Aquatic Biofilms and their Element Enrichment
Sorption Effects of Certain Elements in Seasonal Biofilms of a Drainage Tunnel
Biofilms appear where the smallest micro-organisms enter into synergistic relationships. In some cases they create amazing protection mechanisms against potentially harmful substances, making them interesting subjects for research. How do these organisms manage to survive and grow in water found in a mine and therefore in part, strongly affected by mining activities?
A prerequisite for the cohesion of the micro-organisms is the formation of a polymer matrix - called the "Extracellular Polymeric Substance" (EPS). This is mainly made up of macromolecular polysaccharides and proteins along with small amounts of lipids and nucleic acids. They are produced and excreted by the organisms themselves. The transport mechanism to the nutrient supply depends considerably on the nature and diffusion behaviour of this EPS [1,2]. While predominantly aerobic bacteria establish themselves on the exterior of the EPS, anaerobic bacteria find ideal conditions for easy adaptation in the oxygen poor interior [3,4]. These different requirements promote the building of micro consortiums which can adapt perfectly to the environmental conditions . In aquatic biofilms, the more or less large caverns are permeated with liquid medium and therefore fed with nutrients and oxygen down to the lowest levels.
The Protective Mechanism of Biofilms
The colonisation of every kind of interface enables the biofilms their universal presence, however a stable surface in a moist environment is preferred [1,5,6]. As wave resistant bodies consisting of producers and decomposers, aquatic biofilms are often the single basic nutrient for more highly developed organisms. Making them therefore, an important part of the food chain in aquatic ecosystems. However they can react extremely sensitively to a high increase in element concentration. While some metals such as copper and zinc, depending on their concentration, are essential for aquatic organisms too, others such as lead and cadmium are toxic even in small quantities . Many aquatic biofilms have developed a selective detoxification mechanism as a protective tactic against extremely toxic elements.
They produce intracellular molecules, called phytochelatines, which bind surplus, accumulated heavy metals making them no longer available or harmful. This chelating depends on certain parameters such as nutrients, light and temperature being fulfilled as well as a high concentration of elements in the water.
Previous investigations in surface water and in the aquatic biofilms of mining influenced water (the drainage system of a decommissioned copper mine) showed an increased concentration of the elements Pb, Zn, Cu und Ni . As a result of this, the question was raised - To what extent is the accumulation behaviour of these elements dependent on the different sorption conditions in the biofilm?
According to this, the elements are mainly in a dissolved form in the watery section of the tunnel exit. This surface water offers a surplus of Pb-, Zn-, Cu- und Ni-ions which are more easily available to the biofilms [7,10] and can therefore be more easily accumulated. In addition, as the seasonal temperatures remained constant in the water of the mine shaft there was no reason to expect that any or even a few of the biofilms might die. In accordance with Mages et al. the biofilms were bred on special polycarbonate substrates along the length of the mine shaft exit. Samples were taken at different time intervals of between one and nine months.
Along with the artificially created biofilms from the drainage system of the mine shaft there was also the possibility to investigate the sorption behaviour of several elements in the biofilms which had grown naturally. It is a well-known fact that expiring interactions between solid / liquid interfaces are referred to as sorption processes . In the process, sorbents and sorbed substances can form a chemical compound or be accumulated elsewhere.
The verification of the element distribution, or rather the accumulation pattern in the biofilm, continued to seem particularly interesting. In addition circular poly-carbon substrates with dried growth were enclosed in an epoxy resin then cut and polished. Subsequently parts of the surface were scanned using µ-XRF (micro X-ray fluorescence analysis M4 Tornado, Bruker Nano, Berlin).
Total Reflection X-ray Fluorescence Analysis
The determination of the elements in the biofilms was achieved using total reflection X-ray fluorescence (TXRF). Basic physical principles constitute classic X-ray fluorescence analysis and in the process the use of primary X-rays. In this case X-ray means electro-magnetic radiation with photon energy in the keV range. The resulting X-ray spectra consist of overlapping yet discrete lines which are characteristic for the individual elements of each sample. These lines always stand in the same elementally specific ratio and result in an element spectrum. During simultaneous stimulation of the elements that can be detected, all lines increase in intensity to the same extent .
On applying the TXRF it is possible to simultaneously, qualitatively record and, with a high level of precision into the pg-range, quantify a broad spectrum of elements in the periodic table. The low amount of consumables needed, offers a very good alternative to the usual spectroscopic analysis methods for element determination such as AAS and ICP-MS. On top of this is the high versatility, particularly in the analysis of biological sediment samples which are often in short supply (lower µg range) [13,14].
