Designing a Winogradsky Column to Simulate, Enrich and Predict Microcosm Biofilm Patterns
Building a Tool to Grow Microbes As They Are
Master's Thesis 2006 46 Pages
Review of Literature
Materials and Methods
Marine and freshwater environments create unique niches for many specialized microorganisms needing habitats with a continuous water phase (Fenchel T.,1969).
The mixing and movements of nutrients, O2 , and waste products that occur in freshwater and marine environments are the dominant factors controlling the microbial community. In deep lakes or oceans, organic matter from the surface can sink to great depths, creating nutrient-rich zones where decomposition takes place. Gases and soluble wastes produced by microorganisms in these deep marine zones can move into upper waters and stimulate the ability of other microbial groups. Similar processes take place on a lesser scale in nutrient-rich lakes, and even in microbial mats, where gradients are established on a scale of millimeters (Gorienko et al. 1978).
The Winogradsky Column devised by Sergei N. Winogradsky (1856-1953), forms a microcosm where microorganisms and nutrients interact over a vertical gradient. Fermentation products and sulfide migrate up from the reduced lower zone, and oxygen penetrates from the surface. This creates conditions similar to those in a lake with nutrient rich sediments. Light is provided to simulate the penetration of sunlight into the anaerobic lower region, which allows photosynthetic microorganisms to develop (Anderson et al. 1999).
The basic concept is to establish opposing gradients of sulfide and oxygen in mud that is incubated in the presence of light, thereby giving an opportunity for a variety of phototrophic and chemotrophic sulfur oxidizers to establish themselves at various points along these gradients (Anderson et al. 1999).
A series of reactions occurs as the column begins to mature, with particular members of the microbial community developing in specific microenvironments in response to chemical gradients (Hemanth , 2006).
Biofilms are microbe mats formed when the microbes secrete polysaccharide and get embedded in them. The importance of biofilms is that they are visible and colorful and they can be used to understand the nature of the microenvironment (Gorienko et al. 1978).
Development of Biofilms is an important factor in microbial growth. Depending on particular microbial growth environment, these biofilms can become more complex (H.Chikarmane, 1992).
By producing biofilms in the Winogradsky column, the succession of microorganisms, nutrient cycling, can be studied by noticing changes in the population. Changes in color of soil reflect hydrogen sulfide production and the type of microbes that appear or disappear with changing levels of oxygen and hydrogen sulfide. (Anderson et al, 1999)
1 To device methods to keep track of the changing biofilm and color patterns and obtain mathematical data.
2 To device method to analyse and predict the biofilm patterns.
3 To test the relationship of change in environment factors and the biofilm patterns including degradation of hard substrates.
4 To improve the efficiency of simulation by manipulating the factors.
5 To design a Winogradsky Column that simulates, enriches and predicts Microcosm Biofilm Patterns
6 To analyse the potential of this tool in the field of Microbial Ecology and Biofilm Technology.
REVIEW OF LITERATURE
RELATION OF ENVIRONMENT AND MICROBES
“Everything is everywhere, the environment selects” - (M.W. Beijerinck)
Ecosystem is a community of organisms and their physical and chemical environment that functions as an ecological unit (Fenchel T.,1969).
Microorganisms exist in natural habitats both as populations of similar types of organisms such as micro-colony growing at a localized site, and as communities made up of different types of interactive populations (Overmann J., et al. 1989).
The microbial environment is complex and constantly changing. It is characterized by the presence of overlapping gradients of resources, toxic materials, and other limiting factors. Gradients can form when aerobic and anaerobic regions intersect or when a lighted zone changes into a dark region, creating unique microenvironments. Where the conditions in a microenvironments are suitable, specialized groups of microorganisms can maintain themselves with minimum competition from other microorganisms that have slightly different functional requirements (Gorienko et al. 1983).
Microorganisms will grow in such “microenvironments” until an environmental or nutritional factor limits this process, an expression of Liebig’s Law of the minimum. Factors that can limit microorganisms (and other living organisms) include water, energy in the form of light and chemical compounds, temperature, nutrients, pressure, pH, and salinity. The situations may be more complex than this. Multiple limiting factors can change over time and space. Too much of some factors (metals, salts, hydrogen ions, heat) also can limit microbial activity, an expression of Shelford’s law of tolerance (Lindholm T., 1987)
“Microenvironments” is a critical point that was emphasized by Sergei Winogradsky. Most microorganisms are confronted with deficiencies that limit their activities except when excess nutrients allow unlimited growth. Such rapid growth will quickly deplete nutrients and possibly result in the release of toxic waste products, which will limit further growth (May et al. 1983).
In response to low nutrient level and intense competition, many microorganisms become more competitive in nutrient capture and exploitation of available resources. Often the organism’s morphology will change in order to increate its surface area and ability to absorb nutrients. This can involve conversion of rod-shaped bacteria to “mini” and “ultramicro” cells. Specific and nonspecific attachment to surfaces also will increase, creating biofilms. These biofilms allow microorganisms to use nutrients that often are present at higher localized concentrations on surfaces (Pfenning N., 1978).
