Biopolymers such
Component Function Importance in biofilm
Proteins, polysaccharides, DNA,
amphiphilic molecules adhesion
biotic and abiotic surface colonization by phytoplankton cells, long-term biofilm attachment to the surface
Polysaccharides, proteins, DNA creation of cell clusters
temporary binding of the cells, immobilization of bacterial population, thickening of the structure, identification and com-munication between cells (quorum sensing)
Proteins e.g., lecithin, natural and
charged polysaccharides, DNA biofilm consistency
formation of the biofilm matrix, hydrated polymer network; provides mechanical stability, defined biofilm architecture; inter-cellular communication
Hydrophilic proteins, possibly
proteins water storage
hydrated environment around the biofilm, high tolerance to desiccation or in contact with the environment of water scarcity Polysaccharides, proteins protective barrier tolerance to contamination, anti-microbial agents
Charged proteins or hydrophobic
polysaccharides sorption of organic compounds
accumulation of nutrients, drawing from the environment, sorp-tion of xenobiotics
Proteins containing such inorganic substituents, e.g. phosphates, sul-phates, charged polysaccharides
sorption of inorganic ions formation of a polysaccharide gel, ion exchange, accumulation of toxic metal ions (decontamination)
Proteins enzymatic activity
digestion of exogenous molecules, which are food for microor-ganisms, structural degradation of EPS (release of bacterial cells from the biofilm)
Probably all components of EPS source of nutrients source of carbon, nitrogen and phosphorus DNA exchange of genetic
informa-tion transfer of genes between cells Proteins, probably humic
sub-stances electron donor /acceptor redox reactions in the biofilm matrix
Membrane vesicles (MVs) export of cellular components release of cellular material in the course of metabolism Polysaccharides release of excess energy store of excess carbon in the case of excess in relation to
avail-able nitrogen
Polysaccharides, enzymes enzyme binding collection, storage, stabilisation of enzymes in result of interac-tion with polysaccharides
Table 1. Functions of EPS components in biofilm
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as exopolysaccharides are produced by archaea, bacteria and eukaryotes.
EPS, which has been called the “dark matter of bio-films”, is a combination of cells and plays an important role in the formation and the functioning of structure as a whole. Production of the cellular matrix is a pre-condition for biofilm formation as it accounts for its structure, adhesion to the substrate and protects against dehydration, oxidation, antibiotics or metal cations. EPS together with the cells forms a compact entity; try-ing to isolate this form may result in cell death. Each micro-colony within the biofilm is surrounded by EPS (Kołwzan, 2011; Królasik, 2005). Table 1 lists the basic components of the biological matrix with their func-tions and importance.
Characteristics of micro-organisms colonising
biofilm in wastewater treatment
The presence of biofilms is accompanied by bio-logical processes, such as slow filtration, infiltration, filtration beds and the reactions in the reactors. During their course the biodegradation of organic pollutants, denitrification, nitrification, oxidation or reduction of iron, manganese and sulphur connections may occur (Świderska-Bróż and Kowal, 2009). Biocenosis of the bi-ofilm forms an expanded food chain, in which the first link is the bacteria that can provide nutrition for con-sumers of the first order and which are then available for secondary consumers – predators (Kopec, 2013).
The organisms responsible for production of the mucous EPS shell are Zooglea sp. (fig. 2) and Nitrobac-ter, Nitrosomonas and Sphaerotilus, which contribute to the swelling of the biofilm. The anaerobic biofilm layer also contains sulphur bacteria, Thiothrix and Beggiatoa (Suschka, 2000). The presence of sulphur bacteria often indicates inflow of putrescible sewage to biofilters and insufficient oxygenation (Kopeć, 2013; Świderska-Bróż and Kowal, 2009).
Biofilm properties largely depend on the combina-tion of species. The presence of algae on the surface of biofilters indicates ample oxygenation. Their growth is possible on surfaces exposed to direct sunlight. The most common algae are: Euglena, Chlorella, Stigeoclo-nium, Oscillatoria and Phormidium (Andersson, 2009; Suschka, 2000).
Among the first order consumers, the literature de-scribes filtrators and collectors. The literature includes filtrators and collectors among the first order consum-ers. Some filtrators are sedentary ciliates, while the slow moving ciliates are included among the collectors, the presence of which indicates a stable, trouble-free waste-water treatment process. The most frequently found are Vorticella microstoma, Vorticella convallaria and Oper-cularia. Other organisms generally found include, Epi-stylis, Paramecium, Metopus and Colpidium canpylum (Kopeć, 2013; Świderska-Bróż and Kowal, 2009). Nema-todes (Nematoda sp.) and rollers (Rotaria sp.) are among the predators living in biofilms (fig. 3 ).