Even the results of the distribution of elements in the surface water show very large differences. Although the mine shaft was a drain for copper mining, dissolved Pb at 713 µg l-1 and Zn at 18 mg l-1 are the dominant elements in the aqueous phase. The concentrations of Cu and Ni at 326 and 233 µg l-1 respectively are also high but lower in comparison to Pb und Zn. Moreover, as a result of the tested nutrients, the mine shaft water could be considered oligotrophic.
Figure 2 shows the content of elements in relation to the growth time. The first samples were analysed four weeks after the substrates were distributed. Even at this early stage of the growth period, significant differences in the accumulation behaviour could be confirmed. In comparison with the element concentration confirmed in the filtrate (from Zn via Pb and Cu to Ni in decreasing order), it is clear that the absorption doesn't only depend on the element concentration in the surface water but substantially on the availability of space for sorption .
While Pb und Ni were strongly absorbed form the outset, the absorption of Zn and Cu was more reserved. In contrast, in 2003 Meylan et al. proved in their investigation of element accumulation in algae biofilms that said biofilms accumulate the metals Cu and Zn very quickly and release them then slowly back into the medium .
Investigations into samples taken after a further 14 days of growth lead to the conclusion that element accumulation is subject to strong dynamical development. A relatively fast reduction in the Pb and Ni content occurred thereby favouring the accumulation of Zn and Cu. After a further 14 days the plateau was reached for all sorbents in the waters. Subsequent samples taken showed no further significant changes. Evidence of a very high accumulation of Pb was found in the biofilm independent of deposition or growth time. Based on a concentration of 713 µg l-1 of Pb in the surface water and the comparison with the analysed lead contents in the biofilm of 127 to 531 mg g-1, a temporary maximum accumulation potential with enrichment factors of up to 750,000 becomes clear. On the basis of the element distribution or accumulation pattern shown in figure 3, it can be clearly seen that the elements Al, K, Ca, Si and Mn are either accumulated or stored in either colloidal or particle form. In contrast, the elements Pb, Fe, Cu, S and Zn are organically embedded.
Evaluation of the Sorption Effect in the Biofilms Investigated
The nutrients necessary for the growth of the biofilms were mainly taken in from the dissolved elements available in the mine shaft water. According to Collins und Stotzky the already toxic concentrations of Pb at 713 µg l-1 and Zn at 18 mg l-1 should lead to limitations in cell growth and morphological changes in the cells and as enzyme inhibitors, disrupt photosynthesis . Conversely, investigations by Le Faucher and Sigg revealed that the micro-organisms implement a survival strategy by generating glutathione and therefore phytochelatins which in turn depend on the species composition in the biofilm . These results could be relevant for the biofilms in the mine shaft water and justify the vast growth under partly adverse environmental conditions. According to Wanner und Bauchrowitz, "chemoautotrophic bacteria", which use inorganic substances for their generation, were responsible for the growth of the biofilm. These bacteria are considered "heavy metal tolerant" if strong growth can be observed despite high toxicity. Thereby, the evidently visible strong growth of the EPS (fig. 1) would serve as a protective mechanism and also be at the disposal of the micro-organism as a food depot . Meylan et al. were also able to establish that the absorption of metals relies considerably on the EPS and depending on how quickly this "barrier" is crossed the exchange of free sorption space can take place .
The results of the investigation show the strong dynamics of element enrichment and depletion in aqueous biofilms using waters affected by mining as an example. This illustrates that understanding the accumulation strategy of an organism is paramount to achieving a differentiated evaluation of a bio-available burden on the environment. Hall-Stoodley et al. Hit the nail on the head when they described biofilms as "Dynamic systems with the characteristics of a multifaceted ecosystem."
 Flemming. H.-C. und Wingender J: Biologie in unserer Zeit 31 (3), 169-180 (2001)
 Flemming H-C und Wingender J: Chemie in unserer Zeit 36 (1), 30-42 (2002)
 De Beer D. et al.: Liquid Flow in Heterogeneous Biofilms: Biotechnol. Bioeng., 44, 636-641 (1994)
 De Beer D. et al.: Wat. Res., 30 (11) 2761-2765 (1996)
 Costerton J.W.: J. Antimicro. Agents, 11, 217-221 (1999)
 Flemming. H.-C. und Wingender J.: Nature Reviews Microbiology 8, 623-633 (2010)
 Behra R. et al.: Eawag News: Biofilme, 60d, 16-18 (2005)
 Le Faucheur S. et al.: Eawag News: Biofilme, 60d, 22-23 (2005)
 Mages M. et al.: Spectrochim. Acta B 61, 1146-1152 (2006)
 Meylan S. et al.: Enviro. Sci. Techn. 37, 5204-5212 (2003)
Further literature is available from the author.
Dr. Margarete Mages
Herr PD Dr. Wolf von Tümpling
Helmholtz-Zentrum für Umweltforschung GmbH-UFZ