Biofilms consist of microorganisms immobilized at a substratum surface and typically embedded in an organic polymer matrix of microbial origin. They develop on virtually all surfaces immersed in natural aqueous environments, including both biological and abiological. Biofilms form particularly rapidly in flowing aqueous systems where a regular nutrient supply is provided to the microorganisms. Extensive microbial growth, accompanied by excretion of copious amounts of extracellular organic polymers, thus leads to the formation of visible slimy layers (biofilms) on solid surfaces (Pfenning N.et al. 1989).
Use of waste products of one group of microorganisms by other organisms is a commensalic relationship that often is seen in natural environments. An excellent example of such relationships is the Winogradsky column. On the basis of these interactions, microbial use of available oxidants can be understood, predicted and managed (Gloyna E.F., 1978).
Organic substrates differ in their susceptibility to degradation. Factors influencing degradation are elemental composition, structure of basic repeating units, nutrients present in the environments, abiotic conditions (pH, oxidation - reduction potential, oxygen, osmotic conditions), microbial community present (Bharati P.A.L., 1982).
Hydrocarbons are unique in that microbial degradation, especially of straight-chained and branched forms, involves the initial addition of molecular oxygen. Recently, anaerobic degradation of hydrocarbons with sulfate or nitrate as oxidants has been observed. With sulfate present, organisms of the genes Desulfouihňo sp. are active. This occurs only slowly and with microbial communities that have been exposed to these compounds for extended periods. (Caldwell D.E.. et al. 1975).
Patterns of microbial degradation are important in many habitats. They contribute to accumulation of petroleum products, the formation of bogs, and preservation of valuable historical objects. The situation in natural environments with mixed populations is much more complex. Here, where often only 1 to 10% of observable cells are able to form colonies, the microbiologist is attempting to grow microorganisms that perhaps never have been cultured or characterized. Their lack of growth leads to the use of other terms; that of being “uncultured” or “as yet uncultivated”. In the future it is possible that media or proper environmental conditions for their growth will be developed (Caumette P., 1984).
Microorganisms vary greatly in their tolerance for pH, oxidized versus reduced environments, temperature, pressure, salinity, water availability, and ionizing radiation. Thus environmental factors greatly influence the survival of any particular microorganisms. An extreme environment is one in which physical or chemical conditions become even more restrictive, resulting in a decrease in the diversity of microbial types that can maintain themselves.
Under particularly restrictive conditions this process can continue until only a single type of microorganism, essentially a monoculture exists (Caldwell D.E.. et al. 1975).
Statistics are becoming critical for all these techniques because of increased demands for reliable results, quantitative sampling, and selection of the best sampling size and number of replications (Caumette P., 1984).
Enrichment culture entails devising a culture medium and physical- chemical conditions that favor growth of a class of organisms of interest or even a single species. Physical and chemical factors can determine which organism predominates and can alter the effects of enrichment. The source of inoculum is also important. It has been said that every species of microbe can be found in a single gram of soil. Indeed, many organisms that might not be expected in aerobic soils, such as some obligate anaerobes and thermophiles, are found in sufficient numbers for a positive enrichment. You need only a single viable organism. In practise, it is a good idea to use an inoculum from a habitat likely to contain organism of interest. You can design them rationally but subtle and unknown details, rather than theory, decide which organism turns up (Baas L.G.M., et al. 1956).
In the anaerobic bottom of the column, cellulose is hydrolyzed to oligosaccharides and sugars, which are fermented by Clostridium sp. into organic acids and hydrogen. Clostridium sp. is visible as white patches. The fermentation products (reductant) and sodium sulfate (oxidant) used by sulfate reducing bacteria Desulfouihrio sp. and HnS is produced which diffuses upwards. Gas Bubbles are seen and H2S gradient is established. The mud turns black due to iron sulphide precipitate. Sodium Carbonate establishes a CO2 gradient. Green sulfur chlorobiurn sp. Photoautotroph and Sulphide tolerant, grows in the H_»S dominated anaerobic zone using H2S electron source as reductant for fixing CO2. They form olive green band. Purple sulfur bacteria Chromatium sp. is less sulphide tolerant and grows above. In the anaerobic region where sulphide levels are low, purple non-sulfur bacteria Rhodospirillum sp. and Rhodopseudomonas sp. photoheterotrophs grow using organic matter as electron donor. They are visible as rust coloured bands. At the mud water interface where the Oxygen and sulphide gradients meet, aerobic sulphide oxidizers Thiobacillus sp. and Beggiatoa sp. grow. They use oxygen to oxidize sulphide and produce .sulphate. They are responsible for cloudiness of water. They appear as whitish veil. In the water layer, cyanobacteria and algae and diatoms grow (Anderson et al, 1999) .
Winogradsky Columns are being used to simulate Mars environment’s possible biofilm patterns (NASA QUEST).
Two unexplored areas in science of biofilms are
(1) A natural substitute for Artificial Neural Networks - a tool to predict biofilm patterns.
(2) A tool to degrade hard substances, xenobiotics and antibiotics.
(3) Biofilms show high frequency gene transfer based evolution for degradation (Biofilm Online).
The Family Chromatiaceae bacteria are being used in a number of biotechnological processes like production of single cell protein, sewage and effluent treatment, sulfide removal and sulfur production, production of molecular hydrogen and production of organic molecules (Pfenning, 1986).
A number of bacteria has been isolated and characterized from a Winogradsky Column. (Evans et al, 1999)