In addition to the compounds that are readily bio-degradable, there are also those significantly less sus-ceptible, e.g., fulvic and humic acids, these are natural organic compounds (NOM – natural organic matter). Their decomposition is made possible by aerobic actino-mycetes (Świderska-Bróż and Kowal, 2009).
Fig. 2. A cluster of bacteria Zoogolea sp. Source: http:// www.photo-macrography. net/forum/ viewtopic.php? p=117705&sid= 208e408356a74 b9ff8b341fdab5 8eb4f
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Mechanism of biofilm activity
Biofilm formation on a substrate e.g., biofilters, is af-fected by several factors. Most important is the surface of a carrier or substrate for colonisation by microorgan-isms. The presence of pores and apertures in materials facilitates this process (Miksch et al., 2012). To main-tain a favourable environment for survival and further growth of microorganisms, it is necessary to provide an input of nutrients, including phosphorus, which has a significant impact on cell adhesion capacity. Ensuring a higher temperature allows faster biofilm growth and the presence of liquid phase organic substances, trace elements and oxygen ensures maintenance of biological activity. The principle of biofilm function is the initial adsorption of waste organic substances from water by the mass of microorganisms in which biochemical oxi-dation takes place. The reaction products are discharged from the system (Donlan, 2002; Miksch et al., 2012).
Substances in waste water are often in suspended or colloidal form, which makes direct diffusion difficult. To permit transport and metabolism, these compounds are first hydrolysed to simpler molecules on the surface
of the biofilm. These can then diffuse to the interior. Metabolic end products are removed from the liquid phase in the reverse direction. Diffusive transport oc-curring in the biofilm is divided into two stages. The first covers substrate provision to the surface and the second, conversion by micro-organisms in the film inte-rior (Miksch et al., 2012; Nawrocki, 2010). Oxygen from the atmosphere diffuses into the outer layer of the bio-film, where the processes have the greatest activity and there is highest substrate concentration (Donlan, 2002). The organic substrate used in microbiologically catalysed reactions is transported at the liquid – bio-film phase border in a diffusion layer and transferred inwards. The progressive wear of the substrate lowers its concentration in the biofilm, thus creating the
driv-ing force for further transport. After substrate uptake by the microorganisms, it is metabolised by biochemi-cal oxidation. The residue is assimilated in the form of biomass, which can be partially autooxidised and the metabolic waste products are removed along with ex-cess biofilm (Miksch et al. 2012).
Figure 4 illustrates the structure and flow of a sub-stance within the biofilm.
Biofilters – a tool for biological wastewater
treatment
According to conditions, biological treatments have been classified as natural, semi-natural or artificial. The semi-natural methods include sewage, fish, algal and Fig. 3. A biofilm predator, Rotaria sp.
Source: http://www.microscopy-uk.org.uk/mag/wimsmall/extra/ rotif9.html
Fig. 4. Diffusion of substrates and products within the biofilm
Elaboration from Andersson, 2009; Suschka, 2000.
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aerated ponds, as well as, water-soil devices such as fil-tration ditches, leach drains and irrigation fields. Bio-filters, other than activated sludge devices and digest-ers, have been included with the artificial methods for biological wastewater treatment (Papciak et al., 2005).
A biofilm layer develops on individual packing grains and in the deeper layers of the biofilter. During wastewater purification, weight gain occurs from
sorp-tion of pollutants and microorganisms. Waste water flowing onto the surface of the biofilter causes detach-ment of the excess biofilm from the surface. Sedidetach-menta- Sedimenta-tion of detached fragments can then take place in the secondary settling tank. Wastewater contact with bio-film can be provided as a direct result of the waste water flow over the layer of biofilters or by periodic flooding or immersion (Henrich et al., 2008).
Biofilters can be divided into the following types: trick-ling (aerobic and anaerobic), rotating, fluidised and im-mersed (aerobic – aerated and anaerobic) (Suschka, 2000). The figure 5 shows biofilters permanently immersed under laboratory conditions. The packing material for the biofilters is chemically modified ceramic aggregate (which is aerated).
Biofilters can be used to reduce suspended bacteria and to clear turbidity in purified wastewater. In addi-tion, their adsorption capacities allow removal of or-ganic impurities and coloured compounds.
Characteristics of selected biofilters
Disc biofilters, characterised by the unfolded, smooth surface of discs are a common example of im-mersed biofilters (fig. 6). They are partially imim-mersed by their slow rotation in a cylindrical sewage-filled vat. Biofilm is formed on the surface of the discs and is aer-ated by exposure to air. During the immersion phase, aerobic conditions are provided in the vat (Bever et al., 1997). Excess biofilm is released by lateral forces from rotation of discs. Then, biofilm fragments with the puri-fied wastewater migrate to the secondary settling tank. On the first discs the biofilm reaches a thickness of 1.5 to 3 mm and should be light brown. In the next section it can take on a reddish or golden hue and its thickness is reduced (Miksch et al., 2012). Disc type biofilters are used to remove organic carbon and for nitrification. The disks are typically made of plastic, such as PVC, poly-styrene or polyethylene.
Fig. 5. Biofilter — permanently immersed ceramic packing under laboratory conditions
Photo by E. Łaskawiec.
Fig. 6. Disc type biofilters in a household BioDisc® sewage treatment plant with and without biofilm
Source: http://www.ecoprius.pl/kanalizacje-i-domowe-bio-oczyszczalnie-sciekow/domowe-biologiczne-oczyszczalnie-sciekow/przy-domowe-biologiczne-oczyszczalnie-sciekow-biodisc-ba-bd.html
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Immersed biofilters are characterized by high op-erational stability and their ability to adapt to changing conditions. Lack of need for artificial aeration reduces the water treatment cost. For these reasons, solutions of this type are often used in home sewage treatment plants (Ignatowicz et al., 2011).
Conventional trickling filters represent a large group of methods using fixed biofilters. Originally, the packing was large stones of 5 to 20 cm or even crushed lava due to its high porosity. Currently, the most popu-lar packing is plastic tiles or packages (Heidrich, 2008; Henze 2002). Wastewater is distributed over the sur-face of the biofilter and then it runs down through the packing mass and is then collected and discharged from the base. Nutrients from wastewater reach bacte-ria through diffusion and membrane-exchange. Plastic tiles are characterised by high hydraulic permeability and a highly developed active surface with little car-rier mass (fig. 7). Producers of carcar-riers offer products manufactured from a variety of materials and in many shapes, which allows technology to be tailored to meet the needs of the treatment plant.
Unlike trickling filters, permanently immersed fil-ters use packing material in the wastewater tank. Re-moval of treated wastewater from the biofilter is pe-riodic. As for other biofilters, the packing material is used for biomass growth. Most often it is in the form of packets of plastic material, which provide a large sur-face area for microorganisms. This method can be used for decomposition of carbon compounds, nitrification and denitrification (Papciak et al., 2005). An example of packing used for such a method is the polyethylene tube mesh structure, BIO – NET® (fig. 8). This design ensures free and even flow of air and wastewater. The oxygen supply can be provided by disc membrane diffusers in-stalled in the base of the biofilter. This also allows wash-ing of the biofilter, detachment of biofilm fragments and the removal of excess sludge (Papciak et al., 2005; Heidrich, 2006).
Conclusions
Biofilms are a subject of interest to scientists from many disciplines. They have both positive and adverse effects on the environment and living organisms.
An understanding of the biofilm phenomenon is particularly valuable in wastewater treatment technol-ogy. Monitoring activity of microorganisms populating the biofilter packing can provide much useful informa-tion about their activity. To control wastewater treat-ment effectively using microscopic methods, a detailed knowledge about the microbes living in the biofilm is required. Although this method is labour-intensive, it allows direct observation of changes in species and their numbers, which may indicate operational instability of a biofilter, and warn of failure of the purification process.
A wide variety of biofilter designs allows the choice of their structure to suit virtually any conditions for purification. This allows the increasing use of
biologi-cal methods of sewage treatment and water treatment, thus reducing use of chemical methods. They also offer the advantage of a reduced quantity of sludge produced, which otherwise often requires time-consuming pro-cessing.
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Compatability with
the Polish core curriculum:
Supplementary subject Nature, IV educational stage Educational goals – general requirements:
Understanding the scientific method involving posing hypothe-ses and their verification by means of observations and experi-ments.
Contents of education – detailed requirements: 13. Technologies of the future.
Geography, IV educational stage, extended scope Educational goals – general requirements:
III. Proposing solutions to problems occurring in geographical environment, in accordance with the idea of sustained balance and cooperation principles, international included.
A student indicates proposals for local, regional and global so-lutions of environmental, demographic and economic problems consistent with the idea of sustained balance and based on equal principles of co-operation between regions and countries. Contents of education – detailed requirements:
6. Spheres of Earth – pedosphere and biosphere. A student: 6) discusses basic principles of sustained development and judges
possibilities of their realization on a local, regional and global scale.
